17th International Congress of Virology

ABSTRACT BOOK AS OF 28 AUGUST 2017

17th International Congress of Virology 1

17 July 2017 Oral Presentation

17th International Congress of Virology 2

Speaker Abstract Submission

100 years of Bacteriophages

An outsider’s critical appraisal of prokaryotic virus taxon and virus nomenclature

J. H. H. Kuhn*

Objectives: Taxonomy consists of two subdisciplines: classification and nomenclature. Nomenclature is the process of naming taxa (concepts of the mind) and their physical members (organisms, viruses) in adherence to official taxonomic codes. In virology, nomenclature rules are stipulated in the International Code of Virus Classification and Nomenclature (ICVCN). Unfortunately, these rules are loosely written, thereby allowing different interpretations. Diverse interpretations led to different types of naming in different virologic subdisciplines. Here I will provide specific examples of my concerns with the current prokaryotic virus taxon and virus nomenclature.

Methods: Names of all prokaryotic taxa currently accepted by the International Committee on Taxonomy of Viruses (ICTV) and a representative sample set of taxon member names were analyzed for their adherence to the ICVCN and compared to taxon and taxon member names in use in other virological subdisciplines.

Results: Prokaryotic virus taxon and virus nomenclature still differ in several ways from ‘standard’ (i.e., non-prokaryotic) virus taxon and virus nomenclature. For instance, the majority of prokaryotic taxon names contain typically acronymic letter and number combinations directly fused to rank-specific suffixes (e.g., genus Cd119virus) whereas such combinations are rarely, if ever, used, in eukaryotic virus nomenclature (e.g., genus Adenovirus). Prokaryotic taxon and taxon member names also typically contain references to the virus hosts in the form of species names that are prone to changing, and prokaryotic virus species names often contain letter/number combinations that in eukaryotic nomenclature would indicate sub-virus strains or isolates (e.g., Mycobacterium virus Bxz1 versus Kolente ledantevirus strain DakArK7292). Conlusion: Prokaryotic and eukaryotic virus taxon and taxon member nomenclatures differ considerably, and substantive changes in both subdisciplines will be necessary to homogenize and standardize virus taxonomy.

Keywords: Virus classification, virus nomenclature, virus taxonomy

17th International Congress of Virology 3

Speaker Abstract Submission

100 years of Bacteriophages

Prophage induction: A Trojan Horse Approach to Control Salmonella on foods

Brigitte Cadieuxa, Elizabeth Tompkinsa, Anna Colavecchioa, Julie Jeukensb, Luca Freschib, Jean-Guillaume Edmond Rheaultb, Irena Kukavica-Ibruljb, Roger C. Levesqueb, Lawrence D. Goodridgea aFood Safety and Quality Program, Department of Food Science and Agricultural Chemistry, McGill University, Ste Anne de Bellevue, QC, Canada bInstitut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, QC, Canada

Keywords: Salmonella, fresh produce contamination, antimicrobials, prophage induction

In North America, Salmonella is recognized as the causative agent of almost 50% of outbreaks in which fresh produce is the vehicle. There is an acute need to develop methods to reduce the presence of Salmonella on fresh produce, prior to human consumption. Temperate bacteriophages (prophages) have been found within genomes of the majority of Salmonella enterica. Many of these prophages can be induced, resulting in lysis of their host. The goal of this study was to investigate prophage induction as a novel approach to control Salmonella on fresh produce. Salmonella Typhimurium and Newport isolates were exposed to mitomycin C, and growth of the cells was monitored by measuring the optical density (OD600). Detection of phage-specific integrase genes by PCR was used to confirm the release of induced prophages from their host. The ability of mitomycin C to induce prophages in S. Typhimurium and Newport was evaluated by inoculating tomatoes or spinach with 5x107 and 5x108 cells, respectively. Mitomycin C was sprayed onto each sample before recovering the bacterial cells and performing plate counts. PCR confirmed release of prophages in bacterial lysates. A 1-log reduction in S. Typhimurium and 1.5-log reduction in S. Newport were observed on tomatoes sprayed with mitomycin C compared to tomatoes sprayed with water, while a 2-log reduction was obtained for both serovars on spinach. Since mitomycin C is an antibiotic, it can not be used as a prophage inducer on fresh produce. We therefore developed a high-throughput screening (HTS) method to identify food-grade prophage inducers. The HTS method is based upon the use of a genetically-modified Escherichia coli strain (ATCC 33312) in which the lacZ gene has been incorporated into the phage lambda chromosome. Upon exposure to prophage inducers, the E. coli strain produces elevated levels of β-galactosidase which is measured with a luminescent substrate. In the presence of various inducers, the amount of β-galactosidase produced was monitored over time by mixing the induced cells with the Beta-Glo luminescent substrate and measuring the luminescence produced. Mitomycin C and streptonigrin (known prophage inducers), as well as concentrated coffee and tea, were shown to be potent inducers in the screening assay, and generated high luminescence values. These findings provide a proof of concept that prophage induction can effectively control Salmonella on fresh produce. Future work will focus on identifying additional food-grade prophage inducers using the high-throughput screening method described here.

17th International Congress of Virology 4

Speaker Abstract Submission

100 years of Bacteriophages

The last 30 years of Bacteriophage and Lytic Enzymes

D. Trudil*

Objectives: To determine the effectiveness and utilization of bacteriophage and lytic enzymes with specific bacterial samples and in human, animal and biocontrol applications.

Methods: Various phage lytic enzymes including PlyC, Staph Aureus, Group B Streptoccus and listeria as well as bacteriophage sb1 and phage cocktail preparations where challanged with laboratory samples. Additionally, phage preparations were used in animal use as well as in human therapuetic applications.

Results: The bacteriophage and lytic enzymes demonstrated on average 97% killing in culture samples with Human, animal and biocontrol demostarting 80 -98% effectiveness.

Conclusion: The war between bacteria and other disease causing agents continues. In the face of new and emerging bacterial pathogens, the widespread distribution of anti-biotic resistance, new approaches are needed. Bacteriophage therapies have been used in human therapeutic applications in the Former Soviet Union with thousands of patients for years. Now in the West, under FDA compassionate care use, we have seen equally impressive results. In addition to their rapid and specific lytic activities, lysins are also attractive compounds. They are highly specific and fast acting, usually causing lysing in seconds. Their recognition portion may be more specific than monoclonal antibodies. It can also be noted that due to the modular design of lysins, with their independent functional domains, this makes them ideal for domain-swapping studies in which bacterial specificities and catalytic activities are improved or adapted for use with other pathogens. Also, as the developmental time for bacteriophage and lytic enzymes is relatively short, this technology is ideal for new or emerging bacterial threats. Considering their long history, they are already one of the most successful antimicrobial agents on Earth. The natural, “green” Bacteriophage, in specific applications, have been GRAS (Generally Regarded as Safe) approved by the FDA. The lytic enzyme is not harmful to humans or sensitive equipment. These technology strategies, should continue to be looked at as an effective approach to control bacteria for human, animal and food pathogen applications.

Keywords: Antibiotic resistance, Bacteriophage, lytic enzyme

17th International Congress of Virology 5

Speaker Abstract Submission

100 years of Bacteriophages

Bacteriophages as biological control agents in agriculture, horticulture, husbandry and aquaculture

D.İ. Kurtbӧke

GeneCology Research Centre and Faculty of Science, Health, Education and Engineering, School of Science and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia

Plant and animal diseases caused by bacteria often result in serious economic losses. Disease control is best achieved when integrated pathogen control measures combining few different pathogen control strategies are implemented. One of these strategies involves the use of bacteriophages to eliminate pathogens infesting plants or animals. Bacteriophages are specific viruses only infect closely related bacterial species, their production costs are low and biocontrol preparations can be prepared with ease making them ideal pathogen control agents replacing environmentally unfriendly chemical control agents. The effectivity of bacteriophage control agent however, is defined by surrounding environmental factors as well as the susceptibility of pathogenic bacterial hosts at the infection site. Thus, successful implementation of phage-mediated disease control strategies should take into consideration the dynamic processes of natural settings. This presentation will provide Australian examples where bacteriophages were used as biocontrol agents ranging from enteric bacterial infested strawberries to edible oysters.

17th International Congress of Virology 6

Abstract Submission

Virus entry, fusion, assembly & budding OR156

STRUCTURE OF A PENTAMERIC VIRION-ASSOCIATED FIBER WITH A POTENTIAL ROLE IN ORSAY VIRUS ENTRY TO HOST CELLS

Y. Fan 1, Y. Guo 1, W. Yuan 1, Y. Liu 1, W. Zhong 1, Y. J. Tao* 1BioSciences, Rice University, Houston, United States

Objectives: Despite the wide use of Caenorhabditis elegans as a model organism, the first virus naturally infecting this organism was not discovered until six years ago. The Orsay virus and its related nematode viruses have a positive-sense RNA genome, encoding three proteins: CP, RdRP, and a novel δ protein that shares no homology with any other proteins. δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but the structure and function of both δ and CP-δ remain unknown.

Methods: A combination of electron microscopy, X-ray crystallography, computational and biophysical analyses as well as reverse genetics have been used to probe the structure and function of the δ/CP-δ proteins.

Results: The Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal α-helical bundle, a β-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the δ fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-δ shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-δ fibers were generated by organism-based reverse genetics. These viruses were found to be either non-viable or with poor infectivity according to phenotypic and qRT-PCR analyses. Furthermore, addition of purified δ proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner.

Conclusion: Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ functions as a cell attachment protein to mediate Orsay entry into worm intestine cells.

Keywords: None

17th International Congress of Virology 7

Abstract Submission

Virus entry, fusion, assembly & budding OR157

GENERATION OF SINGLE-ROUND INFECTIOUS ENTEROVIRUSES A SPECIES WITH ENTEROVIRUS A71 REPLICON

S. Zhang 1,*, M. Wang 1, J. Yan 1, Y. Zhang 1 1Scientific Research Center, Shanghai Public Health Clinical Center, Shanghai, China

Objectives: Single round infectious particles (SRIPs) are useful tools for studying viral entry, antiviral drug screening, serology studies and vaccine development etc. However, SRIPs for Human Enteroviruses A species, which are responsible for hand, foot, and mouth diseases (HFMD) pandemics, are not available yet.

Methods: Enterovirus A6, A10 and Coxsackievirus A16 capsid genes were cloned from viral genomic RNAs. trans- encapsidation was assayed by cotransfection of 293T cells with a plasmid expressing T7 RNA polymerase, EVA71 subgenomic replicon cDNA generated by PCR and the plasmids encoding viral capsid proteins. The viral infectious activity and specificity were measured by luciferase activity and neutralizing assay.

Results: Successful SRIPs of Enterovirus A6, A10, A71 and Coxsackievirus A16 were generated through trans- encapsidation and the replication of the SRIPs was verified by luciferase activity in infected RD cells. In addition, the infectious activity of SRIPs was also examined in SCARB2-knockout RD cells. We found SRIPs of EVA6 and EVA10 but not EVA71 and CVA16 could infect knockout cells, which is consistent with the wide-type viruses. To further confirm the specificity of the SRIPs, we incubated SRIPs with corresponding specific antisera or control sera prior to infection. The results showed that infectivity was only abrogated after incubation with serum against the specific capsid protein of the SRIPs.

Conclusion: Trans-encapsidation can occur between Human Enteroviruses A species and enable efficient production of SRIPs, which are useful tools for further HFMD research.

Keywords: Enterovirus, single round infectious particles, trans-encapsidation

17th International Congress of Virology 8

Abstract Submission

Virus entry, fusion, assembly & budding OR158

Vaccinia viral A26 protein contains a pH-sensitive domain that is critical for mature virus endocytosis

H.-W. Chang 1, H.-Y. Cheng 1, D.-L. M. Tzou 2, K. J. D. Carillo 3, W. Chang 1,* 1INSTITUTE OF MOLECULAR BIOLOGY, ACADEMIA SINICA, TAIWAN, 2INSTITUTE OF CHEMISTRY, ACADEMIA SINICA, TAIWAN, 3INSTITUTE OF CHEMISTRY, ACADEMIA SINICA, TAIWAN, Taipei, Taiwan

Objectives: Vaccinia mature virus (MV) enters cells through either endocytosis or plasma membrane fusion, depending on cell types and virus strains. Our previous studies showed that WR strain of vaccinia mature virus enters HeLa cells through fluid phase endocytosis. Interestingly, when we deleted an ORF encoding a MV membrane A26 protein from vaccinia viral genome the resulting deletion virus, WR-ΔA26, infects HeLa cells through plasma membrane fusion. We thus hypothesized that A26 protein may act as an acid-sensitive membrane fusion suppressor protein that controls virus entry pathway specificity. Here we aim to dissect the structure domain of A26 protein in order to understand how it functions as an acid- sensitive regulator of membrane fusion.

Methods: We thus generated a series of recombinant vaccinia viruses that contains various deletions of A26 protein. We then purified these mutant vaccinia MV particles and investigated how these mutant viruses enter cells. In addition, we also generated additional site-directed mutagenesis of A26 ORF in order to specifically address specific residues in A26 protein in virus entry process.

Results: Our genetic data revealed that the N-terminal region of A26 protein is critical for the low-pH-mediated conformational change during virus entry through endocytic pathway. Consistently, a recombinant protein containing the N- terminal portion of A26 protein also exhibited a pH-dependent conformational change in vitro

Conclusion: Together our data provide important genetic and biochemical evidence supporting that A26 protein functions as an acid-sensitive fusion suppressor.

Keywords: A26 protein, entry regulator, vaccinia virus

17th International Congress of Virology 9

Abstract Submission

Virus entry, fusion, assembly & budding OR159

VACCINIA VIRUS PROTEINS A36 AND F12/E2 SHOW STRONG PREFERENCES FOR DIFFERENT KINESIN LIGHT CHAIN (KLC) ISOFORMS

W. Gao 1,*, D. Carpentier 1, H. Ewles, S.-A. Lee, G. Smith 1 1Pathology, University of Cambridge, Cambridge, United Kingdom

Objectives: Vaccinia virus (VACV), the prototypic orthopoxvirus, utilises microtubule-mediated trafficking at three distinct stages of its life cycle, including viral egress. Transport of triple-enveloped intracellular enveloped virions (IEVs) to the cell surface is the best-characterised process, although the regulatory mechanisms and viral and host proteins involved are poorly understood. The IEV-associated proteins A36, F12 and E2 are involved in this process. Deletion of the F12L or E2L genes causes a drastic reduction in IEV egress, while deletion of the A36R gene causes a less severe inhibition. Previous studies have shown that ectopically-expressed portions of A36 interact with kinesin light chain (KLC), and that the F12/E2 complex interacts preferentially with the KLC isoform 2. Here we investigate interactions between all of the IEV-associated VACV proteins and the kinesin-1 motor to better understand VACV egress and the role of kinesin-1 in this process.

Methods: Interactions between IEV-associated viral proteins and KLC isoforms 1 and 2 were studied using co- immunoprecipitation of ectopically expressed KLC and viral proteins expressed at endogenous levels during infection. This identified A36, F12 and E2 as binding partners for KLC but with distinct KLC isoform preferences. KLC1/KLC2 chimeras and truncations were then designed to identify the specificity determining regions in KLC for these VACV proteins using the same techniques. We also tested whether the interaction of A36 with KLC1 was affected by the presence of F12 or E2.

Results: The results support previous observations that F12/E2 interacts preferentially with KLC2, but surprisingly showed that A36 when expressed at endogenous levels during infection interacts strongly with KLC1 and showed almost no binding to KLC2, contrary to previous reports showing interaction with both isoforms. The F12/E2 interaction with KLC2 was mapped to a region of the KLC2 C-terminal tail that overlaps the 14-3-3 binding site. The F12/E2 interaction was shown to be independent of 14-3-3 binding. The specificity-determining region in KLC1 for A36 was mapped to the heptad repeat (HR) region. The F12/E2 complex was also shown to enhance the A36 – KLC1 interaction, suggesting the three IEV proteins act cooperatively to mediate trafficking.

Conclusion: We propose a model where VACV proteins A36 and F12/E2 interact with different KLC isoforms and different regions to mediate activation of the kinesin-1 motor and trafficking of the fully formed IEV to the cell surface. We also show that F12/E2 expression enhances the A36 – KLC1 interaction, to our knowledge this is the first example of a pathogen encoding different proteins that cooperatively interact with distinct domains of KLC, providing insight into host cell regulation of cellular trafficking and suggesting other pathogens may employ similar methods.

Keywords: Egress, Kinesin-1, VACV

17th International Congress of Virology 10

Speaker Abstract Submission

Paramyxoviruses I

COMPARATIVE LOSS OF FUNCTION SCREENS REVEAL COMMON CELLULAR PATHWAYS REQUIRED BY PARAMYXOVIRIDAE AND PNEUMOVIRIDAE

D. Anderson 1,*, S. Y. Kim 2 3, B. Sawatsky 4, K. Pfeffermann 4, J. Pearson 2 3, D. Corcorun 3, L.-F. Wang 1, P. Duprex 5, M. Garcia-Blanco 1 2 3 6, V. von Messling 1 4 1Emerging Infectious Disease, DUKE-NUS GRADUATE MEDICAL SCHOOL, Singapore, Singapore, 2Duke Center for RNA Biology, 3Department of Molecular Genetics and Microbiology, Duke University, Durham, United States, 4Veterinary Medicine Division, Paul-Ehrlich-Institute, Langen, Germany, 5National Emerging Infectious Diseases Laboratories, Boston University, Boston, 6Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

Objectives: Members of the family Paramyxoviridae have recently been re-classified into two families: Paramyxoviridae and Pneumoviridae. Both families include a broad range of respiratory viruses with great relevance for human and animal health, and have similar life cycles and share the respiratory tract as a point of entry. Paramyxoviruses thus exploit aspects of the cellular translation machinery for viral protein synthesis, which may in turn be targeted to specifically treat these viruses.

Methods: To identify cellular proteins involved in the viral life cycle, we performed comparative genome scale siRNA screens with wild type measles, mumps and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line.

Results: Comparative analysis of the top proviral cellular proteins in each screen yielded 42 candidates, which supported growths of all three tested viruses. In addition to coatomer complex I, a known dependency factor, proteasomal degradation, RNA processing pathways and translation were the top pathways required by all three viruses. Among translation factors, ABCE1, a member of ATP-binding cassette transporters was identified as common proviral factor absolutely needed for efficient viral protein translation. ABCE1 knockdown strongly inhibits the translation of MeV mRNAs while only modestly affecting global translation, suggesting that these, and perhaps mRNAs from all Paramyxo- and Pneumoviridae, may be particularly dependent on the function of this protein. Conclusion: This study provides a novel overview of cellular proteins and pathways that impact these important pathogens and identifies potential antiviral targets.

Keywords: ABCE1, Paramyxoviridae, Pneumoviridae

17th International Congress of Virology 11

Abstract Submission

Paramyxoviruses I OR160

DEVELOPMENT OF A NEWCASTLE DISEASE VIRUS VECTOR EXPRESSING A FOREIGN GENE THROUGH AN INTERNAL RIBOSOMAL ENTRY SITE PROVIDES DIRECT PROOF FOR A SEQUENTIAL TRANSCRIPTION MECHANISM

Q. Yu 1,*, Z. Zhang 2, W. Zhao 3, D. Li 2, J. Yang 4, L. Zsak 1 1US National Poultry Research Center, USDA/ARS, Athens, United States, 2College of Life Sciences, Northeast Agricultural University, Harbin, 3Beijing Centre for Disease Control and Prevention, Beijing, 4Chongqing Academy of Animal Sciences, Chongqing, China

Objectives: Newcastle disease virus (NDV), a member of the Paramxoviridae family, has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit (ITU), which potentially attenuates its downstream gene transcription, and subsequently interferes with virus replication and the level of foreign gene expression. To overcome the drawback of the IUT method, in the present study, we developed a novel approach for foreign gene expression by NDV from a second open reading frame (2nd ORF) through an internal ribosomal entry site (IRES).

Methods: The IRES sequence and a red fluorescence protein (RFP) reporter gene were inserted behind the NP, P, M, F, HN, or L gene ORF in a NDV LaSota strain-based infectious clone as a 2nd ORF. Six NDV recombinant viruses vectoring the IRES/RFP gene at each of the six viral genes were rescued using reverse genetics technology. The pathogenicity and growth dynamics of theses six viruses were compared with their parental virus in chicks, embryonating eggs, and cell cultures. The viral mRNAs containing the RFP gene obtained from recombinant virus infected DF-1 cells were detected by quantitative RT-PCR, and the RFP fluorescence intensities in the infected cells were measured by using a fluorescence microplate reader.

Results: The insertion of the 2nd ORF slightly attenuated the virus pathogenicity, but did not affect virus growth ability. Quantitative measurements of the RFP expression from recombinant virus infected DF-1 cells revealed that the abundance of viral mRNAs containing RFP and the red fluorescence intensity were positively correlated with the gene order of NDV 3’ NP>P>M>F>HN>L. The IRES/RFP gene inserted into the 3’ proximal NP gene expressed the highest level of RFP, whereas that inserted into the 5’ proximal L gene expressed the lowest level of RFP.

Conclusion: The results provide direct proof for a sequential transcription mechanism on NDV. The novel NDV vector could be used to express a foreign gene in a controlled manner by selecting a 2nd ORF insertion site relative to the 3’ end of the NDV vector to maximize the efficacy of vaccine and gene therapy.

Keywords: Foreign gene expression, Newcastle disease virus vector, Sequential transcription mechanism

17th International Congress of Virology 12

Abstract Submission

Paramyxoviruses I OR161

MEASLES VIRUS ENTERS BREAST AND COLON CANCER CELLS THROUGH A NECTIN-4 MEDIATED MACROPINOCYTOSIS PATHWAY

S. Delpeut 1, G. Sisson 1, C. Richardson 1,* 1Microbiology & Immunology/Pediatrics, Dalhousie University, Halifax, Canada

Objectives: To investigate the endocytotic mechanisms used by measles virus (MeV) to enter cancer cells that express Nectin-4/PVRL4 on their cell surfaces.

Methods: The receptor-mediated entry process and the contribution of a variety of endocytic pathways, have been examined in cells that express the receptors for measles virus, including SLAM (Signaling Lymphocyte-Activating Molecule) and PVRL4 (Poliovirus Receptor-Like-4, nectin-4). Recombinant MeV expressing either firefly luciferase or the green fluorescence protein together with a variety of chemical inhibitors, well-characterized dominant negative effectors, and siRNA's were used in these studies in conjunction with fluorescence microscopy to evaluate virus uptake. The role of actin in virus uptake was evaluated using Alexa fluor-546 conjugated phalloidin, which binds to filamentous (F)-actin, and inhibitors of actin polymerization, Cytochalisin D and Latrunculin A. Fluid phase uptake of FITC conjugated dextran, another feature of macropinocytosis, was also monitored by flow cytometry. Experiments were performed using Vero-SLAM, Vero- Nectin4, MCF7, HTB-20, and DLD-1 cell lines.

Results: The results showed that MeV uptake was dynamin-independent in the Vero.hPVRL4, Vero.hSLAM cells, as well as PVRL4-positive MCF7 and HTB-20 human breast and DLD-1 colon cancer cell lines. However, MeV infection was blocked in these cells by EIPA, the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. Using phalloidin staining, MeV entry was shown to coincide with actin rearrangements, resulting in a decreased number of F-actin filaments and the formation of protrusions and membrane ruffles on the cell surface. Transient fluid phase uptake of FITC- Dextran was induced over the first 15 min at 37 degrees, following incubation of wtMeV with Vero.hSLAM and Vero.hPVRL4 cells. The siRNA knockdown of PAK1, or use of a dominant negative PAK1 inhibitor, demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner via a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer and DLD-1 colon cancer cells relied upon Rac1 and its effector PAK1, through a PVRL4-mediated macropinocytosis pathway. Entry into these cells failed to be affected by the dynamin inhibitor, Dynasore, but was blocked with the actin inhibitor, Cytochalasin D. Again actin rearrangement and the formation of membrane protrusions coincided with incubation of these cancer cells with MeV. Virus entry was inhibited by knock down of PAK1 expression and the Rac1 dominant negative inhibitor. These results indicate that MeV enters MCF7, HTB-20, and DLD-1 cancer cells in via a classical macropinocytosis pathway that is dependent on the Rac1-PAK1 signaling pathway.

Conclusion: Many cancer cells exhibit increased macropinocytosis as a means of taking up nutrients from the extracellular environment. Contrary to the generally accepted model for MeV infection, where the virus attaches to its receptor, and simply fuses with plasma membrane during the initial stages of uptake, we show that interaction of MeV with Nectin-4 stimulates macropinocytosis along with its characteristic membrane alterations. Once MeV binds to PVRL4, Rac1/PAK1- dependent signaling pathways are activated which trigger vigorous formation of actin-mediated membrane protrusions. This is followed by MeV endocytosis. The observations made during these studies may be of consequence when considering the potential role of measles virus as an oncolytic agent in treating adenocarcinomas that express the tumor marker Nectin- 4.

Keywords: macropinocytosis entry, measles virus, nectin-4 receptor

17th International Congress of Virology 13

Abstract Submission

Paramyxoviruses I OR162

Characterization of Cedar virus cellular tropism and rescue of recombinant Cedar virus

E. Laing 1,*, M. Amaya 1, C. Navaratnarajah 2, S. Petzing 1, S. Da Silva 1, K. Xu 3, D. Fusco 1, G. Marsh 4, H. Field 5, G. Crameri 6, D. Nikolov 3, R. Cattaneo 2, L.-F. Wang 7, C. Broder 1 1Microbiology and Immunology, Uniformed Services University, Bethesda, 2Molecular Medicine, Mayo Clinic, Rochester, 3Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, United States, 4CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, 5Queensland Centre for Emerging Infectious Diseases, Department of Agriculture, Fisheries and Forestry, Coopers Plains, Australia, 6EcoHealth Alliance, New York, United States, 7Programme in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore

Objectives: Cedar virus (CedPV), a recently discovered paramyxovirus isolated from the urine of flying foxes in Queensland, Australia, is proposed to be the first new virus species within the genus Henipavirus. CedPV shares genetic and antigenic homology with the well-characterized henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), and CedPV infection was supported by ephrin-B2 expressing cells. Here, we characterize the function of the attachment (G) and fusion (F) envelope glycoproteins of CedPV, revealing a similar pH-independent membrane fusion process that requires the participation of both the F and G glycoproteins.

Methods: We employed recombinant glycorpotein expression and cell-cell fusion assays, glycoprotein receptor binding assays, and recombinant reverse genetic techniques.

Results: We confirm ephrin-B2 protein interaction with CedPV G as a functional receptor for fusion. However, unlike HeV and NiV, CedPV exhibits a remarkably broader functional ephrin receptor usage profile, as cells bearing CedPV G and F can also facilitate membrane fusion of cells expressing ephrin-B1 and the glycosylphosphatidylinositol (GPI) anchored ephrin-A subclass proteins; ephrin-A1, -A2 and -A5, and this activity correlated with the ability of CedPV G to bind these ephrin proteins. In addition, we have constructed and utilized reverse genetics to rescue a recombinant, replication competent CedPV reporter gene containing virus. Cellular tropism and replication of the recombinant CedPV reporter viruses is currently being evaluated.

Conclusion: Together, these findings suggest the possibility of previously unrecognized potentials for some Henipavirus cross-species transmission because of broader receptor use capabilities. Further examination of this uniquely diverse ephrin receptor use capacity of CedPV will also advance our understanding of the mechanisms of Henipavirus host cell infection and the protein receptor triggered membrane fusion activities of paramyxoviruses. Unlike closely related HeV and NiV, CedPV does not express the interferon antagonist proteins, V and W, and was non-pathogenic in a ferret model of Henipavirus disease. The CedPV reporter viruses have the potential to improve our understanding of Henipavirus pathogenesis and provide important new tools in the development of Henipavirus countermeasures.

Keywords: Cedar virus, Henipaviruses, Virus tropism

17th International Congress of Virology 14

Abstract Submission

Positive strand RNA viruses I OR166

The pandemic SARS coronavirus may have originated from a single horseshoe bat habitat after sequential recombination events

B. Hu 1,*, X.-L. Yang 1, L.-P. Zeng 1, X.-Y. Ge 1, W. Zhang 1, B. Li 1, D.-S. Luo 1, Y.-Z. Zhang 2, M.-N. Wang 1, P. Daszak 3, L.- F. Wang 4, J. Cui 1, Z.-L. Shi 1 1Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases , Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 2Yunnan Institute of Endemic Diseases Control and Prevention, Dali, China, 3EcoHealth Alliance, New York City, United States, 4Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore

Objectives: Horseshoe bats are recognized as the natural reservoirs of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), as an increasing number of SARS-like coronavirus (SL-CoV) have been detected in this bat family since 2005. However, all bat SL-CoVs known to date still show distinct sequence differences from SARS coronavirus (SARS-CoV) in the S gene and/or some accessory genes such as ORF8. Further information is needed to better understand where and how SARS-CoV originated from bat reservoirs.

Methods: We have conducted a 5-year surveillance of SL-CoV in a cave inhabited by horseshoe bats in Yunnan, China. Genomic characterization, phylogenetic analysis and recombination analysis were performed using the full-length genome sequences of 15 bat SL-CoV discovered in this single location. Efficiency of human ACE2 usage was also evaluated for some of the newly identified strains.

Results: Our results revealed that SL-CoVs circulating in this cave are highly diverse in the S gene. Importantly, strains with high sequence similarity to SARS-CoV in the highly variable N-terminal domain (NTD) and receptor-binding domain (RBD) of S protein and the ORF8 region, respectively, were all found in this single location. Meanwhile, compared with other SL-CoVs, strains identified from this cave exhibited higher sequence similarity to SARS-CoV in the non-structural proteins. Evidence supported that frequent recombination events have occurred at multiple genomic sites between different bat SL- CoVs within this cave and may have led to the generation of the direct ancestor of SARS-CoV. Cell entry studies demonstrated that different newly identified SL-CoVs with S variants are all able to use human ACE2 as the receptor.

Conclusion: We have filled the knowledge gap between the currently known bat SL-CoVs and the pandemic SARS-CoV by identifying a gene pool of SL-CoVs where the direct progenitor of SARS-CoV likely originated. These findings provide a clearer understanding towards the geographical and evolutionary origin of SARS-CoV. It also highlights the risk of future emergence of SARS-like disease.

Keywords: bats, SARS coronavirus

17th International Congress of Virology 15

Abstract Submission

Positive strand RNA viruses I OR167

MERS-CoV NUCLEOCAPSID PROTEIN REGULATES TNF mRNA EXPRESSION IN DIFFERENT HUMAN CELL LINES

J. O. Aboagye 1,*, O.-W. Ng 1, Y. J. Tan 1 1Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: Middle East Respiratory Syndrome coronavirus (MERS-CoV), a novel coronavirus, causes severe respiratory distress and systemic infections with high morbidity and death in humans. The MERS-CoV nucleocapsid protein (MERS-N) is a multifunctional phosphoprotein that interacts with viral and host proteins. The terminal domains of N protein of coronaviruses have been reported to have several functions which interfere with several cellular processes including cell cycle, apoptosis, host immune responses and signal transductions. Published data have suggested that MERS virus induces mRNA regulation of several cytokines including TNF in infected cells. Therefore, we aimed to study the role of MERS-N in regulating TNF in several cell lines.

Methods: A549 cells stably expressing MERS-N were generated and used in an apoptosis PCR array. Genes regulated were verified using TaqMan probes in several selected A549 stable clones, transiently transduced (pooled) A549 cells and transiently transfected 293FT and H1299 cells. Furthermore, cells were transiently transfected with N-terminal and C- terminal fragments of MERS-N to identify the domain capable of inducing TNF.

Results: The apoptosis PCR array showed that TNF was upregulated in A549 cells stably expressing MERS-N. The selected A549 stable clones, transiently transduced (pooled) A549 cells as well as transiently transfected 293FT, H1299, and A549 confirmed the upregulation of TNF by MERS-N protein. We further observed that both MERS-N terminal fragments (N- and C- terminal fragments) induced TNF with N-terminal fragment significantly inducing high levels of TNF compared to the C-terminal and the full MERS-N protein.

Conclusion: The data obtained suggest nucleocapsid protein of MERS-CoV regulates mRNA levels of TNF which may possibly play a role in immunity and/or antiviral responses and may be implicated in the pathogenesis of the MERS-CoV in humans. Hence, it will be important to determine the protein expression of TNF as well as genes downstream of TNF regulation.

Keywords: Middle East Respiratory Syndrome coronavirus, Nucleocapsid protein, TNF

17th International Congress of Virology 16

Abstract Submission

Positive strand RNA viruses I OR168

Inhibition of Replication and Pathogenesis of Coronaviruses by Targeting Viral 2'-O-Methyltransferase

Y. Chen 1, C.-M. Li 1, D. Guo 2,* 1Wuhan University, Wuhan, 2Sun Yat-sen University, Guangzhou, China

Objectives: The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. In this study, we tried to screen and develop antiviral inhibitors targeting coronavirus 2'- O-MTase.

Methods: We used biochemical, genetic, virological methodology and animal models.

Results: We showed that the effective 2'-O-MTase activity of coronaviruses (CoVs) is generally relied on the interaction of nonstructural protein 16 (nsp16) and nsp10, forming nsp10/nsp16 complex. The nsp10-derived peptide could broadly inhibit the 2'-O-MTase activities of betacoronavirus and gammacoronavirus in vitro and the viral replication in MHV infection and SARS-CoV replicon models.We further optimized the peptides or other novel inhibitors systematically by using computer- aided drug design and biochemical analysis. Interestingly, all the inhibitors exerted robust inhibitory effects in vivo in MHV- infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection.

Conclusion: As a proof-of-principle study, the current results indicate that coronavirus 2'-O-MTase can be used as a potential target for antiviral drug discovery and development.

Keywords: antiviral, coronavirus, methyltransferase

17th International Congress of Virology 17

Abstract Submission

Positive strand RNA viruses I OR169

STRUCTURAL AND FUNCTIONAL INSIGHTS INTO A CROSS-REACTIVE ANTIBODY THAT RECOGNIZES DENGUE AND ZIKA VIRUS NS1 PROTEIN AND BLOCKS NS1 FUNCTION

D. Watterson 1,*, N. Modhiran 1, L. Liu 1, S. Cheung 1, C. Bletchly 1, K. Stacey 1, P. Young 1 1School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Australia

Objectives: Emerging flaviviral pathogens represent a significant current and projected global health crisis. Dengue virus (DENV) is the leading disease-causing arbovirus in the world, with over 390 million infections annually, resulting in more than 500,000 severe cases requiring hospitalization. Adding to this burden, the recent global spread of the related Zika virus (ZIKV) has caught governments and aid agencies by surprise and resulted in the WHO declaration of a global heath emergency. The co-circulation of these two related viruses poses significant complications for accurate diagnosis as well as vaccine design. However, the similarities between the two viruses also offers the potential for the development of future treatments and vaccines that could offer broad protection against these important human pathogens. We investigated the potential for cross-reactive antibodies that bind the non-structural protein 1 (NS1) from both DENV and ZIKV with the aim of investigating selected antibodies through both structural and functional approaches.

Methods: A panel of antibodies were assessed for cross-reactivity for both DENV and ZIKV NS1 proteins. Previously, we identified that DENV NS1 is a key mediator of disease pathology and functions to induce cellular activation and vascular leak by interaction with the innate immune receptor TLR4 on both monocytes and endothelial cells. The potential for a selected Mab to alter NS1 function was assessed using an in vitro endothelial permeability assay. The recognition site of the Mab was assessed through peptide binding and proteolytic product analysis. The antibody interaction with NS1 was further characterized using single particle reconstruction anaylsis of NS1 and antibody complexes.

Results: A highly cross-reactive Mab capable of binding both DENV and ZIKV NS1 protein was identified. Using an in vitro model for vascular permeability we have shown that this cross-reactive antibody is able to block NS1 induced leak. Peptide mapping and proteolytic product analysis revealed that this antibody recognizes a region within the c-terminal domain of NS1 (NS1c). Using cryo-electron microscopy (Cryo-EM) single particle analysis of both the soluble secreted hexameric form of NS1 as well as the dimeric NS1c subdomain together with the antibody we have revealed the epitope and stoichiometric basis for recognition of NS1.

Conclusion: NS1 is a key component of DENV pathology and we have previously demonstrated that NS1 can mediate both cell activation and endothelial permeability. Our findings presented here reveal a key cross-reactive epitope within NS1 that has significant implications for diagnostic development and also provides the foundation for new antivirals and vaccine design to counter both DENV and ZIKV.

Keywords: Antibody, Dengue, Zika

17th International Congress of Virology 18

Speaker Abstract Submission

Insect viruses

INVESTIGATING THE HOST RESTRICTION OF INSECT-SPECIFIC FLAVIVIRUSES AND THEIR POTENTIAL AS NEW TOOLS IN BIOTECHNOLOGY

J. Hobson-Peters*, B. McLean 1, A. Colmant 1, C. O'Brien 1, J. Harrison 1, T. Piyasena 1, H. Bielefeldt-Ohmann 1, Y. Setoh 1, A. Khromykh 1, R. Hall 1 1The University of Queensland, St Lucia, Australia

Objectives: We recently discovered several insect-specific flaviviruses (ISFs) in mosquitoes from different regions of Australia. Based on our previous observations that ISFs fail to replicate in vertebrate cells but can suppress the replication in and transmission by mosquitoes, we undertook a thorough investigation of these viruses to further define their host range, the molecular mechanisms of their host restriction and their potential as tools for biotechnology.

Methods: Full length viral genome sequences were obtained by deep sequencing and viral host range was determined by inoculating a range of mosquito and vertebrate cell lines with each virus and detecting replication by the appearance of viral proteins, viral replicative RNA and siRNA responses by the host cell. The construction of infectious DNAs of ISFs, as well as chimeric viruses, were generated using circular extension polymerase cloning (CPEC).

Results: Determination of the complete genome sequences of these viruses and phylogenetic analysis revealed we had discovered 9 new ISF species representing two distinct genetic lineages. We observed a considerable variation in the mosquito host range of different ISFs, with some viruses restricted to a single mosquito species. Construction of infectious DNAs and chimeric viruses also allowed us to identify stages at pre- and post-cell entry where virus infection and replication is blocked in the infection cycle of vertebrate cells.

Conclusion: We identified ISFs in two distinct genetic lineages that exhibited variable host range among mosquito species. The availability of full-length ISF genome sequences to construct infectious DNAs of these viruses provided valuable research tools to identify the mechanisms of host restriction and the viral factors responsible. The generation of chimeric ISFs also provides proof-of-concept that these viruses represent a novel platform to produce recombinant viruses for biotechnology.

Keywords: chimeric viruses, Host-restriction, Insect-specific flavivirus

17th International Congress of Virology 19

Abstract Submission

Insect viruses OR170

NEWLY DISCOVERED INSECT-SPECIFIC FLAVIVIRUSES FROM ANOPHELES MOSQUITOES ARE HIGHLY SPECIALISED FOR THEIR HOST MOSQUITO SPECIES

A. Colmant*, J. Hobson-Peters 1, H. Bielefeldt-Ohmann 1, A. van den Hurk 2, S. Hall-Mendelin 2, J. Fros 3, P. Simmonds 3, K. Etebari 4, T. Piyasena 1, R. Hall 1 1School of Chemistry and Molecular Biosciences, University of Queensland, 2Public Health Virology, Forensic and Scientific Services, Department of Health, Queensland Government, Brisbane, Australia, 3Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom, 4School of Biological Sciences, University of Queensland, Brisbane, Australia

Objectives: While investigating the biodiversity of insect-specific flaviviruses (ISF) in Australian mosquitoes, we discovered several genetically-divergent ISFs in different Anopheles species. The prototype of this group, Karumba virus (KRBV), was thoroughly analysed to obtain its complete genomic sequence, determine its phylogenetic position in the genus and assess its host range and identify mechanisms of host restriction.

Methods: RT-PCR and Illumina next-generation sequencing were used to obtain partial or whole viral genome sequences and nucleotide and coded amino acid sequences were aligned and phylogenetic analysis performed. In vitro replication was assessed in a range of arthropod cell lines as well as injecting infected mosquito homogenates into colonised Anopheles mosquitoes. Dinucleotide and codon bias usage was further determined to confirm host restriction patterns. KRBV-specific replicative RNA , viral NS1 and host-induced siRNA responses were also used to confirm KRBV replication in mosquito tissues. A chimeric virus containing KRBV structural genes was also constructed using infectious DNA technology.

Results: We report an unprecedented level of host-restriction of this novel group of flaviviruses for their host mosquito species. KRBV, detected at very high prevalence in Anopheles meraukensis, failed to replicate in vitro and in vivo when cells or tissues from other mosquito species were inoculated. In the absence of An. meraukensis cell lines or laboratory colonies, we detected viral antigen in the midgut epithelial cells in sections of wild-caught An. meraukensis by IHC with anti- KRBV mAbs, providing clear confirmation of viral infection. We also constructed a chimeric virus containing the structural genes (prM-E) of KRBV in the genetic backbone of another mosquito-borne flavivirus to demonstrate that host-cell restriction occurs after the virus has entered the cytosol. The phylogenetic analysis of genome sequences of KRBV, and the other Anopheles-associated viruses that we have discovered, further revealed that these viruses cluster together and form a separate lineage within ISFs. This was supported by the dinucleotide usage pattern observed in the KRBV genome sequence which indicated the virus had evolved to replicate only in insects.

Conclusion: The Anopheles-associated viruses reported here represent a new genetic-lineage within the ISFs and exhibit the most specialised host restriction reported to date for this group of viruses. The detection of a complete genome sequence for KRBV, and identification of viral protein, replicative RNA forms and an siRNA response to the virus in mosquito tissues confirm the existence of a functional replicating virus. These findings represent a significant advance in our knowledge of flavivirus ecology and host-restriction and provide new insights into their evolution. Image:

17th International Congress of Virology 20

Keywords: Evolution, Flavivirus, Host-restriction

17th International Congress of Virology 21

Abstract Submission

Insect viruses OR171

A TALE OF PROVIDENCE: AN RNA VIRUS THAT INFECTS AND REPLICATES IN INSECT, HUMAN AND PLANT CELLS

M. Jiwaji 1,*, R. A. Dorrington 1 1Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa

Objectives: Providence virus (PrV) is a positive-sense, single-stranded RNA virus belonging to the Family Carmotetraviridae. This virus has a T=4 capsid structure that is characteristic of the insect-infecting tetraviruses, but the carmo-like viral replicase is related to those of the plant-infecting Tombusviridae and Umbraviruses. PrV was originally discovered as a covert, apparently persistent infection in an insect midgut (Helicoverpa zea, MG8) cell line, and can infect and replicate in Spodoptera tissue culture cell lines. To date, tetraviruses are believed to have a narrow host range confined to one or a few species of lepidopteran hosts.

Methods: Here we redefine the host range of tetraviruses, presenting evidence that PrV infects and replicates in insect, human and plant cells. We confirm the presence of PrV using western blot analyses, transmission electron microscopy and immunofluorescence confocal microscopy.

Results: We show that PrV readily infects, replicates in and produces infectious viral particles in both human cervical (HeLa) and breast (MCF7) cancer cell lines. We also show that PrV is able to replicate and produce infectious virus particles in Vigna unguiculata (cowpea) plants. These unprecedented data show that PrV is a (+) ssRNA virus that encodes an RNA- dependent RNA polymerase that replicates in insect, mammalian and plant cells with a virus particle that enables PrV to infect insect and human cells. Conclusion: Our findings raise important questions about the potential for RNA viruses to make cross-Kingdom jumps in the laboratory and more importantly, in nature.

Keywords: cross-Kingdom host range, Insect RNA virus, virus evolution

17th International Congress of Virology 22

Abstract Submission

Insect viruses OR172

ESTABLISHMENT OF VIRAL REPLICATION COMPLEXES BY PROVIDENCE VIRUS (FAMILY: CARMOTETRAVIRIDAE)

J. A. Awando 1,*, M. Jiwaji*, R. Nakayingah 2, R. A. Dorrington 1 1Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa, 2Institute of Allied Health Sciences, International Health Sciences University, Kampala, Uganda

Objectives: Providence virus (PrV) is a non-enveloped (+ve)-sense ssRNA virus belonging to the Carmotetraviridae Family. PrV has a monopartite genome, encoding three open reading frames (ORFs); at the 5’ end is p130 (a protein of unknown function) that overlaps with p104 (viral replicase), followed by p81 (capsid protein precursor). A read-through stop in the p104 ORF results in the translation of the N-terminal protein p40. The p104 replicase encodes the RNA dependent RNA polymerase (RdRp) domain at its C-terminus. The PrV RdRp domain is phylogenetically related to those of the plant- infecting Tombusviridae and Umbraviruses. There are also similarities between the N-terminus of p40/p104 and the membrane-spanning and the protein-protein interaction domains of the tombusvirus replicases, suggesting that these domains may have a common function in the initiation of PrV replication. The aim of this study was to investigate the role of these putative functional domains in the formation of viral replication complexes in PrV-infected cells.

Methods: We expressed wild-type and deletion derivatives of p104 and p40 fused to EGFP in Spodoptera frugiperda (Sf9) cells. We also used co-immunoprecipitation assays and immunofluorescence confocal microscopy.

Results: Using the techniques named above, we demonstrate that p40 self-interacts and interacts with p104 via an N- terminal protein interaction domain. We also show the presence of two functional membrane spanning domains that are required for the recruitment of p40 and p104 to the Golgi apparatus and secretory vesicles, the sites of PrV replication.

Conclusion: These findings highlight the similarities between PrV and the tombusviruses, supporting the hypothesis that the PrV non-structural proteins originated from a tombus-like plant virus.

Keywords: Insect RNA viral replicase, protein-protein interaction, tombusvirus

17th International Congress of Virology 23

Abstract Submission

Influenza virus

OR173

A CROSS-CLADE H5N1 INFLUENZA A VIRUS NEUTRALIZING MONOCLONAL ANTIBODY 9F4 BINDS TO A NOVEL EPITOPE WITHIN THE VESTIGIAL ESTERASE DOMAIN OF HEMAGGLUTININ

S. S. Paul 1,*, C.-K. Mok 1, T.-M. Mak 1, O.-W. Ng 1, J. O. Aboagye 1, T. J. Wohlbold 2, F. Krammer 3, Y.-J. Tan 1 1MICROBIOLOGY, NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE, Singapore, 2Graduate School of Biological Sciences, 3MICROBIOLOGY, Icahn School of Medicine at Mount Sinai, New York, United States

Objectives: The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised serious concerns. Previously, monoclonal antibody (MAb) 9F4, generated against the hemagglutinin (HA) of H5N1, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pH-mediated conformational change of HA. Furthermore, chimeric MAb 9F4 expressing human constant domains was found to retain high degrees of neutralizing activity. In this study, epitope mapping was performed to characterize the antibody binding site in HA of A/ Vietnam/1203/2004(H5N1) (VN04).

Methods: Escape mutation experiment was performed for epitope mapping. Furthermore, in-silico epitope prediction analysis was also carried out. To validate these predictions, binding of MAb 9F4 to HA substitution mutants was assessed through immunofluorescence and Western blot assays. A cell-based ELISA was also used for quantitative comparison. To confirm the importance of the observed amino acid residues in forming the epitope site in HA, in vitro microneutralization assay was carried out with live virus.

Results: Through escape mutant generation, it was revealed that MAb 9F4 binds to an amino acid residue Arginine (R) at position 53 in vestigial esterase (VE) sub-domain of HA. In-silico analysis predicted another amino acid tryptophan (W) at position 60, also in the VE sub-domain, to be important in the interaction of MAb 9F4 to HA. Binding and virus neutralization studies suggested that R53 is the critical residue for MAb 9F4 binding whereas W60 seems to cooperate with R53 to stabilize the conformation-dependent epitope. However, the mutations were not observed to confer disadvantage in replicative fitness of the virus in vitro. Furthermore, MAb 9F4 was observed to retain neutralizing efficacy against a clade 2.3.2.1a H5N1 virus consisting of an arginine to lysine substitution at positon 53 in HA.

Conclusion: Our results showed that MAb 9F4 binds in a conformation-dependent manner to a novel epitope present in the VE sub-domain of HA. The epitope consists of at least two non-continuous amino acid residues, namely R53 and W60. To our knowledge, these residues have not been previously reported to be critical for interaction with other MAbs and thus form a novel neutralizing epitope in H5N1 HA

Keywords: Epitope mapping, Influenza H5N1, Neutralizing monoclonal antibodies

17th International Congress of Virology 24

Abstract Submission

Influenza virus OR174

LOCAL STRUCTURE CHANGES OF THE INFLUENZA A VIRUS RIBONUCLEOPROTEIN COMPLEX BY A MUTATION IN THE SPECIFIC RESIDUE INVOLVED IN THE EFFICIENT GENOME PACKAGING

N. Takizawa 1,* 1Laboratory of Virology, Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan

Objectives: The influenza virus genome consists of eight single-stranded negative-sense RNA segments and one set of eight segments is packaged into the virion. Both the noncoding regions located at the 3′- and 5′-ends in each segment are necessary for genome packaging and the terminal coding regions are required for precisely bundling the eight segments into one set. It is assumed that one set of eight segments is bundled by direct RNA-RNA interaction between segments because viral RNA segments can form specific networks of intermolecular interactions in vitro. However, the nucleotide residues involved in genome bundling and packaging and precise role of the segment bundling region in infected cells are not well known.

Methods: To identify the nucleotide residues important for genome bundling or packaging in protein coding region, we constructed the new system utilizing the virus libraries containing both premature termination codon and random sequence. The virus libraries were selected by passage using cells expressing the viral protein. After passage, the inserted sequences in the virus library were determined by next generation sequencing and the nucleotide residues selected after passage were identified. In addition, to analyze the structure of viral ribonucleoprotein containing a single mutation at the identified nucleotide residues, the virion was treated with single-stranded RNA modification reagents, and the modified nucleotides were identified by primer extension assay.

Results: Using the virus libraries, we reveal that wild type sequences are not necessarily required for the efficient viral growth when the protein coding bias is not existed. We also identify the residues involved in the efficient viral growth and reveal that the packaging of viral genome is impaired in the viruses that contain a single mutation at the identified residues. Furthermore, the accessibility of the RNA modification reagents around the mutation sites was different between wild type virus and the viruses containing the single mutation, suggesting that the single mutations altered the local structure of viral ribonucleoprotein complex around the mutation site.

Conclusion: Our results, for the first time, demonstrate that the nucleotide residues required for efficient viral genome packaging are involved in forming a precise structure of viral ribonucleoprotein complex.

Keywords: genome packaging, Influenza virus, segmented genome RNA

17th International Congress of Virology 25

Abstract Submission

Influenza virus OR175

IN VITRO HUMAN NASAL EPITHELIAL CELLS AS A COMPLEMENTARY MODEL FOR POPULATION LEVEL TRANSCRIPTOMIC PROFILING OF EMERGING RESPIRATORY INFECTIOUS DISEASE

K. S. Tan 1,*, Y. Yan 1, C. Li 1, W. L. H. Koh 2, H. W. Choi 2, T. Tran 3, R. Sugrue 4, V. T. K. Chow 5, D. Y. Wang 1 1Department of Otolaryngology, 2Saw Swee Hock School of Public Health, 3Department of Physiology, National University of Singapore, 4School of Biological Sciences, Nanyang Technological University, 5Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: In-vitro and in-vivo research based on cell lines and animals are likely to be insufficient in elucidating certain biological and physiological phenomena at a human population level, especially for generating pre-clinical trial data. There is an obvious demand for a model that can further bridge the gap between experimental data and pre-clinical/clinical trials to elucidate the differential responses towards specific agents at a population level. Therefore the aim of the study is to establish an in vitro nasal epithelial model that is complementary to clinical studies in generating population based transcriptomic data

Methods: Our lab has previously generated an in-vitro differentiated, air-liquid interface culture of human nasal epithelial cells (hNECs) derived from nasal epithelial stem cells of healthy donors that retain the fully functional multi-layer of the nasal epithelium, the primary target of respiratory viruses. We showed that these cells, while retaining the genomes of their donors, are capable of displaying the individuals’ differential responses against respiratory viruses such as influenza A virus (IAV), rhinovirus (RV), and respiratory syncytial virus (RSV). Building on this, we performed microarray analyses on the model of hNECs to ascertain the potential of these cells to elucidate population-based responses against human IAV under controlled and reproducible experimental conditions. The transcriptome obtained from the in vitro model were then further compared to clinical data obtained from GEO databases to assess the clinical information that can be captured with the in vitro nasal model.

Results: Transcriptomic analysis of these hNECs from 5 donors (infected thrice independently) were highly comparable with clinical array data (13 independent influenza transcriptomic array datasets obtained from the GEO database), including datasets with high sample sizes, e.g. Zhai et al. (2015) who analyzed 133 microarray samples from 1610 patients recruited in 2 years. The analyses of up-regulated genes detected in our model exhibited over 80% identity, indicating that the hNECs model is capable of elucidating patient level data at a smaller scale when controlled for variables normally present in clinical data. Additionally, our model also allows analyses of down-regulated genes in the nasal epithelium, which are not feasible with data from blood samples.

Conclusion: In conclusion, this study supports the utility of the hNECs model as a small-scale complementary model to acquire representative population level data on their differential responses against various respiratory virus infections in the nasal epithelium.

Keywords: Human nasal epithelial model, Influenza virus, Transcriptome

17th International Congress of Virology 26

Speaker Abstract Submission

Paramyxoviruses II

The Mòjiāng virus attachment glycoprotein confers a mode of infection distinct from genetically related henipaviruses

I. Rissanen 1, A. Ahmed 2, K. Azarm 3, S. Beaty 3, P. Hong 3, S. Nambulli 4, W. P. Duprex 4, B. Lee 3,*, T. Bowden 1 1Division of Structural Biology, University of Oxford, Oxford, United Kingdom, 2Division of Infectious Disease, Boston Children’s Hospital, Boston, 3Microbiology, ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, New York, 4Microbiology, Boston University School of Medicine, Boston, United States

Objectives: In 2012, cases of lethal pneumonia amongst Chinese miners prompted the isolation of a rat-borne henipa- like paramyxovirus, Mòjiāng virus (MojV) that has since been classified by ICTV as a member of the henipavirus genus. Whilst MojV is genetically related to highly pathogenic bat-borne henipaviruses (HNV), the absence of a conserved ephrin receptor-binding motif in the MojV attachment glyco- (G) protein indicates a differing host-cell recognition mechanism.

Methods: We used crystallographic analysis combined with structure-function studies to probe the nature of the MojV envelope glycoproteins. The globular head of the MojV attachment glycoprotein (MojV-G) was crystallized and the structure was determined to 1.94 Å resolution. Structural comparisons were made with other paramxyxoviral attachment glycoproteins form various genera. Receptor binding and fusion assays were used to characterize the nature of the MojV receptor.

Results: Our crystallographic investigation reveals that the MojV attachment glycoprotein (MojV-G) displays a six-bladed β-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, particularly at receptor-binding surfaces. MojV-G exhibits only a slightly greater resemblance to HNV-G glycoproteins (2.1-2.4 Å RMS deviation upon superposition of equivalent Cα atoms) than sialic acid-binding HN glycoproteins found in members of the Respirovirus (RMS deviation of 2.3 Å from HPIV3-HN), Rubulavirus (RMS deviation of 2.5 Å from PIV5 HN), and Avulavirus genera (RMS deviation of 2.5 Å from NDV HN). Notably, MojV-G also poorly overlays with Morbillivirus H glycoproteins (RMS deviation of 3.1 Å from MV-H). Indeed, MojV-G does not interact with known paramyxoviral receptors in vitro, indicating an independence from well-characterized sialic acid, ephrinB2/B3 and CD150 mediated entry pathways. Furthermore, we found that MojV-G is antigenically distinct from the Asiatic (NiV and HeV) and African clades (GH-M74a virus from Ghana) of extant henipaviruses, indicating that MojV or other “henipa-like” viruses would less likely be detected in existing large- scale serological screening studies focused on HNVs. Conclusion: Altogether, our data indicate a unique infection pathway for Mòjiāng virus that is independent of known paramyxovirus receptors. Although Mòjiāng virus has been classified as a henipavirus, its inability to use ephrinB2—the main receptor for all other henipaviruses that largely accounts for their tissue tropism and pathogenesis—raises the question of how pathogenic profile of MojV might differ from canonical ephrinB2-using henipaviruses.

Keywords: glycoproteins, Henipavirus, receptor

17th International Congress of Virology 27

Abstract Submission

Paramyxoviruses II OR163

THE HIGHLY CONSERVED TRYPTOPHAN RESIDUE IN V PROTEIN PLAYS A CRITICAL ROLE IN NEUROVIRULENCE OF MEASLES VIRUS

S. Uchida*, T. Arai, H. Sato, C. Kai, M. Yoneda

Objectives: Measles virus (MV) in rare cases causes fatal neurological complication including subacute sclerosing panencephalitis and measles inclusion body encephalitis. MV belongs to the genus Morbillivirus, in the family Paramyxoviridae, and its genome encodes six structural genes. The structural P gene additionally encodes two accessory proteins, V and C proteins. It has been reported that the recombinant MV lacking V gene less replicated and was attenuated in rodent brain. However, the role of MV V protein in neuropathogenicity has not been investigated. We previously identified UBXN1, a negative regulator of host IFN production, as a novel interacting protein with MV V protein. We also found that MV V stabilized UBXN1 to suppress IFN induction, and a tryptophan residue in C-terminal domain of V protein was specifically critical for UBXN1 stabilization. In this study, we evaluated the role of the interaction of V protein and UBXN1 in neuropathogenesis.

Methods: We generated the rodent brain-adapted recombinant measles virus whose V protein lost the binding ability to UBXN1 by substituting its W residue to R (rMV CAMPH WR), and examined its neurovirulence in mouse brain. We also performed histopathological analysis and quantitative PCR assay to evaluate the viral propagation and host immune response.

Results: Although the rMV CAMPH WR grew in cultured cells as well as the parental rMV CAMPH, its virulence was attenuated in mouse brain. As a results of histopathological analysis and quantitative PCR assay, the propagation and replication of rMV CAMPH WR were suppressed compared to the parental rMV CAMPH, and type I IFNs were more induced in the brain infected with rMV CAMPH WR.

Conclusion: Our results suggest that the neuropahthogenicity of MV requires the highly conserved tryptophan residue in V protein which is specifically critical for the stabilization of UBXN1.

Keywords: measles virus, V protein

17th International Congress of Virology 28

Abstract Submission

Paramyxoviruses II OR164

STRUCTURAL INSIGHTS INTO STAT1-DEPENDENT INHIBITION OF STAT2 PHOSPHORYLATION BY SENDAI VIRUS C PROTEIN

K. Oda 1,*, T. Oda 2, Y. Matoba 3, T. Irie 1, M. Sato 2, T. Sakaguchi 1 1Department of Virology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 2Graduate School of Medical Life Science, Yokohama City University, Yokohama, 3Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

Objectives: Interferon-α/β (IFN-α/β), produced from virus-infected cells, induces antiviral states in surrounding non-infected cells. However, many viruses have unique countermeasures against the antiviral effects of IFN-α/β. In Sendai virus (SeV) C protein is responsible for the suppression of IFN-α/β signaling by inhibiting tyrosine phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and STAT2. It is known that C protein can associate with STAT1 but not with STAT2. Binding of C protein to STAT1 seems to be important for inhibition of the phosphorylation of STAT1 and STAT2, although the inhibition mechanism is unclear. C-terminal domain of C protein (named as Y3) associates with STAT1ND. We previously demonstrated by using protein crystallography that two molecules of Y3 can bind to the homodimer of N- terminal domains of STAT1 (STAT1ND) (Oda. et. al., J. Virol. 2015). We also showed by small-angle X-ray scattering analysis that STAT1ND has the ability to associate with STAT2ND with the help of the linker peptide (an unpublished observation). C protein may interact with STAT2ND through binding to the heterodimer of STAT1ND and STAT2ND. The aim of this study was to determine the mechanism by which C protein inhibits phosphorylation of STAT1 and STAT2 in the heterodimer.

Methods: IFN-α was added to cells in which an N-terminal domain-deleted mutant of STAT1 (STAT1ΔN) or STAT2 (STAT2ΔN) was co-expressed with C or Y3 protein. Western blotting analysis was then carried out to estimate the amount of IFN-α-induced phosphorylated species. We have also established cells that constitutively expresses a site-directed mutant of STAT2 with a lower affinity to STAT1ND than the wild type. The established cells were transfected with an expression vector of C or Y3 together with pISRE-GFP that possesses an ISRE element upstream of GFP, and the amount of IFN-α-induced GFP expression was evaluated by Western blotting analysis. We also investigated whether SeV weakened the effect of INF-α on the superinfected vesicular stomatitis virus in the established cells.

Results: Transiently expressed C protein almost completely inhibited the phosphorylation of STAT1ΔN or STAT2ΔN. On the other hand, Y3 only weakly inhibited the phosphorylation of STAT1ΔN or STAT2ΔN than that of the full-length forms and rarely inhibited the phosphorylation of full-length STAT2 in the absence of STAT1. In the established cells expressing the STAT2 mutant, the rate of inhibition of the IFN-α signal transduction by Y3 was significantly decreased compared to that in cells expressing the wild type. Additionally, in the mutated STAT2-expressing cells, SeV infection did not stimulate propagation of the superinfected vesicular stomatitis virus.

Conclusion: C protein seems to inhibit the IFN-α-induced phosphorylation of STAT2 by at least two different ways mediated or not mediated through the binding to STAT1ND. Our results indicate that the association of C protein with the heterodimer of STAT1ND and STAT2ND is one of the important factors for blocking the IFN-α-induced phosphorylation of STAT1 and STAT2. We propose that one molecule of C protein associates with the dimeric structure formed between STAT1ND and STAT2ND. Thus, C protein-bound STAT1:STAT2 heterodimer would be induced to take an antiparallel form, which is easily dephosphorylated.

Keywords: C protein, sendai virus, STAT2

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Abstract Submission

Paramyxoviruses II OR165

HENDRA VIRUS V PROTEIN PLASTICITY ENABLES BINDING TO MULTIPLE NUCLEAR TRANSPORT PROTEINS

S. Atkinson 1,*, M. Audsley 1, D. Thomas 1, G. Marsh 2, J. Paxmann 3, S. Heaton 1, K. Wagstaff 1, G. Moseley 4, D. Jans 1, N. Borg 1 1Infection & Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry & Molecular Biology, Monash University, Clayton, 2CSIRO Health and Biosecurity, Australian Animal Health Laboratory (AAHL), CSIRO, Geelong, 3La Trobe Institute for Molecular Sciences and Department of Biochemistry and Genetics, La Trobe University, Bundoora, 4Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Australia

Objectives: Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes severe disease and a high incidence of fatality in infected humans. Despite recurrent outbreaks and potential for human lethality, no vaccine or anti- viral agent is available to prevent or treat human HeV infection. Key to HeV pathogenicity is the co-transcriptional mRNA editing of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The V protein modulates the host response to infection by targeting numerous host proteins. New host proteins are frequently being discovered, including those involved in nuclear trafficking, which we investigate here.

Methods: By combining in vitro and in vivo analyses, we show both nuclear import and export receptors are amongst those targeted by HeV V, and play a role in infection. Circular dichroism (CD) spectroscopy and small angle X-ray scattering (SAXS) were used to investigate changes in the folded state of V upon binding to its nuclear transport partners. Fluorescent cell microscopy and immunoprecipitation analysis was used to determine the cellular localisation and binding partners of both wild-type and mutant versions of HeV V.

Results: We identify the HeV V nuclear export signal and show its importance in both export and binding of HeV to exportin. Structural analysis reveals HeV V is inherently disordered and gains structure upon binding its target nuclear transport receptors; a trait that offers numerous functional advantages. Studies with import and export inhibitors suggest a previously unknown role for HeV in the nucleus during infection.

Conclusion: HeV V is inherently disordered and gains structure upon binding its target nuclear transport receptors. V plays an important role in modulating the host immune response to Hendra infection. These findings broaden our understanding of HeV-host interactions.

Keywords: Hendra virus, intrinsically disordered proteins, nuclear trafficking

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Speaker Abstract Submission

Positive strand RNA viruses II

Effects of Evolutional Substitutions on Antigenicity and Virulence of Enterovirus A71

Jen-Ren Wang1,2,3*, Sheng-Wen Huang1

1Center of Infectious Disease and Signaling Research, 2Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan 3National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan

Enterovirus A71 (EV-A71), as a neurotropic virus, is the major etiology of hand-foot-and-mouth disease and occasionally causes neurological involvement. EV-A71 has increased activity in the Asia-Pacific region with sporadic cases or outbreaks in Asia, Europe, and even Africa. Until now, seven genotypes A to G were identified, and predominant genotypes of EV-A71 continue to change in worldwide especially in Asia. To evaluate the effects of evolutional substitutions on viral antigenicity and virulence, reverse genetics system was applied for investigation.

In antigenic evolution, substitutions at VP1 capsid protein were identified to co-operatively contribute to diverse antigenic properties as well as receptor binding ability among EV-A71 genotypes, which potentially increased viral fitness. Since EV-A71 vaccination program begins, it should be concerned whether EV-A71 will generate antigenic drift and cause another outbreak after virus breaking through the bottleneck in human. We thus investigated potential antigenic escape mutants derived from parental strain through serial passage selections using a human antiserum containing neutralizing antibody against EV-A71. Although antibody escape mutations of EV-A71 were identified by next generation sequencing, they only showed limited change on antigenicity. In contrast to other rapid antigenic evolving viruses such as influenza virus, we thus suggest that EV-A71 is more antigenic stable against human humoral immunity.

In viral virulence, EV-A71 contains diverse variants which may arise viral virulence. From genome comparison of human isolates, C158 identified in 5' untranslated region was found to play a pivotal role of virulence determinant on virus translation and EV-A71 virulence in a mouse model. Substitutions were also identified in capsid protein and non-structural protein regions in EV-A71 evolution. Genotype B5 and C4 accumulate dozens of substitutions in the non-structural protein region. Complete genome analysis reveal intra- and inter-serotypic recombination occurrence in re-emergent genotype C2, B4 and C4, suggesting increase of viral fitness through the processing of EV-A71 evolution. In vitro and in vivo evidences reveal the recombination of EV-A71 and coxsackievirus A8 (CV-A8) generates 3Dpol/251I substitution which increases viral temperature resistance and virulence. In the capsid protein region, mouse adapted strain (MP4) acquires VP2149M and VP1145E mutations which synergistically increase viral virulence in mice through increasing virus binding and RNA release.

EV-A71 acquired several substitutions which might be benefited for viral replication and fitness under selective pressure in the human host. These viral substitutions retrieved from global evolution or in the host collectively contribute to re- emergent EV-A71 viral fitness and pathogenesis.

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Speaker Abstract Submission

Positive strand RNA viruses II

Hand, Foot and Mouth Disease in Vietnam

L. V. Tan*, L. N. T. Nhan, N. T. Hung, T. H. Khanh, H. M. Tuan, S. Whitehorn, P. T. Qui, N. V. V. Chau, H. M. T. Van, H. R. Van Doorn , O. B. O. the Emerging Infections Group

Please specify what speaker category are you in?: Workshop speaker

Objectives: Hand, Foot and Mouth Disease (HFMD) is an emerging infection of the Asia-Pacific region. Our ongoing research program aims to address unanswered questions about clinical, epidemiology, pathogen evolution, cost of illness and host-genetic makers associated with severe HFMD in Vietnam.

Methods: A multi-hospital-based observational study of HFMD was conducted at three referral hospitals for southern provinces in Ho Chi Minh City, Vietnam. Demographic, clinical data and cost of illness were collected alongside clinical specimens from the enrolled patients. Multiplex PCR and next generation sequencing were employed to identify the specific enterovirus serotypes and to study pathogen evolutions, respectively. A genome-wide association (GWAS) based approach was used to explore genetic markers of disease severity.

Results: Between July 2013 and July 2015, 1547 patients (including 590 outpatients and 957 inpatients; median age: 21.2 months, IQR: 12.4 – 25.7) were enrolled. 20 different enterovirus serotypes were detected in 86% of the patients. Enterovirus A71 (EV-A71) was the major cause accounting for 24% followed by coxsackievirus A6 (CV-A6) (22%), CV- A16 (9%) and CV-A10 (8%). Temporally, these four common enteroviruses replaced each other during the study period. Of 100 severe patients, EV-A71 was detected in 61%, while CV-A16, CV-A10, CV-A6, and other enteroviruses serotypes were responsible for the remaining 39%.

Whole-genome phylogenetic analysis revealed a replacement event occurring in 2012 between EV-A71 subgenogroup B5 and C4, and since then B5 has continued to circulate as a dominant subgenogroup with C4 being sporadically detected. In contrast, all the Vietnamese CV-A6 isolates belonged to a single genogroup A, which has been circulating in Vietnam since 2011. Cost of illness varied greatly between disease severities; ranging from $USD 244 (95%CI: 230 – 258) per patient for those with grade 2A (mild HFMD) to $USD 1984 (95%CI: 1752 – 2227) per patient for those with grade 3 (severe HFMD). GWAS analysis identified two genetic markers potentially associated with severe HFMD.

Conclusion: The results have highlighted the importance of active molecular surveillance and understanding long-term pathogen evolution, and are informative for public health in prioritizing the development of intervention strategies. The discovery of potential host genetic markers may have profound implication for disease management.

Keywords: Coxsackievirus , enterovirus A71, Hand, Foot and Mouth Disease

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Speaker Abstract Submission

Positive strand RNA viruses II

System biology approaches in dissecting the replication processes of human enteroviruses that cause HFMD

Justin Jang Hann Chu

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore

The Hand, Foot and Mouth Disease (HFMD) is a febrile illness, which can result from infections by a plethora of human enteroviruses, including Enterovirus 71 (EV71). Although HFMD is generally a mild and self-limiting affliction in young children and immunocompromised adults, a subset of patients develop severe neurological, poliomyelitis-like symptoms, which can result in fatality. By using integrated system-wide approaches including human genome-wide gene silencing profiling, miRNA profiling and proteomics via high-throughput techniques, combined with bio-imaging and computational biology, we attempt to understand the biological complexity of virus-host interactions and translating it into antiviral strategies against human enteroviruses. Given the compact genome of EV71, many cellular proteins are likely to be required for its successful replication. To date, only a handful of these factors have been identified. Here we report the identification of host susceptibility and resistance factors affecting EV71 infection from a genome-wide RNAi screen coupled with miRNAome and proteome profiling in human cell lines and clinical samples. Bioinformatics analyses revealed the involvement of a plethora of cellular processes including transcription regulation, cell cycle, calcium signaling, translation initiation and membrane biogenesis etc. A number of host susceptibility (MINK kinase, NGLY1) and resistance (CDK6 and AURKB) factors were selected for detailed analysis due to its strong inhibitory or enhancement profiles upon gene silencing in EV71-infected cells. Through proteomic analysis and infection inhibition assay, we have mapped out the detail pathway/mechanism on the interaction of host-viral interactions. This study provides a comprehensive map of cellular components involved in EV71 replication that can form the basis for antiviral targeting and our understanding of virus pathology.

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Speaker Abstract Submission

Positive strand RNA viruses II

DYNAMIC HAPLOTYPE SWITCHES IN QUASISPECIES ASSOCIATES WITH VIRAL INVASION INTO CENTRAL NERVOUS SYSTEM AND DISEASE SEVERITY OF ENTEROVIRUS A71 IN HUMAN

S.-W. Huang*, Y.-H. Huang 1, J.-R. Wang 1 2

1Department of Medical Laboratory Science and Biotechnology, 2Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan city, Taiwan

Objectives: RNA virus accumulates mutations to rapid adapt the changes of environment, but most of mutations may have lethal effects on viral fitness. Enterovirus A71 (EV-A71), as a neurotropic RNA virus, causes various clinical manifestations, with severe neurological complications occasionally. However, the association between viral diversity and pathogenesis of EV-A71 in human is not clear.

Methods: We used next-generation sequencing method to examine viral quasispecies of EV-A71 isolates from various tissues and patients with different disease severity.

Results: Here we demonstrated that EV-A71 utilized haplotype switch to maintain population diversity for tissue adaption. Instead of extinction of non-preferred haplotype, virus retained kinetic equilibrium of diverse haplotypes containing VP1-31 substitution in viral quasispecies existing in various tissues. Haplotypes with VP1-31D mainly appeared in orodigestive tract whereas those with VP1-31G dominated in central nervous system tissues. In vitro viral growth and fitness experiment indicated VP1-31G had better growth and fitness in human neuron cells, but VP1-31D had higher growth and fitness in human colorectal cells. Higher proportion of VP1-31G also appeared in viral quasispecies from fatal patients then hand-foot-and-mouth disease patients.

Conclusion: Our study thus demonstrated that EV-A71 dynamically adjusted VP1-31 proportion to flexibly fit various tissue tropism which was also associated with disease severity in human infection. Analysis of the viral haplotypes of clinical isolates provides a potential predictor for the outcome of EV-A71 infection.

Keywords: enterovirus A71, quasispecies

17th International Congress of Virology 34

Speaker Abstract Submission

Plenary: Virus-host Interactions

Coronavirus diversity, phylogeny and interspecies jumping

Patrick Woo

The SARS epidemic has boosted interest in research on coronavirus biodiversity and genomics. Before 2003, there were less than 10 coronaviruses with complete genomes available. After the SARS epidemic, up to August 2015, there was an addition of more than 40 coronaviruses with complete genomes sequenced. These include two human coronaviruses (human coronavirus NL63 and human coronavirus HKU1) and at least 30 other mammalian and avian coronaviruses. Two novel lineages in Betacoronavirus and a novel genus (Deltacoronavirus) have been discovered. The diversity of coronaviruses is a result of the infidelity of RNA dependent RNA polymerase, high frequency of homologous RNA recombination and the large genomes of coronaviruses. Among all hosts, the diversity of coronaviruses is most evidenced in bats and birds, which may be a result of their species diversity, ability to fly, environmental pressures and habits of roosting and flocking. In general, bat coronaviruses are the gene pools of Alphacoronavirus and Betacoronavirus lineages B, C and D whereas bird coronaviruses are the gene pools of Gammacoronavirus and Deltacoronavirus. However, recent evidence also suggested that rodent coronaviruses could be the gene pools of Betacoronavirus lineage A. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed, which were results of recent interspecies jumping and may be the cause of disastrous outbreaks of zoonotic diseases.

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18 July 2017 Oral Presentation

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Abstract Submission

Viral diagnostics

OR179

RAPID VIRAL DISEASE DIAGNOSIS AND MULTIPLEX DETECTION TECHNOLOGY WITH POTENTIAL FOR POINT- OF-CARE USE

J. Macdonald 1 2,*, J. Kristoffersen 1, J. Li 1, A. James 1

1Genecology Research Centre, Inflammation and Healing Research Cluster, and School of Science and Engineering, University of the Sunshine Coast, Sippy Downs, Australia, 2Division of Experimental Therapeutics, Department of Medicine, Columbia University, New York, United States

Objectives: Rapid diagnosis of acute infections is critical for effective treatment of patients and animals, as well as monitoring and containment of disease spread. However, rapid antigen-based point of care assays, such as lateral flow devices, often lack sensitivity and specificity required for best-practice management, particularly for dangerous infectious diseases. Because of this, samples are usually collected and shipped to central laboratories for detection of pathogen genetic materials by real-time PCR. We have investigated use of isothermal amplification technology recombinase polymerase amplification coupled with lateral flow detection (RPA-LF) as a viable alternative for rapid detection of diseases without requiring the extensive laboratory infrastructure required for real-time PCR detection. We also determined if RPA- LF could be expanded to enable multiplex analysis of diseases on a single strip.

Methods: We developed and optimised RPA-LF for diagnosis of Hendra, Ebola, Adenoviruses, Japanese Encephalitis (JEV), Murray valley encephalitis (MVEV) and West Nile viruses (WNV). Analytical sensitivity was tested using quantified synthetic nucleic acids, and analytical specificity was tested using nucleic acid extractions of cultured viruses. Clinical specificity and sensitivity was tested using nucleic-acid extracted from a wide range of samples, including nasal, oral, rectal and urogenital swabs, as well as bloods, and RPA-LF results were compared to the gold standard real-time PCR. For each test, after addition of sample, RPA reactions were incubated at 39 °C for 5-30 minutes (depending on the assay), and subsequently detected by lateral flow (5 minutes). For multiplex reactions, 7-different labelled primers were employed, which bound to corresponding seven different antibodies embedded in a lateral flow strip.

Results: The RPA-LF assays exhibited excellent analytical sensitivity and specificity, detecting down to 100 copies of nucleic acid template, and allowing discrimination between closely related pathogens. The assays also indicated excellent clinical sensitivity and specificity, correctly identifying virus in 100% of samples tested, and did not react with samples confirmed to be negative by real-time PCR. Importantly, some of the assays were extremely rapid, for example, the Hendra assay could amplify nucleic acids in as little as 6 minutes. Multiplex studies indicated that RPA could tolerate up to seven different primer label mixtures in a single tube reaction, and could specifically detect the correct mixtures applied in each case. We also demonstrated this technology could be used to develop a text-display output system using binary and molecular encoding, enabling analysis of multiplex results outputs without requiring an external reader.

Conclusion: The RPA-LF assay appears highly amenable for use at the primary point of care, as it requires only minimal inexpensive equipment – mainly a heating block - for operation. Some technical expertise and a centrifuge may be required to pre-process samples, but this would be similar skills required for the processing of blood samples that are already performed on-site in such clinics. Development of multiplex applications also appears feasible, and would improve efficiency and reduce costs compared to performing multiple assays for single diseases.

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Keywords: lateral flow, multiplex diagnostics, recombinase polymerase amplification

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Abstract Submission Viral diagnostics

OR180

APPLICATION OF NEXT-GENERATION SEQUENCING TO DETECT ACYCLOVIR-RESISTANT HERPES SIMPLEX VIRUS 1 VARIANTS AT LOW FREQUENCY IN THYMIDINE KINASE GENE FROM VIRAL ISOLATES, EMERGENCE IN PATIENTS WITH STEM CELL TRANSPLANTATION

H. Fujii 1,*, S. Kakiuchi 2, T. Yoshikawa 1, S. Yamada 1, N. Omura 1 3, M. Shibamura 1 4, S. Harada 1, M. Saijo 1

1Virology 1, NATIONAL INSTITUTE OF INFECTIOUS DISEASE, 2Pediatrics, The University of Tokyo Hospital, 3Life Science & Medical Bioscience, Waseda University, 4Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Objectives: HSV-1 infections are associated with poor prognosis in patients who receive hematopoietic stem cell transplantation (HSCT). Patients with HSCT usually administered with acyclovir (ACV) prophylactically for prevention of HSV-1 reactivation. However, prolonged antiviral therapy sometimes causes emergence of ACV-resistant mutants, and place the patients at risk for the development of ACV-resistant HSV-1. In recent years, next generation sequencing (NGS) allows the detection of minor variant drug-resistance mutations in HCV, HIV and so on, and also used to study monitoring evolution of drug resistant viruses.

In this study, we focused on the mutation in HSV-1 thymidine kinase (TK) genes, which is the major cause of ACV resistance, and established the NGS system to determine low-abundance variants. Furthermore, we followed the emergence of TK- associated ACV-resistant HSV-1 in the isolates recovered from HSCT patients over time using the NGS system and compared the results with those obtained by using Sanger sequencing method.

Methods: Oral swab was collected regularly once a week from HSCT patients, who were administered with anti-herpesvirus drug for prophylaxis and tested for virus isolation. When HSV-1 was isolated, the viral isolates were tested for ACV- sensitivity with a plaque reduction assay. DNA was isolated from each viral isolates, and 1000 copies of HSV-1 DNA was used to amplify HSV-1 TK gene and its contiguous region with PCR. The purified DNA were used for performing both Sanger sequencing and NGS.

To calculate error rate and sensitivity for variant detection, plasmids contain wild-type or mutated HSV-1 TK gene and its contiguous region were mixed in ratio of 100:0, 99:1, 95:5, 90:10 and 80:20 and performed PCR and following sequencing the same as viral isolates.

Results: The Single Nucleotide Polymorphisms (SNPs) were found to be present at a frequency below 1% in the sample from single plasmid, and the error rates in homopolymer region was higher than those of non-homopolymer region. The calculated frequency of plasmid mixture based on the output data closely resembled the ratios evaluated, which demonstrated that the NGS system had sensitivities for detection 5 – 95 % SNPs in the sample.

The NGS assay established in this study detected not only the all mutations detected with Sanger sequence, but also low- level TK-gene mutations associated with ACV-resistance, which had been missed with Sanger sequencing.

Conclusion: This NGS system allowed to study monitoring evolution of TK gene associated drug resistant HSV-1, which might provide important information for management of patients undergoing therapy. Also, the approach described here might be applicable to study HSV-1 TK mutation diversity and could help to discover drug-resistant viruses rapidly, which might be helpful for proper treatment.

Keywords: acyclovir resistant, herpes simplex virus, thymidine kinase

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Abstract Submission

Viral diagnostics

OR181

A novel 3base assay for the detection of flavivirus/alphavirus in clinical samples

D. Millar*

Objectives: Flavivirus is a genus in the family Flaviviridae that contains a large number of viral agents capable of causing encephalitis and jaundice. Most flaviviruses are transmitted to the human population by a bite from infected mosquitoes or ticks. Individual members such as dengue (DENV), yellow-fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) cause significant morbidity and mortality worldwide with up to 100 million new dengue infections alone per year. Using 3base™ technology we developed a rapid real-time PCR assay that is able to detect the presence of any flavivirus or alphavirus in a clinical sample along with specific primer/probe sets for pan- DENV (1-4), WNV, Zika, YFV, SLEV, TBEV, JEV and the alphavirus chickungunya (CHIKV).

Methods: In order to assess assay sensitivity, synthetic in vitro transcribed (IVT) RNAs were prepared. In addition, whole virus samples (Zeptometrix, Buffalo, USA), RNA (Vircell, Granada, Spain) and proficiency panels (QCMD, Glasgow, Scotland) were obtained. Mock clinical samples consisting of serum, urine and saliva were prepared using whole virus particles to determine assay sensitivity. The assay was further tested using matched serum, urine and saliva of patients from a dengue outbreak on the South Pacific Island of Vanuatu to determine to geographical distribution of flavivirus/alphavirus. RNA extraction and PCR set-up was performed on a GS1 automated extraction platform (Genetic Signatures, Sydney, Australia). PCR was performed on a CFX real-time PCR instrument (Biorad, California, USA).

Results: The assay was originally optimised in singleplex form, then multiplexed using IVTs and synthetic constructs. We observed sensitivity for all targets to be less than 10 copies of target when introduced into the RT-PCR reaction. Results obtained using the 2016 QCMD DENV, WNV and Zika challenges showed 100% concordance with expected results. Spiking experiments using serum, urine and saliva for DENV, WNV and Zika showed a sensitivity of <10 copies in the PCR reaction. No inhibition was observed confirming the assay was compatible with a full range of clinical samples. The testing algorithm used in Vanuatu is shown in figure 1. Results of the Vanuatu study will be presented in detail.

Conclusion: As a result of the genome complexity reduction achieved using 3base™ technology we were able to design a single primer/probe set that detected all flavivirus and alphavirus species. In the most basic form of the assay a single tube duplex RT-PCR consisting of pan-flavivirus and pan-alphavirus can be used to detect the presence of any flavivirus/alphavirus present in the primary patient sample. Positive samples can then be reflexed to determine the exact flavivirus/alphavirus responsible for the disease. The assay can be fully automated and provides healthcare workers with a fast and accurate diagnosis of flavivirus/alphavirus infections.

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Keywords: Alphavirus, Flavivirus, real time PCR

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Speaker Abstract Submission

DNA Viruses I

CHALLENGES AND OPPORTUNITIES IN SEQUENCING COMPLETE HUMAN CYTOMEGALOVIRUS GENOMES DIRECTLY FROM CLINICAL MATERIAL

A. Davison*, N. Suárez 1, G. Wilkie 1, M. Zavattoni 2, P. Hubáček 3, E. Hage 4, T. Ganzenmueller 4, T. Schulz 4, G. Gerna 5 1MRC-University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom, 2Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, 3Department of Medical Microbiology, Motol University Hospital, Prague, Czech Republic, 4Institute of Virology, Hannover Medical School, Hannover, Germany, 5Experimental Research Laboratories-Transplantation Area, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Objectives: Human cytomegalovirus (HCMV) can be a serious problem in congenital or transplant-acquired infections, and also in people infected with HIV. Infections involving multiple strains have been linked to poor clinical outcome in immunocompromised patients, and some studies in congenital cases reflect a similar linkage. The viral genome is 236 kbp in size and has several long-recognised features that present challenges to genomic studies. These include the existence of hypervariable genes, the presence of gene-disrupting mutations, the prevalence of infections involving multiple strains, an evolutionary history characterised by extensive recombination, and a propensity to mutate when isolated in cell culture. As a result, meaningful studies of pathogenesis are bound to include analysing strains directly from clinical material in the context of a sound understanding of the number of strains present, as failure to monitor this aspect of infection may lead to the derivation of genome sequences representing artefactual chimaeras and to overestimates of the intrahost diversity of the individual strains present.

Methods: By using modified Illumina sequencing library preparation and target enrichment techniques coupled to a bioinformatics pipeline for sequence assembly, we have determined the sequences of many complete HCMV genomes from such material and deposited them in GenBank. We have incorporated in this pipeline a means of assessing whether each library is sufficiently diverse to represent the sample adequately, as well as a genotyping approach that utilises several hypervariable genes to identify distinct viral strains and estimate their proportions.

Results: We have thus far applied this pipeline to 158 samples from 60 patients, the majority from transplant situations. We will present our findings in relation to the features of the viral genome listed above. Conclusion: In addition to detailed observations on hypervariation, natural mutation and multiple strain infections, our approach has detected signs of intrahost recombination in some longitudinal samples.

Keywords: genomics, herpesvirus, human cytomegalovirus

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Abstract Submission

DNA viruses I

OR182

THE HUMAN CYTOMEGALOVIRUS IE1 PROTEIN ANTAGONIZES PML NUCLEAR BODY MEDIATED INTRINSIC IMMUNITY VIA THE INHIBITION OF PML DE NOVO SUMOYLATION

E.-M. Schilling 1, M. Scherer 1, N. Reuter 1, J. Schweininger 2, Y. Muller 2, T. Stamminger 1,*

1University of Erlangen-Nürnberg, Institute of Clinical and Molecular Virology, 2University of Erlangen-Nürnberg, Division of Biotechnology, Erlangen, Germany

Objectives: Nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by PML/TRIM19 that is SUMO modified on three lysine residues (K65, K160, K490). This modification may arise from autoSUMOylation, since PML was proposed to act as SUMO E3 ligase itself. Importantly, PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV), however, this intrinsic defense is counteracted by the immediate-early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by mediating a loss of PML SUMOylation. The major objective of this study was to unravel the mechanism of IE1-mediated loss of PML SUMOylation

Methods: In order to differentiate between proteolytic deSUMOylation of PML and inhibition of PML de novo SUMOylation, we made use of a cell-based assay as well as in vitro SUMOylation assays using prokaryotically expressed and purified proteins. The influence of IE1 on specific lysine residues was determined by infection studies using primary human foreskin fibroblasts (HFFs) stably expressing PML SUMO site mutants. PML dimerization was analyzed by co-immunoprecipitation.

Results: Interestingly, we observed that IE1 expression did not affect preSUMOylated PML, however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As2O3-mediated hyperSUMOylation of K160 thereby blocking PML degradation. Due to a structural similarity between the IE1 globular core and the coiled-coil region of TRIM family members, we hypothesized that IE1 may interfere with coiled-coil mediated dimerization of PML and thus autoSUMOylation. However, a decrease in PML dimerization was not observed in the presence of IE1.

Conclusion: The data clearly demonstrate that IE1 selectively prevents de novo SUMOylation of PML at specific lysine residues either by directly abrogating PML SUMO E3 ligase function - and thus autoSUMOylation - or by blocking access to SUMO sites.

Keywords: cytomegalovirus, PML nuclear bodies (PML-NBs), SUMOylation

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Abstract Submission

DNA viruses I

OR183

EXPRESSION OF AN ALTERNATIVELY SPLICED IL-15 ISOFORM IN SKIN ENHANCES NEUROINVASIVENESS AND REACTIVATION IN HERPES SIMPLEX VIRUS-1 ZOSTERIFORM MOUSE MODEL

C.-C. Ku 1,*, Y.-J. Lin 1, C.-N. Yu 1

1College of Immunology, National Taiwan University, Taipei, Taiwan

Objectives: Interleukine15 (IL-15) is the member of IL-2 family of cytokine. The pleiotropic function of IL-15 is not only essential to support effector activity of natural killer cells, memory CD8+ T cells and intraepithelial lymphocytes but also involved in human psoriasis and other inflammatory disorders. Regulating tissue-specific IL-15 activity is a plausible approach to develop new strategies for treating inflammatory diseases. Several IL-15 mRNA isoforms generated by alternatively spliced mechanism in vivo have been identified. Our investigations on IL-15DE7 that has a partial deletion in exon 7 of the IL-15 gene have demonstrated that IL-15DE7 suppresses keratinocyte activation to many kinds of external stimulation and blocks neutrophil recruitment to inflamed skin.

Methods: IL-15DE7-expressed skin was infected with Herpes Simplex Virus-1 (HSV-1). The development of zosteriform rash at the primary and the secondary site of infection were monitored in IL-15DE7-expressed mice compared with C57/BL6 wild type (WT) mice. Paired skin and the innervating dorsal root ganglia (DRG) tissues were collected from the same mouse after HSV-1 infection at various times up to day 28. The expression levels of proinflammatory-related cytokines and chemokines as well as the transcriptional levels of HSV-1 genes in both IL-15DE7-expressed and WT mice were measured by quantitative real-time polymerase chain reaction (qRT-PCR).

Results: Infection of IL-15DE7-expressed mouse skin with HSV-1 resulted in a prolonged and earlier re-emerged zosteriform rash compared with mock expressed WT mice. While HSV-1 significantly induced expression of genes that encode for neutrophils recruiting (Cxcl1, Cxcl2, Cxcl3 and Cxcl5), macrophages recruiting (Ccl2) chemokines, proinflammatory cytokines (Il6, Il1b) and HSV-1 induced T cell activation factor (Tnfsf14), they were all suppressed in HSV- 1 infected IL-15DE7-expressed skin. Transcriptions of HSV-1 transactivator ICP4 and glycoprotein B (gB) genes were also increased in the innervating DGR of IL-15DE7-expressded mice compared with WT mice. These results suggest that IL- 15DE7 modulates immune cell chemotaxis and anti-HSV-1 immunity in skin and affects reactivation of HSV-1 from the innervating neurons.

Conclusion: We have demonstrated that suppression of proinflammatory responses in skin is likely to enhance the neuroinvasiveness and reactivation of HSV-1 from innervating DRG. Whether this is caused by the depressed development of tissue resident memory T cells mediated by IL-15DE7 is under investigation. Results from these experiments will lead to a better understanding on how a natural cytokine isoform generated by alternatively splicing interacts with other cells or molecules of the immune system and regulates tissue-specific immunity.

Keywords: alternatively spliced isoform, Herpes Simplex Virus-1 pathogenesis, HSV-1 zosteriform mouse model

17th International Congress of Virology 44

Abstract Submission

DNA viruses I

OR184

NON-CANONICAL SMAD SIGNALLING PROMOTES SPREAD OF VACCINIA VIRUS

A. Gowripalan 1,*, C. McKenzie 1, T. Newsome 1

1Department of Microbiology, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia

Objectives: Many viruses have the ability to manipulate cellular signalling pathways in order to overcome obstacles which impede viral replication. These pathways often control critical functions such as apoptosis, cell migration and cytoskeletal reorganisation. Some of these same pathways are aberrantly controlled in tumour cells, where they facilitate metastasis and tumour survival. One of the most commonly dysregulated systems in this context is the TGF-β/Smad signalling cascade. Observations in our lab suggest vaccinia virus (VACV), an oncolytic poxvirus, can exploit elements of this same pathway during infection. Our research aimed to investigate how VACV is able to do this, and whether any components of the pathway may modulate infection dynamics, in the hope of finding novel candidates for oncolytic therapy research.

Methods: To determine the influence of VACV on TGF-β/Smad signalling, downstream targets of this pathway were monitored during infection, via luciferase assay, qPCR, immunoblot and immunofluorescence assays. Further work went on to study the effects of TGF-β-associated transcription factors, known as Smad proteins, on elements of infection, including spread and replication. These experiments were carried out using cell lines created via CRISPR-Cas9 and siRNA technologies.

Results: Specifically, we found that VACV can potently activate the R-Smad transcription factors, Smad2 and Smad3, as well as the common Smad, Smad4. In the canonical TGF-β system, R-Smad proteins are activated by the TGF-β receptor, before forming complexes with the common Smad, and then modulating transcription of target genes. In our assays, we detected expression of these targets at both the transcript and protein level following infection. Furthermore, we have demonstrated a role for the common Smad, Smad4, in a number of aspects of viral infection, including viral replication, virus-induced cell invasion and migration. Interestingly, it appears that activation of these proteins occurs entirely independently of TGF-β receptor phosphorylation in the VACV context.

Conclusion: As of yet, no virus is known to stimulate the TGF-β pathway in the same manner as VACV, or lead to phosphorylation of Smad proteins independently of the TGF-β receptor. Understanding how VACV is able to induce this outcome, without TGF-β receptor input, could be critical in understanding dynamics of non-canonical Smad signalling, and aid in the development of targeted oncolytic agents.

Keywords: cell migration, TGF beta, vaccinia virus

17th International Congress of Virology 45

Abstract Submission

Plant viruses I

OR185

Key determinant of viral synergism in Phalaenopsis co-infected with Cymbidium mosaic virus and Odontoglossum ringspot virus

N.-S. Lin 1,*, S.-C. Lee 1, H. Pai 1

1Academia Sinica, INSTITUTE OF PLANT AND MICROBIOLOGY BIOLOGY, Taipei, Taiwan

Objectives: The synergistic effect of co-infection of distinct plant viruses enhances virus accumulation, symptom expression and also economic loss in agricultural crops. The mixed infection of the two most prevalent viruses infecting orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), often has synergistic effects, with intensified viral symptoms in orchids. However, the molecular mechanism of this synergism is still unknown. Here we identified and characterized the molecular determinant of the viral synergism involved in CymMV and ORSV pathogenesis in Phalaenopsis.

Methods: The synergistic pair of CymMV and ORSV was isolated from the natural host Phalaenopsis amabilis. The infectious clones of viruses were constructed into a T7 promoter-driven vector, and their individual infectivity was assayed in the experimental hosts Nicotiana benthamiana and Chenopodium quinoa by RNA blot. The synergistic effects of the infectious clones were also assayed in Phalaenopsis and N. benthamiana plants. To identify the potential viral suppressors of RNA silencing (VSRs), viral proteins were sub-cloned individually into a Agrobacterium-mediated transient expression vector, and VSR activities were examined by RNA and protein accumulation of the transiently expressed reporter, green fluorescent protein. The verified VSR and its attenuated mutant were then transiently expressed in N. benthamiana plants for functional assay. The infectious clones expressing attenuated VSR were generated and assayed for their infectivity and pathogenicity.

Results: The infectious clones of CymMV and ORSV were successfully generated. Co-infection of both clone-derived viruses had a clear synergistic effect of chlorotic ringspot symptoms in P. amabilis although individual viruses did not exhibit any symptoms when inoculated alone at 10 days post-inoculation. Hence, the biological features of infectious clones were indistinguishable from viruses isolated from diseased orchids. Among the eight viral protein genes tested for viral VSR activity, ORSV P126 was the only functional VSR verified. N. benthamiana leaves with transient P126 expression showed enhanced viral titers and systemic spreading of CymMV, which suggests a role for P126 in viral synergism. Moreover, we identified a key amino acid residue in P126 that affects the VSR activity and also the viral accumulation of ORSV.

Conclusion: In this study, we elucidated the CymMV/ORSV pathogenicity and synergism in the natural host P. amabilis and the compatible experimental host N. benthamiana by naturally isolated virions and infectious full-length clones of CymMV and ORSV. In both plant species, intensified symptoms were the most pronounced effects of CymMV and ORSV synergism. By VSR activity and functional assays, we identified ORSV P126 as a key VSR protein involved in the synergistic effect of enhancing viral titers and spreading co-infected CymMV. We also characterized a residue in the helicase domain of P126 associated with the VSR activity and ORSV accumulation. Our findings provide direct evidence to support that viral VSR P126 is a key determinant in the synergistic effects of viral co-infection in Phalaenopsis.

Keywords: Phalaenopsis orchids, Viral suppressor for RNA silencing, Viral synergism

17th International Congress of Virology 46

Abstract Submission

Plant viruses I

OR186

INFECTION STRATEGY OF BROME MOSAIC VIRUS IN HOST AND NON-HOST PLANTS

S. Nakagawa 1, K. Yazaki 1, T. Okuno 1 2, M. Kaido 1, Y. Takano 1, K. Mise 1,*

1Graduate School of Agriculture, Kyoto University, Kyoto, 2Faculty of Agriculture, Ryukoku University, Otsu, Japan

Objectives: In plants, the non-host resistance against viruses remains poorly understood and previous studies suggest RNA silencing as the most likely mechanism. Brome mosaic virus (BMV), the type species of the genus Bromovirus, has three positive-sense genomic RNAs. RNA1 and RNA2 encode 1a and 2a proteins, respectively, which are both required for viral RNA replication. RNA3 is a dicistronic RNA encoding 3a movement protein and coat protein (CP). The CP is translated from subgenomic RNA4 that is synthesized from minus-strand of RNA3. BMV includes many strains, but any of the strains tested cannot infect any ecotypes of Arabidopsis thaliana efficiently, demonstrading that BMV cannot infect A. thaliana because of its non-host resistance against BMV. Our preliminary experiments suggested the existence of multi-layered resistance of A. thaliana against BMV by testing systemic infectivity of a few BMV strains in an RNA silencing-deficient mutant, dcl2 dcl3 dcl4 (hereafter dcl2/3/4). In this study, to clarify the multi-layered resistance mechanisms against BMV, we tested additional strains of BMV and investigated viral factors involved in overcoming the unknown layer of resistance other than RNA silencing.

Methods: BMV virions or in vitro transcripts were inoculated to whole plants or protoplasts of Arabidopsis thaliana, cowpea and Chenopodium quinoa. Systemic infection was assayed by ELISA using anti-BMV antiserum and Northern blot analysis to detect viral RNAs.

Results: We first tested systemic infectivity of 12 BMV strains in an RNA silencing-deficient Arabidopsis mutant, dcl2/3/4. Three BMV strains efficiently infected dcl2/3/4 plants systemically while nine other strains still failed to infect the mutant plants, confirming the existence of multi-layered resistance of A. thaliana against BMV. Meanwhile, one of the nine non- infectious BMV strains, that is a legume-infecting strain M2 (hereafter BMV-M2), infected dcl2/3/4 plants at low frequency due to the generation of mutants with several mutations in the non-coding regions and CP gene within RNA3. These BMV- M2 mutants commonly showed enhanced accumulation of 3a movement protein in dcl2/3/4 protoplasts. Transgenic dcl2/3/4 plants expressing 3a protein to high levels were systemically infected with the wild type of BMV-M2. Thus, BMV-M2 systemically infected dcl2/3/4 plants, no matter whether large amount of 3a protein was expressed from virus or transgene of plants. In either case, BMV-M2 multiplied to higher levels in the inoculated leaves, which may cause efficient movement to upper uninoculated leaves, leading to successful systemic infection. On the other hand, the adaptive mutations conversely reduced infectivity of BMV-M2 in natural hosts, cowpea and Chenopodium quinoa due to enhanced accumulation of 3a protein.

Conclusion: Some BMV strains cannot infect Arabdopsis thaliana due to RNA silencing and other strains are restricted their infection due to additional unknown resistance mechanisms. The high level accumulation of 3a movement protein is a solution to overcome such unknown resistance in a non-host. In contrast, BMV M2 strain adapts natural hosts by controlling 3a protein accumulation at lower levels.

Keywords: Brome mosaic virus, Arabidopsis, non-host resistance

17th International Congress of Virology 47

Abstract Submission

Plant viruses I

OR187

ISOLATION OF AN NB-LRR GENE INVOLVED IN RICE RESISTANCE AGAINST BROME MOSAIC VIRUS

T. Oya 1,*, K. Nitta 1, Q. Xu 1 2, K. Yasuda 1, M. Teraishi 1, M. Kaido 1, T. Okuno 1 3, Y. Takano 1, Y. Okumoto 1, K. Mise 1

1Graduate School of Agriculture, Kyoto University, Kyoto, Japan, 2Shenyang Agricultural University, Shenyang, China, 3Faculty of Agriculture, Ryukoku University, Otsu, Japan

Objectives: Plant viruses infect host plants interacting with and utilizing host plant factors through infection steps, such as multiplication in single cells, cell-to-cell movement, and long-distance movement. On the other hand, plants prevent pathogens from invading and spreading by sensing their existence and activating immune responses. For example, N, a tobacco protein belonging to the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs), recognizes p50, the helicase domain of Tobacco mosaic virus replicase protein, and triggers severe immune responses involving necrosis. Brome mosaic virus (BMV), a plant RNA virus, contains a variety of strains and each has unique host range. One of the strains, F-strain (BMV-F), can infect rice. In this study, we discovered the difference in susceptibility between indica and japonica rice, and investigated the molecular mechanism of rice resistance against BMV-F.

Methods: We inoculated BMV-F to 12 cultivars of rice including indica and japonica rice cultivars to examine the difference in susceptibility. Linkage analysis and positional cloning were conducted with cv. Koshihikari (japonica) and cv. Habataki (indica) to locate resistance/susceptible factors. Referring to RNAseq data, we selected and cloned candidate genes. Transgenic rice lines, originally susceptible to BMV, expressing the candidate genes were produced by Agrobacterium- mediated method. They were inoculated with BMV-F to examine the susceptibility. As for both indica and japonica cultivars, we analyzed cDNA sequence and expression levels of a candidate gene.

Results: Five out of six indica cultivars were susceptible, but all six japonica cultivars were resistant. The resistance factor was located within the 50 kb region containing two candidate genes named NBS1 and NBS2, both of which are NB-LRR genes, on chromosome 8 of Koshihikari. The transgenic lines with NBS2 but not NBS1 showed the resistance, indicating that NBS2 of Koshihikari acts as a resistance factor. Expression levels of NBS2 were similar between Koshihikari and Habataki. However, nucleotide sequencing of cDNAs revealed a stop codon before LRR domain-coding region in the NBS2 of five susceptible indica cultivars. One resistant indica cultivar, Nona Bokra, did not have the stop codon.

Conclusion: These results suggest that NBS2, a rice protein belonging to the class of NB-LRRs, confers the resistance in rice against BMV, and that the difference in susceptibility between japonica and most indica cultivars is caused by the stop codon in NBS2.

Keywords: Rice, Brome mosaic virus, Plant immunity

17th International Congress of Virology 48

Abstract Submission

Arboviruses I OR188

Structure, Maturation, and Antibody Binding of Zika and related flaviviruses

R. Kuhn*

Objectives: The structures of mature and immature Zika virus were determined by cryo-electron microscopy. Furthermore, the distribution of prM content was assessed by particle morphology and mass spectrometry and compared with dengue viruses. The binding of neutralizing antibodies to the virion were also analyzed.

Methods: Mature and immature Zika viruses were grown and purified in large scale as previously described. Antibody Fab fragments were generated by proteolysis and used to saturate binding sites on virions. The samples were cryo-frozen and imaged using a Titan Krios using a K2 direct electron detector. Image reconstructions were done as previously described. prM amounts were determined using Selected Reaction Monitoring mass spectrometry.

Results: We will present the structures of mature and immature Zika viruses and compare these with other flaviviruses, especially dengue virus. The new immature Zika virus has provided us with the first view of an ordered capsid core and suggests that the maturation process induces a conformational change in the core. How this is affected is under investigation and will be discussed. The maturation of Zika (prM content) compared with dengue viruses will also be presented as will a discussion of particle dynamics. Finally, the binding of neutralizing antibodies to Zika virus will be presented and the mechanism by which they neutralize will be proposed. In particular the structure of ZIKV-117, a potent human neutralizing antibody,in complex with Zika virus will be shown. One Fab of ZIKV-117 is shown to bind to each asymmetric unit with two potential sites of binding.

Conclusion: High resolution structures of Zika viruses have provided new insights into flavivirus structure and biology as well as demonstrating unique aspects of Zika virus. These studies lay the foundation for the rational design of vaccines and antiviral therapeutics and for a general understanding of the Zika virus life cycle.

Keywords: Cryo-EM, Dengue virus, zika virus

17th International Congress of Virology 49

Abstract Submission

Arboviruses I OR189

A Mutation identified in Neonatal Microcephaly Destabilizes Zika Virus NS1 Assembly in vitro H. Yang*, D. Wang

Objectives: An unprecedented epidemic of Zika virus (ZIKV) infection had spread to South and Central America. ZIKV infection was recently confirmed by CDC (the Centers for Disease Control and Prevention) to cause neonatal microcephaly, which posed a significant public health emergency of international concern. No specific vaccines or drugs are currently available to fight ZIKV infection. ZIKV nonstructural protein 1 (NS1) plays an essential role in viral replication and immune evasion, of which research could potentially be valuable in understanding ZIKV anti-virus specificities and in the development of antibodies against ZIKV.

Methods: We determined the crystal structure for ZIKV NS1172-352, expressed and purified the T233A mutant proteins of ZIKV NS1172-352. We also expressed and purified the full length parent and T233A NS1s with insect cell system.

Results: The crystal structure for ZIKV NS1172-352, which forms a head-to-head, symmetric dimer with a unique 14- stranded β-ladder conserved among flaviviruses. The assembly of the β-ladder dimer is concentration dependent. Mutation of T233 to Ala disrupts this elaborated interaction network, and destabilizes the NS1 dimeric assembly in vitro.

Conclusion: Our structural study of ZIKV NS1 provided a framework for understanding the assembly and immune recognition of this critical viral protein. In the future, it will be worth to investigate the functional and pathogenic impacts of destabilized ZIKV NS1 assembly and/or possibly conformational variation induced by T233A mutation on the secretion of NS1 as the marker for infection, and on the immune evasion of ZIKV.

Keywords: None

17th International Congress of Virology 50

Abstract Submission

Arboviruses I

OR190

Effects of salivary gland proteins in dengue virus replication in the mosquito Aedes aegypti

C. Sri-in 1,*, S.-H. Shiao 2, W.-C. Tu 1

1Entomology, National Chung Hsing university, Taichung, 2Parasitology, Natioanal Taiwan university, Taipei, Taiwan

Objectives: Dengue fever is the most prevalent arthropod-borne viral disease in humans with no effective medication and vaccine. Previous studies have indicated that proteins from mosquito salivary gland may influence the infections of dengue virus (DENV) on human. However, the functions of salivary gland proteins on DENV infection in the mosquito Aedes aegypti remain largely unknown. Therefore, we hypothesize that some salivary gland proteins of Ae. aegypti may enhance or suppress the replication and transmission of dengue virus. In this study, we showed the effect of salivary gland protein in the regulation of dengue virus replication and transmission.

Methods: We identified several salivary gland proteins in the mosquito Ae.aegypti, namely D7, 34kDa and Apyrase. Next, we examined the tissue specific expression of these salivary gland proteins after DENV infection. These salivary gland proteins were then individually silenced in the mosquito and infected with DENV. To elucidate their effects on DENV replication, we made use of RT-qPCR analysis and Western blot analysis to examine the DENV transcript and E protein expression, respectively. Furthermore, we investigate the effect of 34kDa on DENV infectivity by Plaque assay.

Results: Our results showed that the transcripts of these salivary gland proteins were up-regulated in the mosquito salivary gland post DENV infection. The replication of DENV RNA was significantly inhibited with the absence of D7 and Apyrase. Furthermore, the expressions of NS-1 and E protein of DENV were inhibited when silencing of 34kDa. Interestingly, we showed that silencing of 34kDa inhibited the DENV infectivity.

Conclusion: Taken together, our findings indicated that salivary gland proteins are important for dengue virus replication and transmission in the mosquito Ae. aegypti. Especially, 34kDa plays important role in the replication of DENV structural and nonstructural protein and the infectivity of DENV.

Keywords: Aedes aegypti, dengue virus, salivary gland proteins, gene silencing

17th International Congress of Virology 51

Abstract Submission

Arboviruses I

OR191

THE MICROBIOME COMPOSITION OF AEDES AEGYPTI IS NOT CRITICAL FOR WOLBACHIA-MEDIATED INHIBITION OF DENGUE VIRUS

M. Audsley 1,*, Y. Ye 1, E. McGraw 1

1School of Biological Sciences, Monash University, Clayton, Australia

Objectives: Dengue virus (DENV) is primarily vectored by the mosquito Aedes aegypti, and is the causative agent of dengue fever and the more severe dengue haemorrhagic fever/dengue shock syndrome. A novel method for DENV control involves stable transinfection of Ae. aegypti with the common insect endosymbiont, Wolbachia, which mediates an antiviral effect. However, the mechanism by which Wolbachia reduces the susceptibility of Ae. aegypti to DENV is not fully understood. In this study we considered the impact of Wolbachia infection on the intrinsic microbiome of Ae. aegypti, and whether other bacteria that reside in the mosquito might affect the ability of Wolbachia to limit dengue virus infection.

Methods: We used 16S rDNA sequencing to compare the microbiome profiles of Ae. aegypti mosquitoes stably infected with Wolbachia strain wMel to those without Wolbachia. We further used antibiotic treatment of Ae. aegypti to shift the microbiome profiles and assessed their susceptibility to DENV provided through an infectious blood-meal.

Results: Although Wolbachia had significant interactions with several taxa, causing changes in their relative abundance, the microbiomes of the Wolbachia-infected and uninfected mosquitoes were similarly dominated by Elizabethkingia and unclassified Enterobacteriaceae. Moreover, despite a significant shift in the microbiome due to antibiotic treatment, we found no difference in DENV infection rates or on the DENV load of infected mosquitoes.

Conclusion: Together these data indicate that it is unlikely that specific resident bacteria are required for the fundamental mechanism by which Wolbachia reduces susceptibility of Ae. aegypti to dengue virus.

Keywords: dengue virus, microbiome, Wolbachia

17th International Congress of Virology 52

Abstract Submission

Virus discovery

OR192

THE RISK OF EMERGING ARBOVIRUSES: DISCOVERY OF A DIVERGENT GROUP OF BUNYAVIRUSES IN AUSTRALIAN MOSQUITOES

J. Hobson-Peters*, B. McLean 1, A. Colmant 1, C. O'Brien 1, J. Harrison 1, D. Warrilow 2, A. van den Hurk 2, D. Watterson 1, C. Johansen 1, R. Hall 1

1The University of Queensland, St Lucia, 2Public Health Virology, Forensic and Scientific Services, Department of Health, Queensland Government, Brisbane, Australia

Objectives: Bunyaviruses have recently caused unprecedented outbreaks of morbidity and mortality in man and animals. Indeed, Crimean Congo hemorrhagic fever (CCHFV), Severe fever with thrombocytopenia syndrome and Heartland viruses have been associated with outbreaks of fatal hemorrhagic fever and are seen as a major emerging risk to human health. Furthermore, recent ground-breaking advances in arthropod-borne virus discovery and studies on their evolution, indicates that many pathogenic arboviruses, particularly bunyaviruses, evolved in arthropod hosts. To overcome a fundamental gap in our knowledge of the prevalence and diversity of bunyaviruses in Australian mosquito populations and to gauge the potential of these viruses as emerging vector-borne pathogens, we screened mosquito populations of far northern Australia and Papua New Guinea and uncovered the presence of a divergent group of bunyaviruses.

Methods: Through our development of an innovative sequence-independent RNA virus detection system that targets the double-stranded viral RNA (dsRNA) replicative intermediates, in conjunction with bunyavirus-reactive antibodies and genus- specific PCR primers, we screened hundreds of mosquitoes from populations of Far North Queensland, Papua New Guinea and Northern NSW. New viruses were further characterised by deep sequencing of the viral genomes and phylogenetic analyses, mass spectrometry of viral proteins and infection studies in a range of vertebrate and mosquito cell lines.

Results: We discovered a series of novel bunyaviruses in Australian and Papua New Guinean mosquito populations. These findings underscore the gross underestimation of bunyaviruses currently circulating within Australian mosquito populations. The viruses at the core of our studies represent the largest, most genetically diverse collection of a new group of mosquito- borne bunyaviruses that form a putative genus in the Bunyaviridae family, provisionally named Goukovirus. Our identification of putative markers of vertebrate virulence and vector-borne transmission in these new bunyaviruses, suggest that they may be at a critical evolutionary point with the potential to infect and cause disease in vertebrates. Furthermore, the genomes of this novel group of viruses, including Badu virus from the Torres Strait in North Queensland, exhibit important evolutionary links to pathogenic bunyaviruses including Rift Valley fever virus and CCHFV.

Conclusion: Our discovery of the first goukoviruses in Australia provides a unique opportunity to study the evolution of bunyaviruses, investigate their emergence risk, and determine whether the presence of goukoviruses in mosquitoes modulate the transmission of other significant arboviruses.

Keywords: bunyavirus, Insect-specific virus

17th International Congress of Virology 53

Abstract Submission

Virus discovery

OR193

A PUTATIVE CROSS-FAMILY RECOMBINANT CORONAVIRUS WITH A REOVIRUS GENE IN BATS

W. Liu 1,*, C. Huang 1, Y. Zhao 1, G. Gao 1 1National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China

Objectives: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals.

Methods: We screened and identified potential novel viruses, in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing.

Results: We identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1). Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia.

Conclusion: A putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus was reported, providing insights into the fundamental mechanisms of viral evolution. Image:

Keywords: bat, coronavirus, Reovirus

17th International Congress of Virology 54

Abstract Submission

Virus discovery

OR194

THE META-TRANSCRIPTOMIC DISCOVERY OF PATHOGENS IN NEGLECTED AND UNDIAGNOSED DISEASE SYNDROMES OF NATIVE AUSTRALIAN WILDLIFE

J.-S. Eden 1 2,*, K. Rose 3 4, M. Shi 1 2, J. Ng 5, Q. Wang 5, V. Sintchenko 1 5, E. Holmes 1

1Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life and Environmental Sciences and Sydney Medical School, Charles Perkins Centre, University of Sydney, Sydney, 2Centre for Virus Research, Westmead Institute for Medical Research, Westmead, 3Taronga Conservation Society Australia, Australian Registry of Wildlife Health, Mosman, 4School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, Townsville, 5Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research-Pathology West, Westmead Hospital, Westmead, Australia

Objectives: In contrast to disease affecting humans and livestock, wildlife disease outbreaks are often neglected and their causes remain elusive. This has clear implications for efforts to preserve global biodiversity but importantly, also represents a significant risk to human health as most novel emerging diseases are spill overs from zoonotic sources. In this study, we investigated a number of neglected and undiagnosed disease syndromes affecting native Australian birds and marsupials over the last 20 years. Some of these conditions include clenched claw disease in rainbow lorikeets, black and white bird disease and wobbly possum disease in brushtail possums.

Methods: To identify potential infectious agents, total RNA from affected tissues was extracted and sequenced using an unbiased, high-throughput RNA sequencing approach. RNA samples were pooled for library preparation based on tissue and disease type. The data was then assembled and screened for viral, bacterial and fungal pathogens by comparisons to existing databases using blast.

Results: A number of candidate viral pathogens were identified including highly divergent picornaviruses affecting black and white birds and rainbow lorikeets, a novel avian paramyxovirus in cases of clenched claw disease, as well as, other novels viruses from the family Arteriviridae, Flaviviridae, Hepadnaviridae and Polyomaviridae. Using this approach, we also identified two cases of tularemia in ringtail possums, that was then confirmed following the isolation of F. tularensis ssp. holarctcica biovar japonica from diseased liver tissue. This is of particular importance as it confirms the existence of this notorious pathogen in the southern hemisphere.

Conclusion: This study has identified a number of candidate pathogens but also reveals important aspects of viral diversity, emergence and evolution.

Keywords: metagenomics, virus discovery, zoonotic pathogens

17th International Congress of Virology 55

Abstract Submission

Virus discovery

OR195

Next-generation Sequencing Analysis of Cerebrospinal Fluid from Adults with Unexplained Central Nervous System Infections

N. Thi Thu Hong*, T. Tan Thanh 1, N. To Anh 1, N. Thi Hoang Mai 2, H. Dang Trung nghia 2, N. Phu Huong Lan 2, P. Phu loc 2, N. Hoan Phu 2, L. Hong Thai 2, N. Van Vinh Chau 2, T. Tinh Hien 1, J. Day 2, T. Hong Chau 1, G. Thwaites 1, L. Van Tan 1

1Oxford University Clinical Research Unit, 2Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam, Ho chi Minh, Viet Nam

Objectives: We employed an in-house metagenomics approach to look for the pathogen contents in cerebrospinal fluid of adults with unexplained Central Nervous System infections admitted to an infectious ward of Hospital for Tropical Diseases in Ho Chi Minh city between 2010 and 2011.

Methods: A total of 57 patients were included for the analysis using a previously published in-house MiSeq-based metagenomics approach. Any metagenomics results suggestive of (un)known pathogens were then confirmed by specific PCR analysis of the original CSF specimen.

Results: The selected CSF specimens were processed and sequenced in a single MiSeq run. Analysis of the obtained sequences followed by pathogen specific PCR confirmation test determined the presence of human pathogen in 14% (8/57) of the specimens. Detected pathogens included Streptococcus suis serotype 2 (n=4, 7%), Streptococcus pneumoniae (n=2, 3.5%), Haemophilus influenzae (n=1, 1.8%) and hepatitis B virus (n=1, 1.8 %). Antibiotic use prior to lumbar puncture was recorded in 2; 1 Streptococcus suis serotype 2 and 1 Haemophilus influenzae. The detection of three common causes of pyogenic meningitis suggested that our pipeline could detect both viral and bacterial pathogens. Evidence of previously uncharacterized viruses was absent.

Conclusion: In this study, the data highlights the potential of next-generation sequencing based method as a pan-pathogen assay for diagnostics of infections caused by a wide range of pathogens as CNS infections, while research to evaluate its clinical sensitivity, specificity and utility is needed.

Keywords: Central Nervous System Infections, Metagenomics, Next-generation sequencing

17th International Congress of Virology 56

Abstract Submission

DNA viruses II

OR196

RECOGNITION OF ADENOVIRUSES THAT LIKELY HAVE SWITCHED HOST

M. Benkő 1,*, M. Z. Vidovszky 1, I. I. Podgorski 1, T. Görföl 2, B. Harrach 1

1Institute for Veterinary Medical Research, Hungarian Academy of Sciences, Centre for Agricultural Research, 2Department of Zoology, Hungarian Natural History Museum, Budapest, Hungary

Objectives: To estimate the diversity and distribution of adenoviruses (AdVs) especially among exotic and wild living animals, we carry on PCR screenings continuously on different samples of live or dead vertebrate specimens. Recently, bats emerged as a particularly rich source of hitherto unknown AdVs. Phylogeny inference revealed several unexpected and interesting close relationships between certain bat AdVs and well-known pathogens of domestic animals. AdVs occur commonly in humans and different vertebrate animals all over the world. The majority of infections result in mild self-limiting disease of the respiratory or alimentary tract, whereas other organs are less frequently affected. Therefore AdVs are generally considered as facultative pathogens. Among the few exceptions is canine adenovirus type 1, the causative agent of the so-called infectious canine hepatitis also known as Rubarth’s disease, which might be fatal and regular vaccination of puppies is used for its prevention.

Methods: Nested PCRs with consensus primers targeting the gene of the viral DNA-dependent DNA polymerase (pol) or of the IVa2 protein were used to detect the presence of AdVs in guano or organ samples of live-captured or dead bats, respectively. The PCR products were sequenced, and the sequences were used in phylogeny reconstructions. The genetic content of some fully-sequenced genomes were submitted to comparative analyses.

Results: On the phylogenetic trees, the AdVs, detected in representatives of the different bat families appeared in usually well-separated although not necessarily monophyletic clades. For example the family Vespertilionidae which includes by far the largest number of small bats, seemed to harbour viruses from at least two major AdV lineages. Interestingly, canine AdV type 1 and 2 did not only cluster among some vesper bat AdVs, but a particular bat AdV appeared between the two canine AdVs proving their very close common ancestry. Another AdV that may cause fatal infection, namely in horses, is equine AdV-1, which also appeared among the AdVs of microchiropteran bats. These perceptions were reinforced by the identical gene composition of the E3 region of the canine AdV-1 & -2, equine AdV-1, and bat AdV-2 & -3. Finally, bovine AdV type 10 seemed to be most closely related to an AdV discovered in megachiropteran bats, i.e. in Indian flying foxes.

Conclusion: The number of presently known bat AdV types based on the different pol sequences soon will be reaching 100 which is probably the tip of the iceberg only since more than 1200 extant bat species have been described and every species might harbour one or more distinct AdV types. The phylogeny reconstruction of bat AdVs strengthened our hypothesis on the co-evolution of AdVs with their vertebrate hosts. Previously, we have also described several suspected host switches between larger groups of animals such as from reptiles to ruminants. In the present study, novel possible host-switch events of smaller scale among mammals could be glimpsed. The AdVs exceptionally pathogenic to dog, horse and cattle, respectively, most likely have close common ancestry with certain chiropteran AdVs. The good news is that no bat AdV seems to fall close to the clade of the primate (including human) AdVs. The grants (OTKA K100163 & K112440) provided by the Hungarian Scientific Research Fund are highly appreciated.

Keywords: adenovirus, bat, host switch

17th International Congress of Virology 57

Abstract Submission

DNA viruses II

OR197

IDENTIFICATION AND APPLICATION OF AN EARLY FOWL AVIADENOVIRUS 9 (FADV-9) PROMOTER

Y. Pei 1, P. Krell 2, E. Nagy*

1Pathobiology, 2Molecular and Cellular Biology, University of Guelph, Guelph, Canada

Objectives: The dsDNA genomes of fowl aviadenoviruses (FAdVs) are about 10 kb larger than those of mastadenoviruses and have unique left and right end sequences and genes. Our previous studies demonstrated that some regions of these unique ends of FAdV-9 are dispensable in vitro but play a role in vivo. This allowed us to target some non-essential regions for foreign gene expression. Moreover, little is known about the promoters of these FAdV-specific genes. We wished to understand the function of the unique sequences/ORFs at the very left end of the genome. We also wanted to improve our FAdV-9 based vector system by generating recombinant viruses expressing two genes in tandem, mCherry and EGFP, separated by an IRES and driven by an FAdV-9 native promoter.

Methods: To test their efficiency as a “native promoter” FAdV-9 nucleotides 1-874 (EP874), 73-574 (EP502) and 575-874 (EP299) were amplified by PCR and the fragments were cloned into pEGFP-N1 replacing the CMV promoter driving EGFP in the vector but retaining EGFP and the SV40 polyadenylation site. Three plasmids pEGFP-EP874, pEGFP-EP502 and pEGFP-EP299 were generated. To monitor the relative level of promoter activity in these three fragments, the EGFP expression in chicken hepatoma (CH-SAH cell line) cells following transfection with the transient expression plasmids was analyzed by fluorescence microscopy and quantitated using a microplate multimode reader. Two recombinant viruses in which the very left end of the FAdV-9 genome was altered were generated using the lambda Red recombinase system: i) open reading frame 1 (ORF1; nts 847-1338) was replaced with an EGFP coding sequence (FAdV-9∆ORF1-EGFP), and ii) the ORF1-ORF2 (nts 847-2753) region of an FAdV-9 infectious clone was replaced with sequences in the following order: native promoter (EP502 above), mCherry RED, internal ribosome entry site (IRES) which was derived from an Ontario avian encephalomyelitis virus (AEV), and EGFP (FAdV-9∆ORF1-2RED-EGFP). Expression of both mCherry RED and EGFP was investigated by fluorescence microscopy in infected CH-SAH, DF1, Huh7.5 and MDCK cells.

Results: In CH-SAH cells transfected with the three gene expression plasmid DNAs the highest EGFP expression was noted for pEGFP-EP502, which started as early as 12 hours post-transfection (p.t.) and peaked at 3 days p.t. These results indicated that the region from nts 73-574 has functional promoter function and we dubbed it as the FAdV-9 “native promoter”. The expression of EGFP or EGFP and mCherry of the two recombinant viruses respectively were confirmed by fluorescence microscopy. Moreover both recombinant FAdV-9 viruses were genetically stable up to passage 4. FAdV-9∆ORF1-EGFP infected not only avian cells but also human liver cells Huh7.5.

Conclusion: FAdV-9∆ORF1-EGFP expressed EGFP demonstrating that the very left end of the FAdV-9 genome upstream of ORF1, specifically nts 73 to 574 (EP502), has promoter function. These results provide an FAdV-9 specific native promoter for future constructs which may be more efficient than the standard foreign CMV promoter.Moreover, this native promoter (nts 73-574) allows the transcription of an mRNA with two ORFs, the second preceded by an IRES, that allows translation of both the 5’ ORF and the ORF following the IRES. This was demonstrated by expression of both mCherry (at the 5’ end of the mRNA) and EGFP (following an IRES) respectively by the double recombinant virus FAdV-9∆ORF1-2RED- EGFP. These results provide an improved platform for the development of multivalent vaccines.

Keywords: foreign gene expression , fowl aviadenovirus , native promoters

17th International Congress of Virology 58

Abstract Submission

DNA viruses II

OR198

THE DIVERSITY AND MOLECULAR EVOLUTION OF MONKEY ADENOVIRUSES

B. Harrach 1,*, I. I. Podgorski 1, L. Pantó 1, M. Biđin 1, T. Papp 1, G. L. Kaján 1, K. Böszörményi 1, M. Benkő 1

1Institute for Veterinary Medical Research, Hungarian Academy of Sciences, Centre for Agricultural Research, Budapest, Hungary

Objectives: Our aim was to study the molecular diversity of adenoviruses (AdVs) occurring in the more ancient lineages of primates. To this end, we screened samples mainly from monkeys and prosimians, but also from less studied apes for the presence of AdVs. Complete or partial genome sequences were determined and analysed for the exploration of the evolutionary past of AdVs.

Methods: Consensus nested PCRs, designed for the genes of the IVa2 protein and DNA-dependent DNA polymerase were used for screening fecal or organ samples, collected from captive or wild-living animals. To acquire larger genome parts, PCRs with specific primers were used and the DNA fragments sequenced. Most of the previously isolated Old World monkey (OWM) AdVs were sequenced by NGS. Genome organization differences were studied by detailed genome annotations and comparisons. The phylogeny of the AdVs was inferred by ProtDist in case of short partial sequences and by ML on full protein sequences.

Results: Full genome sequences were obtained from all OWM AdVs originating from the American Type Culture Collection but not sequenced earlier. Additionally, a near complete genome sequence was gained from a novel New World monkey (NWM) AdV by using Sanger sequencing. Several novel AdVs were discovered in different NWM and prosimian hosts. Partial DNA sequences were obtained from AdVs newly-detected in prosimians, NWMs, OWMs and from some apes. The phylogeny inference showed that the AdVs group largely accordingly to the phylogenetic relationship of their hosts. This observation was further supported by some shared common characteristics deduced from the full genome analyses of AdVs, namely the number of fibre gene/s (found to be between 1 and 3), and the gene content of the E3 region. A cluster of evolutionary closely related OWM AdVs was found to have almost universally two fibre genes, three of these AdVs even having 3 fibre genes, while other primate AdVs have a single fibre gene. The OWM AdVs had an almost uniform E3 region but the novel NWM AdV had an E3 region with considerably different gene content (two new genes).

Conclusion: Numerous novel AdVs could be detected and partially characterized from prosimians, NWMs and OWMs relatively easily by consensus PCRs. Prosimian AdVs have not been described before, and the number of published NWM AdV sequences had also been very limited. The results of our phylogenetic calculations showed clear signs of virus–host co-evolution. The prosimian AdVs formed the most basal branch followed by that of the NWM AdVs then by OWM AdVs, finally by gibbon and orangutan AdVs. The gorilla/chimpanzee/bonobo/human AdV groups seemed to be the most recent AdV lineages. Only a single OWM lineage acquired (and preserved) a second fibre gene. While the ape AdVs all seem to have a single fibre gene they possess an additional gene in their E3 region (19K) compared to AdVs of the more ancient simians. Our results, obtained by scrutinizing a smaller group of mammals provide additional evidences that underpin the theory on the co-evolution of AdVs with their respective vertebrate hosts. (Support: AD-VEC FP7-324325, OTKA NN107632)

Keywords: adenovirus, diversity, monkey

17th International Congress of Virology 59

Abstract Submission

DNA viruses II

OR199

INTERACTIONS OF HUMAN ENTERIC ADENOVIRUSES WITH HOST CELLS

M. Brown 1 2,*, C. Mangroo 2

1Molecular Genetics, 2Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Objectives: Human enteric adenoviruses, species F types 40 and 41 (HAdV-F40/41), infect the small intestine in vivo. They have distinctive structural features that include both long and short fibres, and a penton base lacking the RGD motif used by other adenoviruses to bind host cell integrins and initiate endocytosis. Indeed, we have identified a partial block in uptake of virions by host cells in culture. For those virions that do enter the cell, it was of interest to determine the relative importance of the two fibres, and host cell integrins, before and after exposure of virions to conditions that mimic passage through the stomach. Initial experiments were done with 293 (human embryonic kidney) cells typically used for propagation and assay of HAdV-F40/41. Before doing comparable experiments in more physiologically relevant Caco-2 intestinal epithelial cells, it was important to establish appropriate culture conditions for optimal infection of Caco-2 cells with wild- type HAdV-F40/41.

Methods: Purified HAdV-F41-EGFP virions, with and without exposure to gastric conditions, were used to infect 293 cells. Soluble knobs from HAdV-F40/41 long and short fibres were used in competitive binding experiments as was antibody against CAR (Coxsackie and adenovirus receptor) and blocking peptide for RGD-binding integrins. Cells were harvested 21 hours post infection for analysis by flow cytometry. To compare HAdV-F40/41 infection in Caco-2 intestinal epithelial cultures at different stages of differentiation ranging from young non-polarized cells to mature differentiated cells, cultures were infected at 2 days as well as 1,2,3,and 4 weeks post seeding and examined by light microscopy, to assess cytopathic effect, by fluorescence microscopy for immunodetection of infected cells and by electron microscopy to assess virion production.

Results: The proportion of 293 cells infected with HAdV-F41-EGFP virions not exposed to gastric conditions was reduced ~ 5-fold by polyclonal antibody against CAR (Coxsackie and adenovirus receptor) and ~ 10-fold by soluble long fibre knob but not by short fibre knob. Surprisingly, after exposure to gastric conditions, virions were blocked mostly by short fibre knob with less inhibition by long fibre knob and by anti-CAR antibody. Despite an IGDD rather than RGD motif on penton base, HAdV-F41-EGFP virions were blocked to the same extent as the control HAdV-C5-EGFP by the blocking peptide GRGDSP, whether or not HAdV-F41 virions were exposed to gastric conditions. Experiments are in progress with blocking antibodies to determine the integrins involved. In experiments with Caco-2 cells, young cultures of undifferentiated non- polarized cells were more susceptible than older, polarized and differentiated cells to infection with HAdV-F41 but cells in older cultures could be infected if cultures were pre-treated with EGTA to disrupt tight junctions.

Conclusion: Under normal culture conditions, HAdV-F41 infection of 293 cells is mediated by long fibre binding to CAR but, after exposure to gastric conditions, use of the short fibre becomes predominant. In both cases, RGD-binding integrins are important. The data suggest a model in which long fibres are lost or damaged during passage through the stomach, with short fibres surviving to mediate binding to primary target cells in the intestine. Long fibres may then mediate subsequent spread of progeny virions to neighbouring intestinal cells. Caco-2 cells at different stages of differentiation will be useful as a model to represent intestinal epithelial cells from the crypt to the tip of intestinal villi.

Keywords: Caco-2 , enteric adenovirus, fibre switch

17th International Congress of Virology 60

Abstract Submission

Plant viruses II

OR200

CONTRIBUTION OF NGS-BASED APPROACHES TO THE ANALYSIS OF FRENCH WINTER BARLEY CROPS VIROLOGICAL STATUS

M. Rolland 1, J. Villemot 2, A. Marais 3, S. Theil 3, C. Faure 3, V. Cadot 2, R. Valade 4, C. Vitry 4, F. Rabenstein 5, T. Candresse 3,*

1ANSES, Angers, 2GEVES, Beaucouzé, 3UMR 1332 BFP, INRA, Villenave d'Ornon, 4Arvalis, Thiverval-Grignon, France, 5Julius Kuehn Institute, Quedlinburg, Germany

Objectives: Despite the widespread use of resistant varieties carrying the rym4 gene, the recent years have seen an increase in the impact of viral mosaic diseases in winter barley crops in France. Surveys showed that yield losses could reach up to 20% in some cases. This novel worrisome situation could result from a change in the populations of the two most widespread Bymoviruses, Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), or from the emergence of (an)other viral agent(s).

Methods: In order to separate between these hypotheses, the MOSA-Hordeum project performed a complete viral inventory on affected barley crops using classical virological diagnostics approaches and next-generation sequencing (NGS) dsRNA- based viral indexing.

Results: Besides Barley yellow dwarf virus, which was sometimes observed in coinfection, these efforts revealed the frequent presence of BaYMV and BaMMV but failed to detect the presence of any other virus, ruling out the involvement of a potential novel viral agent. These results were confirmed by large scale testing of symptomatic samples using specific PCR assays, which showed the high prevalence of BaYMV in rym4 varieties. Analysis of the NGS data showed the existence of various resistance-breaking mutations in BaYMV isolates from resistant plants. Phylogenetic analyses demonstrate that these resistance-breaking mutations independently emerged in a wide range of BaYMV isolates and regions, likely as a consequence of the very widespread use of the rym4 resistance. They also shed light on the genetic structuration of BaYMV and BaMMV at the European scale and on the contribution of pseudo-recombination to the evolution of these viruses. Lastly, the results obtained also demonstrate the presence, but in a more limited fashion, of BaMMV isolates able to overcome the rym5 resistance and allowed the tentative identification of resistance breaking mutations.

Conclusion: Taken together these results illustrate the power and interest of NGS-based approaches to clarify novel pathological situations and demonstrate that the degradation of the virological situation in French winter barley crops is linked with the widespread and independent emergence of variants overcoming the widely used rym4 resistance.

Keywords: next generation sequencing, resistance breaking, viral populations

17th International Congress of Virology 61

Abstract Submission

Plant viruses II

OR201

HOW TO ENGINEER USEFUL MILD STRAINS FOR CROSS PROTECTION, USING PAPAYA RINGSPOT VIRUS AS AN EXAMPLE

S.-D. Yeh 1,*, T.-T. Lin 1, C.-P. Chang 1

1Plant Pathology, National Chung Hsing University, Taichung, Taiwan

Objectives: Worldwide production of papaya is seriously limited by the ringspot disease caused by aphid-borne Papaya ringspot virus (PRSV). A nitrous-acid induced mild strain HA 5-1, derived from Hawaii severe strain HA, has been widely used in Taiwan and Hawaii for control of PRSV by cross protection since 1984. However, strain-specific protection rendered the application of HA 5-1 drawn back in Taiwan and difficult to be applied in other geographic regions.

Methods: In this study, based on the identified essential motifs for potyviral pathogenicity, molecular manipulation at the critical residues of PRSV HC-Pro was attempted to generate useful attenuated strains for cross protection. Singly or in combinations, four conserved motifs of PRSV HC-Pro, including the N-terminal FWKG, highly conserved FRNK, and two amino residues at the central (F206) and C-terminal regions (D397), were modified by site-directed mutagenesis to generate attenuated mutants from the cDNA infectious clone of a Taiwan severe strain PRSV YK.

Results: Two useful attenuated mutants, YK F7I and YK F7I+F206L, were selected out based on the criteria of their mildness, stability, titer dynamic and cross-protection effectiveness. The two mild PRSV mutants provide complete cross protection (100%) on papaya plants against Taiwan severe strain YK, superior to that provided by HA-5-1 (10%).

Conclusion: Our results indicate that these mutants solve the problem of strain-specific protection and have great potential to be used in Taiwan for control of papaya ringspot disease. By this approach, useful mild mutants for control of PRSV by cross protection in Thailand, Vietnam and Mainland China are being generated.

Keywords: attenuated virus, cross protection, plant virus control

17th International Congress of Virology 62

CapsidAbstract Submission

Plant viruses II

OR202

GENETIC DETERMINANTS OF CAPSICUM CHLOROSIS VIRUS AND ITS PLANT HOSTS DURING COMPATIBLE AND INCOMPATIBLE INTERACTIONS

S. Widana Gamage 1,*, D. McGrath 2, D. Persley 3, R. G. Dietzgen 4

1Department of Botany, University of Ruhuna, Matara, Sri Lanka, 2Agriscience Queensland, Qld Department of Agriculture and Fisheries, Gatton Qld, 3Agriscience Queensland, Qld Department of Agriculture and Fisheries, Dutton Park Qld, 4QAAFI, University of Queensland, St. Lucia, Qld, Australia

Objectives: Nature of molecular interactions between viruses and their hosts determines susceptibility or resistance. Capsicum chlorosis virus (CaCV) (genus Tospovirus, family Bunyaviridae) has emerged as an important pathogen of capsicum and tomato crops due to a lack of resistant cultivars in Australia and South-east Asia. Current understanding of molecular interactions between CaCV and its plant hosts is lacking. In this study we aimed to identify viral and host genes that are involved in CaCV infection and that may be used as potential targets in future disease control efforts.

Methods: Function and localisation of CaCV non-structural protein NSm was determined by transient expression in Nicotiana benthamiana using agroinfiltration and confocal live cell imaging. NSm was expressed as fluorescent fusion protein in epidermal leaf cells and protoplasts to determine subcellular localization and tubule formation. Cell-to-cell movement of NSm was investigated using a tobamovirus replicon trans-complementation system. RNA silencing suppression activity of CaCV non-structural NSs protein was demonstrated using a GFP reporter assay in transgenic N. benthamiana line 16c. To determine avirulence factor of CaCV, individual CaCV proteins were transiently expressed in CaCV-resistant and susceptible capsicum to observe leaf necrosis. Comparative transcriptome analyses of CaCV-resistant and susceptible capsicum lines were used to determine detailed molecular responses of capsicum to CaCV infection. CaCV- responsive genes were identified and annotated. Expression levels of selected host genes were validated by quantitative RT-PCR.

Results: We provide several lines of evidence to show that CaCV NSm functions as a viral movement protein that facilitates cell-to-cell movement within plants: NSm localized to the cell periphery forming punctate spots at plasmodesmata and self- assembled to form tubular structures protruding from protoplasts. NSm was able to trans-complement intercellular movement of a movement-defective tobamovirus replicon. CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of the silencing signal. NSs was also identified as avirulence determinant that triggers a hypersensitive response (HR) in CaCV-resistant capsicum, associated with a burst of reactive oxygen. Comparative transcriptome analyses of capsicum showed CaCV resistance was underpinned by strong up- regulation of defence-related genes and down-regulation of energy consuming cellular processes such as photosynthesis. Furthermore, a candidate NB-LRR resistance gene was identified in a highly CaCV-resistant breeding line. This study also identified capsicum genes that responded to CaCV infection at an early stage of a compatible interaction. Majority of down- regulated early response genes were involved in plant defence and defence signalling.

Conclusion: CaCV NSm and NSs proteins support virus intercellular movement and suppression of plant antiviral RNA silencing defences, respectively during a compatible interaction. NSm facilitates tubule-guided cell-to-cell movement of CaCV through plasmodesmata. NSs also functions as avirulence determinant that triggered HR in CaCV-resistant capsicum. We identified capsicum genes that have potential roles in CaCV susceptibility and resistance and may be useful as future novel disease control targets.

Keywords: non-structural proteins, tospovirus, transcriptome 17th International Congress of Virology 63

Abstract Submission

Plant viruses II

OR203

TRANSMISSION OF BAMBOO MOSAIC VIRUS IN BAMBOOS BY DIPTERAN INSECTS

K.-C. Chang: 1, L.-T. Chang 1, Y.-W. Huang 1, Y.-C. Lai 1, C.-W. Lee 1, J.-T. Liao 1, N.-S. Lin 2, C.-C. Hu 1, Y.-H. Hsu 1,*

1Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 2Institute of Plant and Microbial Biology,, Academia Sinica, Taipei, Taiwan

Objectives: Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the major threat to bamboo cultivation. The transmission of BaMV, as well as most potexviruses, by insect vectors has not been documented previously. However, field observations of BaMV disease incidences suggested that insect vectors might be involved, which might pose a risk to current disease management systems. In this study, we aimed to investigate the possibility of insect-mediated transmission of BaMV among bamboo clumps to provide further insights into the infection cycles of BaMV.

Methods: Potential insect vectors were collected from BaMV-infected bamboo plantations. The presence of BaMV genomic RNA on the interior or exterior sides of the insects was detected by reverse transcription-polymerase chain reaction using specific primer pairs. BaMV-free dipterans were fed with medium containing green fluorescent protein (GFP)-tagged or wild- type BaMV virions to track the distribution of BaMV inside the insects or to perform transmission assays in bamboos, respectively.

Results: From the major insects collected, BaMV genomic RNAs were detected inside the bodies of two dipteran insects, Gastrozona fasciventris and Atherigona orientalis, but not in thrips. Artificial feeding assays using GFP-tagged BaMV virions revealed that BaMV could enter the digestive systems and survive in the regurgitant and excretion of the dipterans. BaMV RNA could be retained in the dipterans for up to 4 weeks. Insect-mediated transmission assays indicated that both dipterans could transmit BaMV to bamboo seedlings. These results demonstrated that dipterans with sponge-type mouthpart may also serve as vectors for at least one potexvirus, BaMV, among bamboo plants.

Conclusion: The finding suggested that dipteran insect control should be integrated into the disease management measures against BaMV infections.

Keywords: Insect transmission, dipterans, Potexvirus

17th International Congress of Virology 64

Abstract Submission

Arboviruses II

OR204

CAPSID PROTEIN OF WEST NILE VIRUS IS RESPONSIBLE FOR ACCUMULATION OF UBIQUITINATED DENATURED PROTEINS AND NEUROLOGICAL DISEASE

S. Kobayashi 1 2,*, K. Yoshii 1, W. Phongphaew 2, M. Muto 1, M. Hirano 1, Y. Orba 2, H. Sawa 2, H. Kariwa 1

1Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido university, 2Division of Molecular Pathobiology, Hokkaido university Research Center for Zoonosis Control, Sapporo, Japan

Objectives: West Nile virus (WNV) and Japanese encephalitis virus (JEV) belong to the Flaviviridae family. WNV has emerged as a significant cause of viral encephalitis in birds and animals including humans. The viral genome is translated as a single polyprotein, which is cleaved into three structural (C, prM and E) and seven nonstructural (NS) proteins. The WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of denatured proteins that are often ubiquitinated was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of denatured proteins in the WNV-infected cells.

Methods: Human neuroblastoma SK-N-SH cells were transfected with plasmids expressing the C, prME or NS proteins of WNV or the C protein of JEV. Immunocytochemistry assay was conducted to detect the ubiquitinated denatured proteins in the plasmid-transfected cells. Mutations related to the accumulation of the ubiquitinated denatured proteins were introduced into WNV and the recombinant virus was inoculated into C57BL/6 mice. After inoculation, viral titer and histopathological features of the mice were analyzed.

Results: To identify the viral protein inducing the accumulation of denatured proteins, intracellular accumulation of ubiqutinated proteins was examined in the cells expressing the C, prME or NS proteins, and it was only observed in the cytoplasm of the cells expressing the WNV C protein, but not the WNV prME, NS proteins or the JEV C protein. The analysis of the expression of the chimeric C protein between WNV and JEV revealed that the Leu51 and Ala52 of WNV were responsible for the accumulation. The mutations did not affect viral replication and histological distribution in mouse brains, however, the survival rate of the mice inoculated with mutant WNV significantly increased than that of the WT WNV- inoculated mice.

Conclusion: These results suggested that C protein is responsible for the accumulation of ubiquitinated proteins and the accumulation is related to the pathogenesis of WNV.

Keywords: pathogenesis, ubiquitin, West Nile virus

17th International Congress of Virology 65

Abstract Submission

Arboviruses II

OR205

LABORATORY ANALYSIS OF DENGUE VIRUS INFECTION IN THE EYE

J. Carr 1,*, J. Calvert 1, L. Ashander 2, Y. Ma 2, L. Ong 3, S. Alonso 3, S. C. Phaik 4, J. Smith 2

1Microbiology and Infectious Diseases, 2Flinders University, Bedford Park, Australia, 3National University of Singapore, 4Singapore National Eye Centre, Singapore, Singapore

Objectives: Prior clinical cohorts have associated human dengue infection with ocular presentations, including retinopathy. Although not a dengue-defining presentation or life threatening, ocular manifestations may occur in a significant number of patients, and outcomes may adversely impact visual acuity in the long term. We have investigated the ability of dengue virus (DENV) to infect the eye in cell culture models of infection using two cell populations with important physiological roles in maintaining the ‘blood-retinal barrier’, and mouse models of systemic infection.

Methods: Human retinal pigment epithelial (RPE) and retinal endothelial cell (REC) lines were infected with DENV-2, and viral replication measured by plaque assay and immunolabelling. Host cell responses were assessed by qRT-PCR and functional assays by cell impedance. Eyes were harvested from AG129 mice systemically infected with DENV-2, with or without prior infection of mothers with DENV-1, in an in vivo model of antibody dependent enhancement (ADE) of infection. Eyes were either fixed or total RNA was extracted for analysis by immunolabeling or qRT-PCR, respectively.

Results: Both RPE and REC support productive DENV infection, with demonstration of dsRNA, viral antigen, and release of infectious virus. In culture, DENV infects the majority of RPE and is cytopathic, while only a small percentage of REC are infected with no visual cytopathic effect. Cellular responses include production of innate type I interferon and inflammatory responses, alterations in junctional protein and adhesion molecule expression, with associated reduced permeability of cell monolayers. Complementary studies in the DENV-AG129 mouse model demonstrate DENV RNA in the eyes of all infected mice. The intraocular level of virus is comparable in all samples, and not affected by tissue perfusion prior to harvest or an ADE infection model. Further studies will assess the innate immune responses in this infection model and the cell type(s) supporting DENV infection in the eye.

Conclusion: Outcomes have established essential knowledge that forms a foundation to understand the virological and immunological events likely to contribute to ocular presentations during DENV infection in patients.

Keywords: dengue virus, Interferon, ocular infection

17th International Congress of Virology 66

Abstract Submission

Arboviruses II

OR206

DEVELOPMENT OF AN ANIMAL MODEL TO STUDY OCULAR ZIKA DISEASE

S. Rossi 1,*, Z. Zhao 2, M. Yang 2, S. Azar 3, B. Long 2, Y. Huang 2, L. Soong 4, S. Weaver 4, Y. Chen 2, J. Cai 2

1Pathology, 2Ophthalmology & Visual Sciences, 3Institute for Translational Sciences, 4Microbiology and Immunology, University of Texas Medical Branch, Galveston, United States

Objectives: Zika virus infection in the eye is one of the lesser known manifestations of Zika illness in patients but little has been described to try to characterize the mechanism of this damage.

Methods: A129 mice deficient in type I-interferon signaling were used to develop an in vivo model of ocular Zika disease. Two Zika viruses of differing virulence, FSS13025 and Mex 1-7, were injected into mice and a variety of techniques were used to characterize both acute disease and long-term infection, including immunofluorescence, cell enrichment, in situ hybridization and siRNA silencing. Cell culture ex vivo was also used to confirm results.

Results: Three-week-old A129 mice are readily infected by both Zika strains FSS13025 and Mex 1-7. The virulent FSS13025 strain caused severe acute eye infection but mice succumb to illness by day 6 post infection. The less pathogenic Mex 1-7 strain resulted in long-term infection characterized by a "persistent" Muller cell infection, expression of inflammatory cytokines and the lack of severe cell death. Similar infection with the FSS strain could also be seen in C57Bl/6 mice infected as less than a week old.

Conclusion: We have characterized the eye disease caused by two Zika strains of varying virulence in adult A129 mice. This mouse model may be used for analyzing the efficacy of therapeutics and vaccines to combat Zika virus-induced ocular disease. Further studies are needed to measure the effect of the damage on vision in these mice, which is a necessary next step to corrlating this model to human disease.

Keywords: Mouse model, Pathogenesis, Zika virus

17th International Congress of Virology 67

Abstract Submission

Arboviruses II

OR207

EXPERIMENTAL ZIKA VIRUS INFECTION OF NEOTROPICAL PRIMATES

N. Vasilakis*, J. Ruiz 1, A. Brady 1, T. Kuehl 2, S. Rossi 3, S. Azar 3, S. Weaver 3, J. Simmons 1, C. Abee 1, J. Vanchiere 4

1The University of Texas MD Anderson Cancer Center, Bastrop, 2Baylor Scott and White Clinic, temple, 3University of Texas Medical Branch, Galveston, 4Louisiana State University Health Sciences Center, Shreveport, United States

Objectives: The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density and their exposure to the human mosquito-borne transmission cycle.

Methods: To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to ZIKV infection and to determine whether in utero exposure of fetal squirrel monkeys to ZIKV would recapitulate the central nervous system lesions observed in human fetuses and newborns, we inoculated four animals of each species with ZIKV from the current epidemic

Results: Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days post-inoculation and two at 14 days post- inoculation. Three of the four infants born to ZIKV -inoculated squirrel monkey dams had neuropathologic abnormalities consistent with those seen in human infants. The placentas of all four squirrel monkeys had abnormalities and one was PCR-positive for ZIKV at delivery, greater than 100 days post-inoculation.

Conclusion: This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a sylvatic ZIKV cycle in South America. Furthermore, this study provides evidence that congenital ZIKV neuroinvasive disease like that seen in humans can occur in Neotropical primates, offering a unique model for the study of ZIKV pathogenesis and the effects of ZIKV on neurodevelopmental outcomes. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

Keywords: neotropical primates, pregnancy, zika virus

17th International Congress of Virology 68

Speaker Abstract Submission

Bridging session – Microbial Evolution and Diversity

Using Metagenomics to Redefining Virosphere and Disease Emergence

E. Holmes*

Objectives: Virology is entering a new discovery phase. Bulk DNA and RNA shotgun sequencing, including RNA-Seq based techniques, provide a uniquely powerful means to rapidly reveal the microbial composition of any sample without bias. These metagenomic techniques provide important new information on the composition of the virosphere, the fundamental patterns and mechanisms of virus evolution, and are able to determine disease agents on clinically actionable time-scales.

Methods: Here, I will show how metagenomics, and particularly meta-transcriptomics, has advanced all these areas, providing new information of the viromes (and microbiomes) of a diverse array of invertebrate and vertebrates, including iconic Australian species as well as invasive pests, and how they are able to identify emerging pathogens.

Results: In particular, I will show how newly identified viruses filled major gaps in the RNA virus phylogeny, and reveal an evolutionary history characterized by both host switching and co-divergence that likely extends for more than a billion years. These metagenomic studies also reveal that the invertebrate virome is characterized by remarkable genomic flexibility, including frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Conclusion: Together, these data present a view of the virosphere that is more phylogenetically and genomically diverse than depicted in current classification schemes, and represent a major step forward in diagnostic technologies.

Keywords: Evolution, Metagenomics, Virosphere

17th International Congress of Virology 69

19 July 2017 Oral Presentation

17th International Congress of Virology 70

Speaker Abstract Submission

Plenary – Virus Structure and Macromolecular Assemblies 1

Neutralization mechanism of a highly potent antibody against Zika virus

S.-M. Lok*

Please specify what speaker category are you in?: Plenary speaker

Objectives: The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signal an urgency to identify therapeutics. Recent efforts to re-screen dengue virus human antibodies for ZIKV cross-neutralization activity, showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extra-cellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments.

Methods: We used cryo-electron microscopy to determine the structures.

Results: The 4.0Å resolution pH8.0 complex structure shows the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting it prevents the structural rearrangement of the E proteins during the fusion event - a vital step for infection.

Conclusion: This suggests antibody C10 could be a good therapeutic candidate.

Keywords: None

17th International Congress of Virology 71

Speaker Abstract Submission

Plenary – Virus Structure and Macromolecular Assemblies 2

Structural biology of the flavivirus virulence factor NS1: the enigma continues

J. Smith*

Please specify what speaker category are you in?: Plenary speaker

Objectives: Viruses employ a variety of molecular strategies to carve out an existence in their host and to thwart host defenses. Crystal structures of viral proteins and of the host proteins deployed as molecular weapons contribute enormously to our understanding of viral pathogenesis and our efforts to combat viral infection. Mosquito-borne flaviviruses, including the dengue, Zika, yellow fever, Japanese encephalitis and West Nile viruses, cause serious human diseases in much of the world. A growing number of diverse functions has been discovered for the enigmatic virulence factor known as NS1: it is an essential cofactor in viral genome replication, a mediator of the host immune response, and a trigger of host vascular leakage. NS1 lacks precedent in the structure and sequence databases, so an accurate 3D structure was key to dissecting and probing its diverse functions.

Methods: A high-throughput survey of expression conditions in baculovirus-infected insect cells was essential to identifying conditions for production of recombinant NS1 in its natural glycosylated, disulfide-linked form. The 2.9- Angstrom crystal structure of the NS1 protein from West Nile virus was solved from the anomalous scattering of the native sulfur atoms. Structures of the NS1 protein from dengue virus (3.0 Angstrom) and Zika virus (1.9 Angstrom) were subsequently solved.

Results: The initial West Nile virus NS1 structure revealed a new protein fold with a fundamentally dimeric architecture, permitting assignment of functions to two of the three structural domains. The subsequent structures of NS1 from dengue virus established the basis for its lipid encapsidation when secreted, and structures of Zika virus NS1 led to a fuller understanding of its membrane association in cells.

Conclusion: The crystal structures have inspired several follow-up studies that revealed new functions for this remarkable - but still enigmatic - virulence factor.

Keywords: flavivirus NS1

17th International Congress of Virology 72

Abstract Submission

Virus vaccines

OR208

MICRORNA-BASED SUPPRESSION OF NEUROTROPIC FLAVIVIRUS REPLICATION IN THE ARTHROPOD VECTOR AND IN THE BRAIN OF VERTEBRATE HOST: APPLICATION FOR THE DEVELOPMENT OF ENVIRONMENTALLY SAFE, LIVE ATTENUATED VACCINE.

A. Pletnev*, K. Tsetsarkin 1, N. Teterina 1

1DHHS, NIH, NIAID, LID, Bethesda, United States

Objectives: The mosquito- and tick-borne viruses within the Flaviviridae family comprise important emerging and re- emerging pathogens [Japanese encephalitis (JEV), tick-borne encephalitis (TBEV), West Nile (WNV), St. Louis encephalitis (SLEV) and Zika (ZIKV) viruses] that have caused outbreaks of severe neurologic disease in humans in many regions of the world and had severe impact on wildlife.

Methods: To restrict flavivirus replication in vertebrate and vector hosts, we inserted target sequences for arthropod vector- specific and/or host tissue-specific microRNAs individually or in combination into several distant regions of viral genome.

Results: We demonstrated that genome targeting of neurotropic flaviviruses or their chimeras (TBEV, WNV, SLEV or ZIKV) for cellular microRNAs expressed in the brain results in selective restriction of viral neurovirulence and neuroinvasiveness and complete abolishment of virus neurotropism in highly permissive newborn mice. Simultaneous microRNA co-targeting of two or three distantly located regions of viral genome (ORF and 3’NCR) had an additive effect on reduction of virus neuropathogenesis in the CNS and resulted in a more potent attenuation of the virus compared to targeting of a single region. Moreover, we demonstrated that dual targeting of the viral genome for brain-enriched microRNAs and for tick- or mosquito-specific microRNAs in these regions can silence viral replication in two evolutionally distant species: mice and ticks or mosquitoes, respectively.

Conclusion: We believe that microRNA-mediated silencing of virus replication in vertebrate and invertebrate hosts can reinforce the safety of newly developed and existing vaccines for environment and for use in humans.

Keywords: Flavivirus, miRNA, vaccine

17th International Congress of Virology 73

Abstract Submission

Virus vaccines

OR209

CANDIDATE DENGUE VIRUS SUBUNIT VACCINES DELIVERED TO THE SKIN BY THE NANOPATCH IN A LETHAL MOUSE CHALLENGE MODEL.

D. Muller*, A. Depelsenaire 1, A. Shannon 2, D. Watterson 2, C. Agyei-Yeboah 1, N. Owens 1, S. Corrie 1, M. Kendall 1, P. Young 2

1Australian Institute for Bioengineering and Nanotechnology, 2School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia

Objectives: Dengue is the most significant disease-causing arbovirus infection in the world. Indeed the World Health Organisation has listed dengue vaccine development as a priority area for the last 40 years, with the increasing numbers and size of epidemics in recent years only highlighting the importance of vaccine availability. Currently there are estimated to be 390 million dengue infections annually leading to 25,000 – 30,000 deaths. Dengue virus is transmitted by the bite of an infected female Aedes aegypti mosquito and may result in anything from an asymptomatic infection through to a range of clinical manifestations, including a self-limiting febrile illness, dengue fever (DF) to the more severe forms of disease, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The envelope (E) protein contains the majority of antibody neutralizing epitopes present on the virus making it an ideal candidate for a subunit vaccine approach.

Methods: Truncated recombinant E proteins (sE) of all 4 serotypes of dengue virus were expressed in a Drosophila culture (S2 cells) system and delivered to AG129 mice, with or without Quil A as an adjuvant via subcutaneous (SC), intradermal (ID) injection or via Nanopatch delivery (3 immunizations spaced 4 weeks apart). The Nanopatch contains an ultra high- density array (21,025/cm2) of short ~100 μm microprojections delivering vaccine directly into the skin.

Results: With only 10% of the dose used for the SC immunizations, mice immunized with the recombinant protein coated Nanopatch showed >1000 fold increase in IgG titre and balanced virus neutralization (PRNT80= 1/800) to all serotypes of dengue virus. Upon lethal challenge in the AG129 mouse model 100% of mice receiving the Nanopatch vaccine were protected showing no signs of infection or experiencing any break through virema.

Conclusion: Immunization of mice with tetravalent sE by the Nanopatch resulted in potent balanced neutralizing antibody response. Using the AG129 dengue mouse model, 100% of mice survived lethal challenge showing no signs of disease when vaccinated by Nanopatch. Overall, this is the first study to demonstrate the effective delivery of tetravalent sE by a microarray patch device producing a balanced immune response to all dengue serotypes with protection demonstrated in the AG129 mouse model. The highly effective nature of this delivery system warrants further evaluation in larger animals models.

Keywords: Dengue virus, Nanopatch, vaccine

17th International Congress of Virology 74

Abstract Submission

Virus vaccines

OR210

EVALUATION OF INFLUENZA H7N9 VACCINE CANDIDATES FORMULATED WITH ALUMINUM HYDROXIDE OR SQUALENE EMULSION ADJUVANTS IN FERRETS

M.-H. Huang 1, Y.-C. Hu 1, M.-Y. Chia 1, C.-T. Yeh 2, C.-Y. Lin 1, P.-L. Chen 1, I.-J. Su 1, M.-S. Lee*

1National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, 2Institute of Preventive Medicine, National Defense Medical Center, New Taipei, Taiwan

Objectives: Vaccination strategy is expected to be effective in preventing pandemic influenza; however, antigen sparing is the major challenge in pandemic preparedness. Here we assessed the immunogenicity of inactivated influenza H7N9 whole- virion vaccines (A/Anhui/1/13, NIBRG-268) adjuvanted with aluminum hydroxide or squalene-based emulsions.

Methods: The H7N9 vaccine used was a formalin-inactivated whole virus vaccine, NIBRG-268 (kindly supplied by the UK National Institute of Biological Standard and Control, NIBSC), derived from a reassortant H7N9 vaccine strain A/Anhui/1/2013 virus. The viruses were propagated in serum-free media and in the microcarrier-based MDCK cell culture technology. Formalin-inactivated vaccines were prepared with 0.1 % formalin at 37°C for 24 hr. After the sterilization through a 0.22 µm filter membrane, the HA protein concentrations of the inactivated virus vaccines were measured using the standard single radial diffusion (SRD) assay with the standard antigen and antiserum obtained from the US FDA. Aluminum hydroxide (Al(OH)3, Alhydrogel®) and aluminum phosphate (AlPO4, Adju-Phos®) wet gel suspensions were purchased from Brenntag AG (Frederikssund, Danish) and given as a 300 µg per dose. MF59 is a squalene-in-water emulsion accomplished by using a combination of a lipophilic Span®85 emulsifier and hydrophilic Tween®80 [8]. It is a kindly gift from Novartis Vaccines. PELC is a squalene emulsion stabilized by Span®85 (Sigma-Aldrich, Steinheim, Germany) and a bioresorbable polymer, poly(ethylene glycol)-block-poly(lactide-co-ε-caprolactone) (PEG-b-PLACL). Ferrets were immunized intramuscularly with 2 doses of vaccine antigens at a 2-week interval. Five treatment groups (4 male ferrets per group) with different HA and adjuvant dosages were compared, as follows: Group 1 received 0 μg HA with 300 μg Al(OH)3; Group 2 received 1.5 μg HA with 300 μg Al(OH)3; Group 3 received 3.0 μg HA with 300 μg Al(OH)3; Group 4 received 1.5 μg HA with MF59; and Group 1 received 1.5 μg HA with PELC. Sera were collected at days 0, 14, and 28 for measuring HI and neutralizing antibody titers following WHO’s standard procedures.

Results: Serum HI antibodies were less than 20 in ferrets vaccinated once with 1.5 µg HA of Al(OH)3-adjuvanted inactivated virus. We also found that when the same amount of inactivated vaccine antigen was co-administered with PELC, the induced anti-NIBRG-268 HI titers were higher than those induced by Al(OH)3-adjuvanted inactivated vaccine. 1.5 μg HA of PELC- formulated inactivated virus was even capable of inducing higher antibodies than those obtained from 3 μg HA of Al(OH)3- adjuvanted virus. For Al(OH)3-adjuvanted vaccines, when the antigen dosage was increased to 3 µg HA, the HI titer was slightly enhanced after the first administration. Overall, the Al(OH)3-adjuvanted inactivated virus vaccines could induce significant antibody responses after two doses of vaccination. The MF59-adjuvanted and PELC-adjuvanted vaccines were both highly immunogenic and could induce about 4-fold higher antibody titers than the Al(OH)3-adjuvanted vaccine. This is the first investigation on the immunogenicity of a degradable emulsion, named PELC, as an influenza vaccine adjuvant in ferrets. These results demonstrated that PELC or MF59 (a microfluidized squalene-in-water emulsion adjuvant) could be used as antigen-sparing tools in preparation of prophylactic vaccines against pandemic influenza.

Conclusion: The findings from this preclinical immunogenicity study provide support for further evaluation of the adjuvanted vaccines in clinical trials in humans.

Keywords: adjuvant, Influenza A, vaccine

17th International Congress of Virology 75

Speaker Abstract Submission

Virus vaccines

A Single-Round Infectious Particle Zika virus vaccine

K. B. Sundstrom 1,*, W. A. Saron 1, A. St John 1, E. E. Ooi 1 1EID, Duke-NUS, Singapore, Singapore

Objectives: Zika virus (ZIKV) has emerged to become a major health concern, spreading from Africa to Southeast Asia and, more recently, through the Pacific Islands to South America. Transmitted by the same mosquito vector as dengue virus, the global distribution of ZIKV will likely and perhaps inevitably mirror that of dengue virus, which is now endemic throughout the tropical region and encroaching into the subtropical areas. Although acute ZIKV infection is characterized by mild, self-limiting illness, infection with ZIKV in pregnant mothers is associated with congenital deformations in the unborn child, such as microcephaly. The exact mechanism of how ZIKV causes congenital malformations is still unknown, but both in vitro and in vivo studies show that the virus can infect and replicate in the placenta and can cause fetal damage in animal models. Preventing the spread of ZIKV is thus critical if we are to reduce the health impact of ZIKV. In this context, a vaccine that is able to engender long term immunity without any possibility of persistent infection and onward transmission would have clear and sustainable advantages. Long term immunity has been best achieved by live attenuated vaccines that trigger robust B and T cell responses although live virus infection raises the concern of vaccine persistence in the recipient. Here we explore a strategy that combines the safety of inactivated/subunit vaccines with the immunogenicity of live attenuated vaccines by using a single-round infectious particle (SRIP) approach.

Methods: The ZIKV-SRIP (zSRIP) contains the genetic material coding only for ZIKV non-structural proteins, packaged inside a Zika virus-like particle (VLP). The VLP packaging will ensure uptake of the vaccine by ZIKV susceptible cells. The genome consisting of non-structural genes will be self-replicating and would express the non-structural proteins that contain MHC class I restricted epitopes to elicit CD8+ T cell memory response. Without the structural genes, however, no new infectious particles will be created in the zSRIP infected cells: hence the infection will be limited to a single round in the vaccine recipient. B-cell responses (antibodies) will be activated by uptake of zSRIP by antigen presenting cells.

Results: In vitro results indicate that zSRIP can be produced to sufficient titers required for in vivo testing. We also show that the zSRIP infected cells does not produce virus that can spread to other cells. Study to investigate immunogenicity and protection by the zSRIP in vivo has started and results from this will be presented. Conclusion: zSRIP can be successfully produced to offer a safe and rapid approach to ZIKV vaccine development.

Keywords: Flavivirus, Vaccine, Zika virus

17th International Congress of Virology 76

Abstract Submission

Antivirals I

OR211

EVOLVING RESISTANCE TO IMATINIB IN VACCINIA VIRUS: A CASE STUDY OF A HOST-TARGETED ANTIMICROBIAL

M. Duncan 1,*, J. Horsington, T. Newsome 1

1School of Life and Environmental Sciences, University of Sydney, Sydney, Australia

Objectives: Global incidence of resistance to current antimicrobial drugs is on the rise, highlighting the urgent need to develop novel antimicrobials. Antimicrobials currently in use typically target pathogen encoded proteins or pathways, leading to selective pressure to acquire resistance. Resistance mechanisms, both acquired and innate, are generally elicited by the cellular stress response. A recent approach targets host factors that the invading microbes rely on for their spread, virulence or survival. It is proposed this approach will produce antimicrobials that are much less likely to select for resistance. This study uses vaccinia virus (VACV) to model the development of resistance to host targeted antimicrobial imatinib, which inhibits the release of enveloped virus.

Methods: A putative imatinib resistance allele has been isolated through culturing virus in the presence of imatinib. A mutation in this isolate was identified in the outer envelope viral protein A34. This study aims to characterise the novel A34 resistance mutation as well as another published mutation in A34 (A34K151E). Both mutations lead to higher virus release from cells than the parental WR strain.

Results: Both the isolated and published mutations show resistance to imatinib treatment. In the presence of imatinib enveloped virus is release is almost entirely inhibited in the parental WR; in the mutated strains virus release is inhibited but remains relatively high. Interestingly, the published mutation shows a similar fold reduction in release following imatinib treatment as the parental strain, while the isolated mutation has a much lower fold reduction.

Conclusion: While the two mutations both confer resistance to imatinib, they do so to differing extents and potentially represent different modes of resistance. The similar fold reduction of the published mutation compared to the parental WR suggests the high release is simply compensating for the reduction following treatment. The novel mutation however has a significantly lower fold reduction following treatment, suggesting a potentially different mode of resistance.

References:

Blasco, R., Sisler, J. R., and Moss, B. 1993. Dissociation of progeny Vaccinia Virus from the cell-membrane is regulated by a viral envelope glycoprotein-effect of a point mutation in the lectin homology domain of the A34R gene. Journal of Virology, 67, 3319-3325.

Reeves, P. M., Bommarius, B., Lebeis, S., McNulty, S., Christensen, J., Swimm, A., Charroudi, A., Chavan, R., Feinberg, M. B., Veach, D., Bornmann, W., Sherman, M., and Kalman, D. 2005. Disabling Poxvirus pathogenesis by inhibition of Abl- family tyrosine kinases. Nature Medicine, 11, 731-739

Keywords: antiviral resistance, poxvirus

17th International Congress of Virology 77

Abstract Submission

Antivirals I

OR212

IDENTIFICATION OF NATURAL-BASED INHIBITORS TARGETING ENTEROVIRUS 71 VP1-RECEPTOR INTERACTION

C.-F. Hsieh*, J.-T. Horng 1

1Biochemistry and Molecular Biology, Chang Gung University, Taoyuan, Taiwan

Objectives: Enterovirus A71 (EV-A71) is an important pathogen that can cause severe neurological symptoms and even death. Our laboratory focuses on identifying potent anti-enterovirus 71 compounds from synthetic chemical libraries or natural products and studying their underlying antiviral mechanisms.

Methods: Time course (time-of-addition assay)

RD cells (5 ´ 105 per well) were seeded in 6-well tissue culture plates overnight. Cells were washed with PBS, and then infected with EV71/2231 (moi of 10) for 1 h (–1 to 0 h pi). An aliquot of 50 uM CGUV-007 was added at –3, –1, 0, 2, 4, and 6 h pi. At 8 h pi, the cells and culture medium were collected for determining the titre by plaque-forming assays.

Direct inhibition of EV71 by CGUV-007 determined by centrifugal filtration assay

An aliquot of 5 ´ 106 pfu of virus was mixed with 1 mL DMEM in the presence of CGUV-007 or an equal volume of DMSO, and then incubated at 37 °C for 1 h. The mix was then transferred to Microsep centrifugal filters with a 100kDa cut-off (Pall Corporation, Port Washington, NY, USA), centrifuged at 1900 g at 4 °C for 20 min, and then washed with PBS three times to remove unbound chemicals. The titre of the virus in the concentrator reservoir was then determined by plaque-forming assays.

Results: Results of the time-of-addition assay suggest that CGU-V007 affects an early stage of virus infection. Further experiments demonstrated that CGU-V007 might target viral particles directly, thereby interfering with virus-receptor interactions. Consistently, sequencing of the plaque-purified CGU-V007-resistant viruses showed two mutations in the nonstructural protein VP1, P246A and E98G located near the five-fold axis of EV-A71, which is associated with virus-PSGL1 and virus-heparan sulfate interactions. Since the VP1 E98G and P246A residues are conserved in EV-A71 strains, we proposed that CGU-V007 could potentially inhibit EV-A71 infections of emergent variants.

Conclusion: We demonstrated that CGU-V007 could protect against EV-A71 replication.

Keywords: None

17th International Congress of Virology 78

Abstract Submission

Antivirals I

OR213

IDENTIFICATION OF OBATOCLAX AND BERBERINE AS BROAD-SPECTRUM ANTIVIRALS AGAINST CHIKUNGUNYA AND OTHER VIRUSES

T. Ahola 1,*, F. Varghese 1, K. Rausalu 2, B. Thaa 3, S. N. Amrun 4, B. Kümmerer 5, G. McInerney 3, L. Ng 4, A. Merits 2

1University of Helsinki, Helsinki, Finland, 2University of Tartu, Tartu, Estonia, 3Karolinska Institutet, Stockholm, Sweden, 4Singapore Immunology Network, Singapore, Singapore, 5University of Bonn Medical Centre, Bonn, Germany

Objectives: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus, which is now found throughout the world in tropical and subtropical areas, causing in humans high fever, body rash, headache and severe joint pain that can persist for months or years. We set out to discover novel antivirals against CHIKV and other alphaviruses. To gauge their wider potential, the compounds were tested against other positive-strand RNA viruses. Furthermore, we aimed to define the antiviral mechanisms of action for the two most interesting antivirals we discovered, obatoclax and berberine.

Methods: We identified berberine as antiviral in a high-throughput screen of 3000 compounds using a CHIKV replicon cell line, whereas obatoclax was previously known to be an inhibitor of influenza virus replication in the literature. Their anti- CHIKV action was verified by studying virus production (titer) as well as viral gene expression in infected cells. Mechanisms were established by multiple means, including phosphokinase array, time-of-addition and virus entry experiments.

Results: Berberine was antiviral against a variety of CHIKV strains and against several alphaviruses, but it did not demonstrate activity against flaviviruses. Berberine did not affect the early stages of virus replication. Mechanistically, it inhibited CHIKV-induced mitogen-activated protein kinase (MAPK signaling), which may influence virus assembly. Significantly, berberine provided protection against CHIKV-induced disease in a mouse model of joint inflammation. In contrast, obatoclax was strongly antiviral against CHIKV and another alphavirus, Semliki Forest virus (SFV), even when applied for very short periods during virus entry. We specifically established that obatoclax could neutralize the acidic environment of endosomes, thereby preventing virus entry. Thus, it was also active against other viruses requiring endosome acidification, including flaviviruses such as Zika virus. Unexpectedly, this mechanism is entirely distinct from the previously established anticancer activities of obatoclax. Partially obatoclax-resistant mutants of SFV had alterations in the viral fusion protein E1.

Conclusion: We have identified berberine as an antiviral for alphaviruses, which can confer benefits in a mouse disease model. We propose obatolax as a potent broad-spectrum antiviral, as it generally inhibits virus entry through acidic endosomes at submicromolar concentrations.

Keywords: Alphavirus, Virus entry, zika virus 17th International Congress of Virology 79

Abstract Submission

Antivirals I

OR214

HOST DEFENSE TEMPORIN 1 PEPTIDES AND THEIR POTENTIAL AS ANTIVIRAL AGENTS

L. Palomba 1, F. Merlino 2, C. Zannella 1, G. Franci 1, S. Galdiero 2, P. Grieco 2, M. Galdiero 1,*

1Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, 2Department of Pharmacy, University of Naples ‘Federico II’, Naples, Italy

Objectives: Antimicrobial peptides (AMPs) are widespread in all kingdoms of life and have been shown to exert a direct antimicrobial activity on a large number of invading microorganisms. They represent the most ancient and fast-acting elements of the host’s innate defence system against microbial pathogens.

Methods: In this scenario, AMPs have emerged as good candidates for the generation of new anti-infective agents. While the antibacterial activity has been deeply investigated, the notions available for antiviral activities are still underexplored. Furthermore, due to their chemical and biological instability only few AMPs are currently in clinical trials as antimicrobial agents. The amphibian temporins represent one of the largest families (more than 100 members) and are among the smallest-sized AMPs (10-16 amino acids) found in nature to date. Temporins are known to be active particularly against Gram-positive bacteria, with minimal inhibitory concentrations ranging from 2.5 μM to 20 μM. In details, we focused on Temporin 1 (TL1) analogues.

Results: Those peptides have been modified in order to increase the peptide stability and antimicrobial activity. We then performed a structure-activity relationship study of these peptides with the aim to improve their antimicrobial efficacy without altering their non-hemolytic character.

Conclusion: Among the peptides generated, several of them have shown higher in solution stability, low toxicity and a significant decrease of Herpes Simplex virus type 1 infectivity. Our results show novel possible applications of Temporin 1 peptides in the field of antivirals.

Keywords: Antiviral peptides, Herpes simplex virus type 1, Temporins

17th International Congress of Virology 80

Speaker Abstract Submission

Hepatitis viruses I

Contribution of Macrophage migration Inhibitory Factor (MIF) to Hepatitis C virus replication

Y.-J. Tan*, C. W. Sze 1

1Microbiology and Immunology, NATIONAL UNIVERSITY OF SINGAPORE, Singapore , Singapore

Objectives: Hepatitis C virus (HCV) replication is a complex process involving an intricate interplay between viral components and cellular factors. The aim of this study is to determine if Macrophage migration Inhibitor Factor (MIF) is essential for efficient replication of HCV.

Methods: Using a human apoptosis-pathway specific siRNA library, an important immune regulator MIF was found to be essential for efficient replication of HCV. Subsequently, HCV cell-culture infection system was used with MIF inhibitors to determine the impact of MIF inhibition at different step(s) of virus life cycle.

Results: We showed that HCV infection induced secretion of MIF from the cellular storage without up-regulation of MIF transcript. Through the use of MIF inhibitors, ISO-1 (4,5-dihydro-3-(4-hydroxyphenyl)-5-isoxazoleacetic acid, methyl ester) and 4-IPP (4-Iodo-6-phenylpyrimidine), we showed that the intrinsic tautomerase of MIF played a more significant role than its secretion in HCV replication. Inhibition of MIF tautomerase suppressed HCV replication at the transcript and protein expression. Depletion of MIF secretory partner, p115, showed only minimal impact on HCV replication but a significant reduction in virion production.

Conclusion: Our results indicated that HCV infection induced secretion of MIF from the intracellular storage and the tautomerase activity/active site is required for a successful viral replication.

Keywords: Hepatitis C virus, Virus-host interaction

17th International Congress of Virology 81

Abstract Submission

Hepatitis viruses I

OR215

HEPATITIS C VIRUS REPLICATION REQUIRES RIBONUCLEOTIDE REDUCTASE M2 TO STABILIZE NS5B PROTEIN, AN RNA-DEPENDENT RNA POLYMERASE

B. Kitab 1,*, M. Satoh 2, M. Sudoh 3, M. Kohara 4, K. Tsukiyama-Kohara 1

1Transboundary Animal Diseases Centre, Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan, 2Virology I, National Institute of Infection and Diseases, Shinjuku-Ku, Japan, 3Research Division, Chugai Pharmaceutical Co., Ltd., Kajiwara, Kamakura-City, Kanagawa, Japan, 4Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

Objectives: The pathogenesis of hepatitis C virus (HCV) infection is highly dependent on host cellular factors. We try to identify a new essential factor that participates in HCV pathogenesis.

Methods: Using a genome-wide microarray analysis in humanised chimeric mouse quiescent hepatocytes, we found that expression of the M2 subunit of ribonucleotide reductase (RRM2) was highly upregulated by HCV infection. To clarify the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes in chimeric mice and hepatoma cells. Function of RRM2 was characterized using short interfering RNA (siRNA).

Results: We infected HuH-7/K4 cells with the HCV-JFH1 strain. In both HCV-infected quiescent hepatocytes in chimeric mice and HCV-infected JFH/K4 cells, increased RRM2 mRNA transcription and translation were observed. Furthermore, RRM2 mRNA was significantly decreased when HCV siRNA was used in persistently HCV-infected JFH/K4 cells, and in turn, silencing of RRM2 by siRNA suppressed HCV replication and infection. To address the role of RRM2 in HCV replication, we examined its interactions with two factors that are important for HCV replication: HCV RNA polymerase NS5B and human homolog 1 of protein linking integrin-associated protein and cytoskeleton (hPLIC1), which was reported to ubiquitinate NS5B. Immunoprecipitation analysis showed that, although RRM2 and NS5B were partially co-localised in cells, they interacted directly with each other. Overexpression of hPLIC1 increased the interaction of RRM2 and NS5B, whilst the silencing of RRM2 by siRNA decreased the interaction of NS5B with hPLIC1. Moreover, silencing of RRM2 accelerated the degradation of NS5B by upregulating ubiquitination.

Conclusion: These results collectively demonstrate that RRM2 has a crucial role in persistent HCV infection, and provide new insights for the use of RRM2 as a therapeutic target for HCV-related liver diseases.

Keywords: Hepatitis C virus , NS5B, Ribonucleotide reductase M2

17th International Congress of Virology 82

Abstract Submission

Hepatitis viruses I

OR216

HEPATITIS C VIRUS: FROM RESEARCH ON TRANSLATION TO TRANSLATIONAL RESEARCH

G. Sharma, S. Das*

Objectives: Hepatitis C Virus (HCV) infection is a serious health problem that leads to liver cirrhosis and hepatocellular carcinoma. HCV is a positive-sense, single-stranded RNA virus that delivers its genomic content into the host cytoplasm upon infection. Following this, translation of the viral-encoded proteins is an essential prerequisite for viral replication. Since HCV RNA is naturally uncapped, its translation is guided by an ‘internal ribosome entry site’ (IRES) element present in its 5’UTR. Ribosome binding to the HCV-IRES is unique and fundamentally distinct from the cellular mRNA, and is influenced by a number of trans-acting host proteins and cis-elements on the HCV RNA. We would like to understand the molecular mechanism to identify targets for therapeutic interventions.

Methods: Using various molecular biology and cell biology based techniques we have identified several sequence / structural features of the HCV RNA and host factors important for viral translation and replication.

Results: Several host factors have been identified, among which the human La protein plays a pivotal role in this regulation. La interacts with a conserved GCAC-element at the 5’UTR of HCV RNA and promotes ribosome assembly at the HCV- IRES, leading to viral translation. Interestingly, La also interacts with 3’UTR of the viral RNA, mediating a translation- replication switch and helping in circularization of the viral genome for efficient replication. The other cellular factors that directly or indirectly assist La in these processes include the HuR protein and the microRNA miR-125b. Successful establishment of viral infection can eventually modulate cellular gene expression (such as p53, MALAT1) leading to hepatocellular carcinoma. In parallel, we have identified several plant-based natural products that inhibit different steps of HCV infection for therapeutic intervention.

Conclusion: Our findings provided important leads for rational designing of synthetic peptides and small RNAs that inhibit viral translation. We aim to continue our basic research on HCV infection that would eventually help us design more effective vaccine as well as therapeutic molecules towards better management of HCV infection.

Keywords: None

17th International Congress of Virology 83

Abstract Submission

Hepatitis viruses I

OR217

INVESTIGATING THE CLINICAL UTILITY OF TESTING FOR HEPATITIS C VIRUS RESISTANCE-ASSOCIATED VARIANTS TO UNDERSTAND TREATMENT FAILURES.

S. May 1, F. Kurmoo 2, S. Kaye 3, E. Adamska 1, T. Delahooke 4, M. Wiselka 5, J. Tang 1,*

1Clinical Microbiology/Virology, 2Medicine, University Hospitals of Leicester, Leicester, 3Molecular Diagnostics Unit, Imperial College London, London, 4Hepatology, 5Infectious Diseases Unit, University Hospitals of Leicester, Leicester, United Kingdom

Objectives: To investigate the presence of hepatitis C virus (HCV) resistance-associated variants (RAVS) in 5 patients who have failed directly-acting antiviral (DAA) therapy. Baseline HCV RAV testing has now been recommended in recently updated (2016) guidelines from the USA (American Association for the Study of Liver Diseases – AASLD) and Europe (European Association for the Study of the Liver – EASL). We aim to compare the HCV RAV profile of patients who have failed HCV therapy with their HCV RAV profiles to understand possible reasons for the their treatment failure.

Methods: Five patients were included in this case series. HCV RAV testing was performed at Imperial College using a Sanger-based sequencing assay targeting HCV genes NS3, NS5A and NS5B, depending on their treatment histories.

Case 1 (55-yr old male): G3a (baseline: 2,405,204 IU/ml), previously relapsed on pegylated interferon alpha-PEG- IFN/ribavirin-RBV, started Sofosbuvir-SOF/Daclatasvir-DCV/RBV, suppressed to 37 IU/ml at 12 wks, failed 3-mths post- end of therapy (EOT) at 617,287 IU/ml.

Case 2 (62-yr old male): G1 (baseline: 2,179,958 IU/ml), previously relapsed on PEG-IFN/RBV/telaprevir-TVR, started Harvoni (SOF/Ledipasvir-LDV)/RBV, suppressed to <12 IU/ml at 12 wks, failed 3-mths post-EOT at 2,013,656 IU/ml.

Case 3 (64-yr old female): G3 (baseline: 75,730 IU/ml), previously relapsed on PEG-IFN/RBV, started Harvoni /RBV, suppressed to <12 IU/ml at 12 wks, failed 3-mths post-EOT at 25,023 IU/ml.

Case 4 (64-yr old female): G3 (baseline: 366,188 IU/ml), previously relapsed on PEG-IFN/RBV, started SOF/DCV/RBV, suppressed to <12 IU/ml at 12 wks, failed 10-wks post-EOT (G3a) at 523,740 IU/ml.

Case 5 (63-yr old male): G1a (baseline: 2,2999,576 IU/ml), previously non-responder on PEG-IFN/RBV, discontinued wk 24. Started Viekarax/Dasabuvir-DAS (without RBV due to severe rash, previously), suppressed to <12 IU/ml at 12 wks, failed 3-mths post-EOT at 1,741,661 IU/ml.

Results: The results of the HCV RAV testing for these patients are shown below. Note that where RAVs have been detected, these have all appeared in the NS5A region with none in the NS5B gene. This suggests that all the DAA treatment failures in this case series were due to NS5A resistance, rather than any NS5B resistance.

Case 1: NS5A inhibitor DCV/Elbasvir-EBV/LDV resistance, Ombitasvir-OMB susceptible: 30K. NS5B inhibitor DAS, SOF both susceptible: none

Case 2: NS5A inhibitor DCV/EBV/LDV/OMB resistance: 31M, 93H. NS5B inhibitor DAS possible resistance, SOF susceptible: 316N

17th International Congress of Virology 84

Case 3: NS5A inhibitor DCV/EBV/LDV susceptible: none. NS5B inhibitor SOF susceptible: none

Case 4: NS5A inhibitor DCV resistance, EBV/LDV susceptible: 93H. NS5B inhibitor SOF susceptible: none

Case 5: NS5A inhibitor DCV/EBV/LDV/OMB resistance, Velpatasvir susceptible: 30R. NS5B inhibitor SOF/DAS susceptible: none

Conclusion: Although these Sanger (majority viral population) sequenced HCV NS5A RAV profiles can explain the eventual treatment failure in Cases 1, 2, 4, and 5, this is not explanatory for Case 3. However, all the samples tested for HCV RAVs were taken at 10-13 wks post-EOT, when any more resistant (and potentially less fit) viral populations may have been outgrown by wildtype HCV (originating from as yet unidentified reservoirs) by this time. Sanger sequencing is unable to detect such minority (<15-20%) resistant viral populations, supporting the case for the use of deep sequencing methods for the investigation of HCV RAVs both at baseline and in cases of treatment failures.

Keywords: hepatitis C, resistance-associated variants, treatment failure

17th International Congress of Virology 85

Speaker Abstract Submission

Double Strand RNA Viruses

The TRiC Chaperonin Is Required for Reovirus Assembly

Jonathan J. Knowlton,1 Paula F. Zamora,1 Isabel Fernández de Castro,2 Cristina Risco,2 and Terence S. Dermody1,3,4 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA 2 Cell Structure Laboratory, Centro Nacional de Biotecnología (CNB-CSIC), Campus UAM, Cantoblanco 28049 Madrid, Spain 3 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA 4 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA

As obligate intracellular parasites, viruses use a diverse array of cellular proteins to accomplish each step in a replication cycle. Studies with mammalian orthoreovirus (reovirus) have defined numerous host factors required for viral attachment, entry, and uncoating. Despite our knowledge of many of the cellular genes required for early steps in reovirus replication, our understanding of those required for later replication steps, including assembly and egress, remains limited. To identify host factors required for late steps in reovirus replication, we conducted a two-step RNAi-based screen targeting 7,776 human genes. RNAi knockdown of numerous subunits of the TCP1-ring complex (TRiC), a cellular chaperonin required for folding cytosolic proteins, substantially diminished the quantity of reovirus released from infected cells. siRNA knockdown of the CCT2 and CCT5 TRiC subunits resulted in a 100-fold reduction in reovirus replication and altered the size and morphology of viral replication organelles. Immunoprecipitation of the CCT2 TRiC subunit yielded a complex containing the reovirus σ3 outer-capsid protein and no other reovirus proteins. Phosphorimaging of the native 35S- methionine-labeled σ3 polypeptide, translated in vitro using rabbit reticulocyte lysates, revealed a high molecular weight species corresponding to a TRiC-bound form of the protein. Our data indicate that TRiC forms a complex with the reovirus σ3 outer-capsid protein in infected cells and in vitro. Future studies will assess whether TRiC directly folds σ3 into its functional conformation and if this folding reaction is required for the assembly of mature viral particles. These findings demonstrate a new interaction between a host chaperone and a viral substrate and provide insight into the assembly of a complex viral capsid.

17th International Congress of Virology 86

Speaker Abstract Submission

Double Strand RNA Viruses

ESTABLISHMENT OF LETHAL PTEROPINE ORTHOREOVIRUS INFECTION BALB/C MOUSE MODEL

M. Saijo 1 2,*, K. Egawa 1 2, M. Shimojima 1, S. Taniguchi 1, N. Nagata 3, H. Tani 1, T. Yoshikawa 1, T. Kurosu 1, S. Fukushi 1 1Department of Virology 1, National Institute of Infectious Diseases, Musashimurayama, 2United Graduate School of Veterinary Sciences, Gifu University, Gifu, 3Department of Pathology, National Institute of Infectious Diseases, Musashimurayama, Japan

Objectives: Cases of acute upper respiratory tract infection (URTI) caused by Pteropine orthoreovirus (PRV), genus Orthoreovirus in the family Reoviridae, are reported as an emerging virus infection in Southeast Asia. Since PRVs have been isolated not only from human patients but also from bats in Southeast Asia, there is a potential probability that PRVs infections in humans might be prevalent in Southeast Asia. To study virulence of PRVs as well as prophylactics and therapeutics against PRVs, small animal model for PRV infection is necessary. The infection model using BALB/c mouse model for PRV infection was developed in this study. Furthermore, the pathogenicity of PRV in mice were evaluated, and then the usefulness of this model in assessment of some therapeutics’ efficacy in treatment against PRV.

Methods: The PRV strain isolated from a human patient (Human MB) and the strain isolated from a bat (Bat Samal) were used. BALB/c mice were inoculated via intranasal route with 103 to 106 plaque forming units (PFU) of PRV in 20µl of 2%FCS-containing DMEM. The mice were monitored daily and 50% lethal dose (LD50) of PRV for mice was determined. The mice were sacrificed and the tissues were collected at 1, 3 and 5 days post-infection. The viral loads were determined by qRT-PCR. Histopathological and immunohistochemical analyses were also performed. To study the immunological protective effect against PRV, neutralizing antibody responses in mice with non-lethal dose PRV infection were determined. These mice were then challenged with lethal dose PRV, and clinical symptoms were observed for 14 days. The effect of antisera treatment after infection of lethal dose of PRV was also examined.

3.9 Results: Human MB caused fatal diseases in BALB/c mice. LD50 of Human MB was 10 PFU/head. A high level of viral replication was found in lungs, and pathological changes were demonstrated in the bronchiole and alveoli. Viral antigens were detected in the bronchial epithelium and in the pneumocytes at early and late stages, respectively, indicating that Human MB caused lethal lower respiratory infection in BALB/c mice. All mice pre-infected with non-lethal dose of PRV survived after infection with lethal dose PRV. Furthermore, the mice treated with antisera after infection with lethal dose of PRV showed reduced fatality rate. Conclusion: This PRV infection BALB/c mouse model would be a useful in evaluation of the efficacy of vaccines and drugs for PRV infection. Since pathogenicity (LD50 and the level of viral RNA and pathological lesions in each tissues) of bat-borne PRV (Bat Samal) was similar level to that of Human MB. There is a possibility that bat-borne PRV might be a potential pathogen for respiratory infections in humans.

Keywords: pteropine orthoreovirus, animal model, emerging virus infections

17th International Congress of Virology 87

Abstract Submission

Double Strand RNA Viruses

OR219

DECIPHERING OF CELLULAR RECEPTOR RESPONSIBLE FOR PTEROPINE ORTHOREOVIRUS GAINING ENTRY INTO HOST

Y. F. Tan*

Objectives: To determine which cellular receptor(s) that Pteropine orthoreovirus binds to in order for it to gain entry into host. To determine the potential difference in cellular receptor(s) used by different isolates of Pteropine orthoreovirus to gain entry into host.

Methods: Virus overlay protein binding assay of Pulau and Kampar virus (isolates of Pteropine orthoreovirus) was performed. Briefly, cell lysates of Hek293 were run on a SDS PAGE gel and transferred to a PVDF membrane. Live Pulau and Kampar virus were then introduced onto the membrane. Anti Pulau and anti Kampar were then introduced followed by anti rabbit with HRP (secondary antibody). The membrane was then covered with ECL substrate and an image was captured using ChemiDoc (Biorad). A parallel SDS PAGE gel was run with Hek293 lysates. The bands present on the PVDF membrane were matched with the SDS PAGE gel and the bands on SDS PAGE gel were excised and sent for LCMS sequencing.

Results: After performing virus overlay protein binding assay, two bands were found for both Pulau and Kampar virus. One is at 70kDa while the other is at 65kDa. The 70kDa band was identified as human cytokeratin 1 and the 65kDa band was identified as pyruvate kinase. Human cytokeratin 1 is a cytoskeleton protein that extends to cell surface whereas pyruvate kinase is a protein found only in the cytoplasm and nucleus of a cell. Due to its location, pyruvate kinase cannot be responsible for viral cell entry. Based on the work of Iswara et al., HeLa cells are less permissive to Pulau and Kampar virus infection. It is found out that HeLa cells do not express human cytokeratin 1. This supports the notion that human cytokeratin 1 is responsible for Pulau and Kampar virus cell entry.

Conclusion: Human cytokeratin 1 and pyruvate kinase are two proteins found to interact with Pulau and Kampar virus. Human cytokeratin 1 is a likely candidate for viral cell entry based on our preliminary result and the work of Iswara et al. Pyruvate kinase is not responsible for viral cell entry due to its cellular location however its interaction with Pulau and Kampar virus remains to be investigated.

Keywords: pteropine orthoreovirus, emerging virus infections

17th International Congress of Virology 88

Abstract Submission

Double Strand RNA Viruses

OR220

WHOLE GENOME ANALYSIS OF PORCINE ROTAVIRUSES H DETECTED IN JAPAN IN RECENT YEARS

T. Suzuki*

Objectives: RVH was first detected in fecal sample of piglets under 30 days old collected in Japan during 1991-1995. Human RVH has been identified in epidemic or sporadic diarrhea cases in adults in China and Bangladesh during 1997- 2002. In recent years, porcine RVH has been detected in the United States, and they have co-infected with other RV species (RVA, RVB, and RVC) in swine farms. After discovery of the RVH strain, SKA-1, the prevalence of porcine RVH has been unidentified in Japan. Recently, we have detected several porcine RVHs together with other porcine RV species in fecal samples collected at swine farms in Kyushu. In this study, we performed whole genome analysis of the porcine RVH strains, and compared and analyzed them with those of other RVH strains detected in past and recent years.

Methods: Multiple fecal samples were collected from several different farms at Kyushu in Japan from 2013 to 2015. Viral RNA was extracted from 10% stool suspensions according to manufacturer’s instructions. RT-PCR was performed using sets of specific primers originally designed. The PCR products were sequenced using an automated sequencer. Comparative sequence and phylogenetic analyses were performed by using the Lasergene and the MEGA6 software.

Results: Comparative sequence analyses reveal that the porcine RVHs indicate low sequence identities to human RVHs. Phylogenetic analyses demonstrate that RVHs might be classified into different genotype according to host species, and especially porcine RVHs might be differentiated into multiple genotypes.

Conclusion: Twenty years later from discovery of the porcine RVH strain, SKA-1, there were multiple porcine RVH strains which were genetically distinct from the classical strain in Japan. Our findings indicate that the porcine RVH strains are often co-infected with other porcine RV species (RVA, RVB, and RVC) in swine farms.

Keywords: Japan, Porcine rotavirus H, Whole genome analysis

17th International Congress of Virology 89

Abstract Submission

Viral evolution & population genetics

OR221

CROSS-SPECIES TRANSMISSION OCCURS MORE FREQUENTLY THAN CO-DIVERGENCE AMONG VIRAL FAMILIES

J. Geoghegan 1 2,*, S. Duchêne 3, E. Holmes 2

1School of Biological Sciences, Macquarie University, 2School of Life and Environmental Sciences, University of Sydney, Sydney, 3Centre for Systems Genomics, University of Melbourne, Melbourne, Australia

Objectives: The cross-species transmission of viruses from one host species to another is responsible for the majority of emerging infections. However, it is unclear whether some virus families have a greater propensity to jump host species than others. If related viruses have an evolutionary history of co-divergence with their hosts there should be evidence of topological similarities between the virus and host phylogenetic trees, whereas host jumping generates incongruent tree topologies.

Methods: By analyzing co-phylogenetic processes in 19 virus families and their eukaryotic hosts we provide a quantitative and comparative measure of the relative frequency of virus-host co-divergence versus cross-species transmission among virus families.

Results: Notably, our analysis reveals that cross-species transmission is a near universal feature of the viruses analyzed here, with virus-host co-divergence only apparent in a small number of families and always involving a subset of viruses. Despite the overall high topological incongruence among virus and host phylogenies, the Hepadnaviridae, Polyomaviridae, Poxviridae, Papillomaviridae and Adenoviridae, all of which possess double-stranded DNA genomes, exhibited more frequent co-divergence than the other virus families studied here. At the other extreme, the virus and host trees for all the RNA viruses studied here, particularly the Rhabdoviridae and the Picornaviridae, displayed high levels of topological incongruence, indicative of frequent host switching.

Conclusion: Overall, we show that cross-species transmission plays a major role in virus evolution, with all the virus families studied here having the potential to jump host species, and that increased sampling will reveal more instances of host jumping.

Keywords: Codivergence, Cross-species transmission, Evolution

17th International Congress of Virology 90

Abstract Submission

Viral evolution & population genetics

OR222

DINUCLEOTIDE COMPOSITION IN ANIMAL RNA VIRUSES IS SHAPED MORE BY VIRUS FAMILY THAN HOST SPECIES

F. Di Giallonardo 1,*, T. Schlub 2, M. Shi 1, E. Holmes 1

1Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Life and Environmental Sciences and Sydney Medical School, 2Sydney School of Public Health, Sydney Medical School, The University of Sydney, Sydney, Australia

Objectives: Nucleotide and dinucleotide frequencies differ between viruses. Hence, the processes that shape the virus genome composition are central to the understanding of virus evolution and emergence. It has been proposed that the dinucleotide composition of viruses should match that of their host species, as viruses use the cellular machinery of their hosts for replication. In this study we want to determine if the dinucleotide composition can be used to accurately predict host species or virus family.

Methods: We performed a comparative analysis of 20 RNA virus families from 15 host groupings and determined the dinucleotide odds ratios for the 16 possible dinucleotides. We implemented a discriminant analysis to evaluate the capability of the odds ratios to predict the correct virus family or host taxon from which it has been isolated.

Results: In general, the dinucleotides CpG and UpA are under-represented, and CpA and UpG over-represented. However, there are marked differences between the different virus families. Remarkably, Hepe- and Reoviridae did not show a bias in any dinucleotide. The prediction analysis revealed that only 62% of the 900 virus species analysed were assigned to its correct virus host. In contrast, 81% of the data was predicted to the correct virus family.

Conclusion: We show that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species, indicating that the host has a relatively small impact on the dinucleotide composition of a virus genome. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone.

Keywords: odds ratios, Phylogeny, Virus evolution

17th International Congress of Virology 91

Abstract Submission

Viral evolution & population genetics

OR223

EVOLUTION OF THE RESTRICTION GENE FV1 IN THE WILD MICE POPULATION OF SOUTH EAST ASIA TO PROTECT AGAINST MULTIPLE RETROVIRUSES

M. Yap*, G. Young 1, R. Varnaite 1, S. Morand 2, J. Stoye 1

1Retrovirus–host interactions Laboratory, The Francis Crick Institute, London, United Kingdom, 2CIRAD-CNRS, Centre d'Infectiologie Christophe Mérieux du Laos, Vientiane, Lao People's Democratic Republic

Objectives: Viruses and their hosts are entwined in a continuing evolutionary battle where the hosts acquire resistance to viral infections while viruses develop strategies to overcome the host defences. Forming part of the host anti-virus arsenal are restriction factors that act in a cell autonomous manner. Fv1 is the prototypic restriction factor that was first described to protect mice from infection by the murine leukaemia virus (MLV). We have found that Fv1 from wild mice can restrict non-MLV retroviruses, suggesting that it plays an important role in protection against multiple viruses. As mice have a short generation time, they provide a useful system of studying the evolution of restriction factors.

Methods: To begin to understand how restriction factors are selected for in the wild, we sampled the Fv1 gene from wild mice that were trapped in South East Asia and tested their restriction activities against a panel of retroviruses.

Results: We found a duplicated copy of the Fv1 gene on the chromosome 6 of three closely related mice (Mus caroli, Mus cervicolor and Mus cookii). The duplication contains the Fv1 promoter and open reading frame and is flanked by repeats, suggesting it arose from a LINE-mediated retrotransposition event. We have named the duplicated gene Fv7. There is a high degree of natural variation in both Fv1 and Fv7 in these mice. We observed several single nucleotide polymorphisims. There are also insertions and deletions which change the lengths of the reading frames. In Mus cervicolor and Mus cookii, an insertion by a B1 element in the 3-prime end of the gene resulted in a shorter reading frame. Many of the Fv7 and some Fv1 in Mus cookii contained premature stop codons which produced truncated proteins.

In all of the Mus caroli we studied, the Fv1s restricted feline foamy virus (FFV) while the Fv7s restricted equine infectious anemia virus (EIAV). In addition, some of the Fv1s also restricted MLVs. This suggested that these mice are protected against infection by multiple viruses. Most of the Fv1s and Fv7s in Mus cervicolor and Mus cookii restricted MLVs. In addition, many of the Fv7s in these mice restricted EIAV.

Conclusion: These results suggest that FFV-like, EIAV-like and MLV-like viruses are endemic in the population. Site- directed mutagenesis studies were undertaken to map the specificity determinants of EIAV and MLV restriction in these mice. Interestingly, different residues were found to be involved in each of the species. This suggested a convergent evolution where each species developed independently to restrict the same virus.

Keywords: Evolution, Fv1, Restriction factor

17th International Congress of Virology 92

Speaker Abstract Submission

Viral evolution & population genetics

Genome size dynamics and gene linkage in macroevolution of nidoviruses

A. Gorbalenya 1,*, I. Kuznetsov 2, C. Lauber 3, A. Leontovich 4, I. Sidorov 1, A. Gulyaeva 1 1Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands, 2Faculty of Bioengineering & Bioinformatics, Lomonosov Moscow State University, Moscow, Russian Federation, 3Institute for Medical Informatics and Biometry, Technische Universität Dresden, Dresden, Germany, 4Belozersky Inst. of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation

Objectives: Exceptionally high rates of mutation of RNA viruses limits the utility of multiple sequence alignments in studies of macroevolution of these viruses to very few most conserved proteins. To address this challenge, our group has recently introduced a new approach that is largely independent of residue conservation while examining region-specific dynamics of change of the net sum of deletion and insertion across the genome. This approach exploits strong conservation of genomic layouts in large monophyletic lineages of RNA viruses for partitioning their genomes into spatially equivalent regions. Assuming that all analysed viruses followed a common trajectory of genome size change, it then estimates the contribution of size change in different genome regions to the overall change of genome size using non- linear regression analysis within a rigorous statistical framework. By applying this approach to the order Nidovirales, our group has previously modelled the dynamics of genome size change from 12.7 to 31.7 kb, the largest known RNA genome at the time. Under the genome expansion hypothesis, genome size might have changed in three overlapping waves that, consecutively, involved protein-encoding regions controlling genome replication and expression, and virus dissemination. They are known as ORF1b, ORF1a, and 3’-ORFs, respectively, and collectively account for >95% of genome size. In this report, we extend and refine this analysis using an improved method of regression fitting, a two-fold larger dataset of nidovirus species, and various partitions of the nidovirus genome into regions.

Methods: Our dataset included >3500 genome sequences of nidoviruses of diverse vertebrate and invertebrate hosts, which were analysed using profiles of multiple sequence alignments, reconstruction of phylogenetic trees, weighted hierarchical clustering of pairwise patristic distances, and modelling of the relationships between sizes of the genome and its protein-encoding regions using splines regression and adjusted R2 statistics.

Results: We selected genomes representing >70 (tentative) species from major phylogenetic lineages of nidoviruses and split the coding part of nidovirus genomes into five regions, N-ORF1a, C-ORF1a, N-ORF1b, C-ORF1b and 3’-ORFs, which were analysed in various combinations. Our analysis strongly supported the wave-like dynamics of genome expansion. We further observed a particularly good fitting of viruses of all lineages into the dynamic trajectory for C- ORF1a and N-ORF1b, and slightly less so for C-ORF1b, the regions that led genome expansion in the 15-20 kb size range. Overall the fitting was good for viruses of the family Arteriviridae, and most viruses of the subfamily Coronavirinae and several established and putative families of invertebrate nidoviruses. One coronavirus species, a new invertebrate nidovirus, and majority of the subfamily Torovirinae deviated most in the N-ORF1a and 3’-ORFs from the nidovirus dynamic trajectory in the opposite directions, region- and lineage-wise. Conclusion: Based on the obtained results we conclude that C-ORF1a, N-ORF1b, and C-ORF1b genome regions, encoding replicative proteins released by 3CL protease from polyproteins, are under similar and strong size constraint indicative of their linkage and functional interaction in all nidoviruses. In contrast, size expansion of N-ORF1a and 3’- ORFs might have proceeded in lineage-specific manner under constraint on the cumulative size of these regions, indicative of a functional overlap between these two regions and a role of mutation in the choice of a region to expand most.

Keywords: Nidovirales, Coronavirinae, Arteriviridae, Torovirinae, Mesoniviridae, Roniviridae, evolution, RNA genome size, constraint, gene linkage, gene acquisition & loss

17th International Congress of Virology 93

Abstract Submission

Antivirals II and Late-breaking Talk

OR224

HEPARAN SULFATE MIMETIC COMPOUNDS IN MODULATING RRV AS POTENTIAL THERAPIES

A. Supramaniam 1,*, V. Ferro 2 3, L. Herrero 1

1Institute for Glycomics, Griffith University, Gold coast, 2School of Chemistry & Molecular Biosciences, 3Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Australia

Objectives: Arthritogenic Alphavirus, such as Ross River virus (RRV) and Barmah Forest virus (BFV), are transmitted by mosquito vectors and cause musculoskeletal manifestations. Patients experience excruciating pain and inflammation of their joints and surrounding muscle tissues. Current treatments for arthritogenic alphaviruses are non-specific and include the use of non-steroidal anti-inflammatory drugs (NSAIDs) which only provide temporary or partial relief for these debilitating conditions. Therefore, the need to explore other classes of compounds for alphaviral treatment is imperative. A recent study has reported the efficacy of pentosan polysulfate (PPS), a sulfated polysaccharide and heparan sulfate (HS) mimetic that reduced the recruitment of inflammatory infiltrates and protected the cartilage matrix from degradation in RRV infected PPS treated mice.

Methods: Herein, we describe the use of novel HS mimetics for the treatment of RRV induced arthritogenic disease. We evaluated the treatment efficacy of a representative compound in both a mouse model of RRVD and an in vitro cell based system. We first assessed drug efficacy through a 50% plaque reduction (IC50) in vitro and further evaluated drug efficacy in mice, by evaluating their joint dysfunction, expression levels of both host soluble factors and components of the cartilage matrix, viral titer and histopathology in viral specific target organs.

Results: Increasing concentrations of the HS mimetic compound inhibited plaque formation in both RRV (field isolates and laboratory adapted prototype; T48) and BFV suggesting an anti-viral effect. Furthermore, compound treated RRV infected mice had significantly reduced viral loads in target organs corresponding to a reduction in their clinical scores of limb weakness and immune infiltrate recruitment. At peak disease, compound treated RRV mice had lower expression levels of host factors IL-6 and CCL-2. In addition, treatment also demonstrated a reduction in the matrix degrading enzyme heparanase in both calf and ankle samples with key protection in muscle fibres and hyaline cartilage structure.

Conclusion: Taken together these findings suggest that the HS mimetic compound may have a direct inhibitory effect on both RRV infection as well as the RRV-induced inflammatory disease in host organisms. This suggests a dual mode of action in its efficacy to treat RRV infection and disease indicating a potential to treat patients who suffer both acute and chronic symptoms.

Keywords: Alphavirus, Heparan sulfate mimetics

17th International Congress of Virology 94

Abstract Submission

Antivirals II and Late-breaking Talk

OR225

Single Chain Variable Fragment-1G1 inhibits H5N1 Avian Influenza Virus Growth via Neutralization of NS1

C.-K. Mok 1,*, Y.-J. Tan 1 2

1Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, 2Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore

Objectives: A new strategy to prevent the infection of H5N1 avian influenza A viruses is needed due to the emergence of drug resistant mutants. We developed a strategy targeting the viral non-structural protein 1 (NS1), a highly conserved and indispensable component for viral replication. A monoclonal antibody (mAb) 1G1 targeting the H5N1 NS1 effector domain (ED) has been identified and found being capable of inhibiting viral growth. A single chain variable fragment (scFv) of 1G1 would be used for further characterizations.

Methods: Epitope mapping of mAb-1G1 was performed through mutagenesis screening assay. The 1G1-scFv was cloned into a CMV vector and expressed in H1299, 293T, and A549 cells. The localization of NS1 co-transfecting with 1G1-scFv was validated using immunofluorescence assay. A recombinant H5N1 NS1 virus was used in viral inhibition assay and the effect of 1G1 on viral protein expression as well as virus titer were determined using immunoblot and plaque assay, respectively.

Results: MAb-1G1 was identified to bind to amino acid P210 and N212 in the H5N1 NS1-ED domain. By transiently co- transfecting NS1-ED and 1G1-scFv into H1299 cells, the translocalization of NS1-ED was found to be blocked, and it remained in the cytoplasm. When 1G1-scFv was intracellularly expressed in 293T or A549 cells, the viral replication of H5N1 showed significant reduction. Based on the expression of viral M1 protein, it was reduced by 53.09% at 10 hours post-infection (hpi) in single-step growth kinetics, and 0.784 log of the forming progeny virus was inhibited at 48 hpi in multiple-step growth kinetics. We further transiently co-expressed 1G1-scFv with a known 2H6-scFv, which binds to the NS1 RNA-binding domain, in 293T cells. The combination of both scFvs reduced the M1 protein by a high 87.14% and 72.72% at 8 and 10 hpi respectively. These reductions were ~3.4-fold higher than that achieved by single scFv neutralization.

Conclusion: Our findings demonstrated that 1G1-scFv might bind to the nuclear localization sequence at the C-terminal end of ED, subsequently inhibiting viral growth. When combined with 2H6-scFv, the multi-targeting neutralization of H5N1 was better than using single-targeting neutralization through inhibiting the functions of viral NS1 protein.

Keywords: Antiviral antibodies, Influenza H5N1, Single chain variable fragment

17th International Congress of Virology 95

Abstract Submission

Antivirals II and Late-breaking Talk

OR226

CARDIOTONIC STEROIDS SUPPRESS ADENOVIRUS REPLICATION IN HUMAN RESPIRATORY EPITHELIAL CELLS

M. Brown 1 2,*, F. Grosso 2, A. Cochrane 1

1Molecular Genetics, 2Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Objectives: We have shown that digoxin and digitoxin reduce adenovirus yield by 2-4 logs in human lung epithelial cells (A549) by suppressing viral DNA replication at one or more steps beyond immediate early E1A expression. The current study aims to characterize the nature of the antiviral block by analyzing the effect of digoxin and digitoxin on specific viral proteins normally expressed prior to DNA replication. In addition, experiments were done to examine the effect of digoxin and digitoxin on adenovirus replication in cultures of primary human nasal epithelial cells (hNEC) grown at the air-liquid interface (ALI).

Methods: A549 cells were infected with human adenovirus C5 under conditions sufficient to infect more than 90% of the cells. After one hour adsorption, virus inoculum was removed and replaced with fresh medium with and without 100 nM digoxin or digitoxin. Cells were harvested at different times post infection for analysis of specific viral proteins by immunoblot and by fluorescence microscopy. Polarized hNEC cultures grown at ALI were infected by application of concentrated adenovirus C5 to the apical surface. After two hour adsorption, inoculum was removed, the apical surface washed and the basal medium replaced with fresh medium with and without 150 nM digoxin or digitoxin. Cells were fixed at 3-7 days post infection for immunodetection of infected cells using antibody against hexon, the major capsid protein of adenovirus.

Results: In studies with A549 cells, immunoblot analysis at 14 hours post infection showed only a modest reduction in expression of E2 72K DNA binding protein (DBP), in contrast to E1B 55K protein which was barely detectable in treated cells. Despite appreciable levels of E2 72K DBP, its staining pattern was altered. Though still exclusively nuclear, the signal in most cells was diffuse rather than clustered at viral replication centres. A small proportion of cells showed some clusters within a diffuse nuclear signal but the clusters appeared to be smaller and fewer in number than in untreated cells. An antiviral effect was also seen in hNEC cultures. Fluorescence microscopy showed isolated infected cells in the treated cultures compared to small foci of infected cells in untreated cultures, consistent with a block in spread of progeny virus.

Conclusion: Decreased adenovirus DNA replication in A549 cells treated with digoxin and digitoxin reflects reduced expression and/or mislocalisation of key viral proteins, including E1B 55K and the E2 72K DBP. In contrast, when the drugs are applied to hNEC cultures after virus adsorption, they appear not to block the first round of virus replication but prevent spread of progeny virus to neighbouring cells.

Keywords: adenovirus DNA binding protein, digoxin, primary nasal epithelial cells at ALI

17th International Congress of Virology 96

Abstract Submission

Antivirals II and Late-breaking Talk

OR227

RIG-I Activation and Viral Gene Silencing a Potent Combinatorial Drug Design for HCV Therapy

M. I. Alam*, G. Hartmann 1, M. Schlee 1

1Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany

Objectives: Retinoic acid inducible gene I (RIG-I) is a cytosolic viral radar which senses RNA viruses in infected cells leading to the induction of interferon (IFN) that antagonizes virus replication. However, Hepatitis C virus (HCV) NS3-4A protein negatively targets IPS1 adaptor molecule of RIG-I to evade innate immunity that hampers the production of IFN. Recombinant IFN together with ribavirin therapy has clinical restrictions because of its short half-life, frequent dosing and expensive with great side effects. Although recent new class of expensive and potent anti-HCV drug Sofosbuvir is in the market but viral resistance associated with it may be predicted upon time. Therefore, inexpensive new class of anti-HCV drug must be invented before it happens. Here, we have investigated antiviral impact of bi-functional 3p-siRNA (tri- phosphate as RIG-I activator and siRNA as viral silencer) on HCV replication. We used HCV sub genomic replicon and autonomous replicating Huh-Lunet cells contained with firefly luciferase as a replication indicator.

Methods: Firefly luciferase measurement, type 1 interferon measurement by ELISA, Transfection

Results: Our data shows that RIG-I activator 3pRNA alone inhibits about 50 % HCV replication in transiently replicating cells. siRNA a potent HCV silencer shows synergistic antiviral impact together with RIG-I activator at same dose. Non- synergistic and synergistic antiviral impact of 3pRNA and 3p-siRNA respectively is dose dependent.

As expected siRNA alone do not show IFNβ production in HCV replicating cells but 3p-siRNA does. Interestingly, removing phosphate group from 3p-siRNA using alkaline phosphatase loses the production ability of IFNβ therefore, synergistic antiviral effect was strongly reduced.

Remarkably, our further data shows that RIG-I activator 3pRNA alone is not able to suppress HCV replication in autonomous replicating cells at any extent however siRNA does. Subsequently synergistic antiviral effect of 3p-siRNA was also achieved in autonomous HCV replicating cells. This interesting results indicate that in transient HCV replication the function of RIG-I seems to partially remain intact while in autonomous replicating cells RIG-I function seems to be strongly hampered by HCV viral protein NS3-4A as a causal factor of evasion of innate immunity for the successful virus replication leading to chronic infection.

Conclusion: Altogether, 3p-siRNA as a combinatorial RNA molecule could might be used as anti-HCV drug after meeting proof of concept.

Keywords: HCV therapy, Immune evasion, RIG-I activator

17th International Congress of Virology 97

Late breaking abstract

Antivirals II and Late-breaking Talk

OR228

Repurposing of signal transduction inhibitors to fight the flu – MEK inhibitors efficiently block influenza virus replication in mice with a prolonged therapeutic window

S. Ludwig*, S. Pleschka 1, O. Planz 2

1University of Giessen, Giessen, 2University of Tuebingen, Tuebingen, Germany

Objectives: Influenza A virus (IAV) infection results in the activation of a variety of intracellular signaling responses. IAV exploit some of these activities to support efficient replication. This dependence of IAV on cellular signaling factors provides opportunities for a novel antiviral strategy that targets essential host factors instead of viral components.

Methods: We have identified the cellular mitogenic Raf/MEK/ERK kinase cascade to be suitable for antiviral intervention. We have employed several inhibitors, which block the pathway on the level of MEK and that are under advanced clinical evaluation or even licensed for clinical use for other diseases. We have analyzed their antiviral potential on a broad range of influenza viruses in vitro and in vivo, including studies on resistance development and therapeutic treatment window, and have unraveled their antiviral mode of action.

Results: We could demonstrate that inhibition of this pathway efficiently blocked virus replication in cells and animals. MEK inhibitors are now under advanced clinical evaluation or even licensed for clinical use for other diseases. We show that these novel signaling blockers (a) efficiently inhibit influenza virus replication in vitro and in vivo, (b) are broadly active against all influenza A and B viruses analysed so far, (c) are active against oseltamivir resistant viruses, (d) are not toxic for cells or animals in the concentration and time line used, (e) display an enhanced therapeutic window compared to standard of care, and (e) confirm the postulated mode of action: blockade of the export of viral genomes from the nucleus.

Conlusion: Repurposing of clinically tested MEK inhibitors is a promising approach to develop safe and efficient anti- influenza viruses with a prolonged treatment window and a high barrier towards development of resistance

Keywords: Influenza virus, Antiviral drugs, MEK inhibitors, therapeutics

17th International Congress of Virology 98

Abstract Submission

Hepatitis viruses II

OR229

THE DISTRIBUTION OF HEPATITIS B VIRUS SUBGENOTYPES AND THE TIME OF THEIR EMERGENCE IN THE NATIVE POPULATION OF SIBERIA, RUSSIA

V. Manuylov 1, V. Chulanov 2, I. Karandashova 2, A. Karlsen 3, S. Netesov 4,*, Y. Ostankova 5, A. Semenov 5, T. Tallo 6

1Sechenov First Moscow State Medical University, 2Central Research Institute of Epidemiology of The Rospotrednadzor, 3Russian Medical Academy of Postgraduate Education, Moscow, 4Novosibirsk state university, Novosibirsk, 5Pasteur Research institute for epidemiology and microbiology, Saint-Petersburg, Russian Federation, 6Public Health Agency of Sweden, Stockholm, Sweden

Objectives: Hepatitis B virus (HBV) is a highly variable DNA-virus that is classified genetically at 10 major genotypes and at least 34 subgenotypes that complicates its molecular diagnostics and is significant for the proper clinical treatment and outcome of hepatitis B. HBV also has at least 9 main serotypes (subtypes) of its surface antigen – HBsAg, that may affect the efficacy of the immunization by recombinant vaccines and the sensitivity of the ELISA tests. Studying of the geographical distribution of HBV genotypes and HBsAg subtypes has an important epidemiological significance, and also is crucial for investigating of the evolutional history of the HBV.

Methods: In this study, we investigated genetic and serological variability of HBV among multiple groups of native population of Siberia: Kazakhs, Altaians, Teleuts and Tyva at the South-West of Siberia; Nenets, Komi, Khants and Selkups (Yamalo-Nenetsky Region), Dolgans and Nganasans (Taimyr Peninsula) and Kets in the Northern Siberia; Buryats, Russian (rural), Yakuts and Chikchi at the East of Siberia. It was shown that these groups differ statistically by the prevalence of HBsAg-positive persons with levels range from 2-4% of population of YNAR and Russians to 10-13% in Altaians, Teleutes, Tyva, Taimyr, Yakuts and Chukchi. Total 340 HBV DNA sequences (Pre-S/S genome region) were studied.

Results: Most of strains in the total group belonged to the genotype D (82%), although genotypes A (5%) and C (13%) were also found. Prevalence of the main subgenotypes of the genotype D – D1, D2 and D3 and HBsAg subtypes were dissimilar in different groups of native populations: D1 (mainly HBsAg subtype ayw2) prevailed in the groups of Kazakhs (89%), Russians (88%), Teleutes (100%), joint group of Taimyr (72%) and Tuva (100%); D2 (subtype ayw3) – in Yakuts (50%) and group of Yamalo-Nenetsky Region (48%); D3 (ayw2) – in Altaians (76%), Buryats (40%) and Chukchi (51%). Subgenotype A2 (adw2) was common for Yakuts (22%), while C1 (adrq+) often occurred among Chukchi (27%) and the population of Taimyr (19%). Statistical analysis of the obtained data has shown that the studied groups were epidemiologically isolated from each other and may be considered as local epidemiological reservoirs with circulation of different HBV variants.

Discovery of genotype C and adrq+ subtype (which are endemic for the Southeastern Asia and previously were not reported as a common HBV types in Russia) at the Taimyr peninsula and Chukotka – the regions at the far North of Siberia is an important result since the clinical outcome for the genotype C is different comparing to the genotype D that is the most prevalent genotype in Russia.

To estimate the date of the emergence of the HBV subgenotypes Siberia we used the evolutional model of (Osiowy et al. Molecular evolution of hepatitis B virus over 25 years. J. Virol. 2006. Vol. 80, No. 21. P. 10307-314) which gives maximum HBV mutation rate 7,9 × 10-5 substitutions per site per year. The most recent common ancestors were reconstructed as the consensuses of strains of the same subgenotype in a given native group. The analysis shown the mean times of the of the HVB subgenotypes in Siberia: the D1 and D3 emerged not later than 180 years ago, D2 and A2 – 160 years, C1 – 200 years ago.

17th International Congress of Virology 99

Conclusion: The HBV genotype D is the most common genotype in Siberia. The prevalence of the HBV subgenotypes statistically differ in the groups of Siberian native populations that is a result of their epidemiological separateness. The HBV emerged in the Siberian native population not later than in the beginning of the 18th century.

Keywords: Hepatitis B virus, Siberia, Subgenotypes

17th International Congress of Virology 100

Abstract Submission

Hepatitis viruses II

OR230

TRANSMISSION OF HBV GENOMIC DNA MEDIATED BY EXTRACELLULAR VESICLES

T. Sanada 1,*, Y. Hirata 1, Y. Naito 2, N. Yamamoto 1, Y. Kikkawa 3, Y. Ishida 4, C. Yamasaki 4, C. Tateno 4, T. Ochiya 2, M. Kohara 1

1Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, 3Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, 4PhoenixBio Co., Ltd., Hiroshima, Japan

Objectives: An extracellular vesicle (EV) is a nano-vesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. Recently, intensive studies have shown that EVs are involved in infectious biology, influencing on host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection.

Methods: We investigated the EV-mediated transmission of HBV infection by using human hepatocytes derived from humanized chimeric mouse (PXB-cells). Purified EVs were isolated by ultracentrifugation. HBV-DNA level in EVs was quantified by real-time PCR. Immune precipitation was performed using antibodies to EV marker molecules, which is CD63, and CD81. To determine whether inhibition of EV secretion affect the HBV-DNA level in culture fluid of HBV-infected PXB- cells, PXB-cells were treated with GW4869, which is known as an inhibitor of EV secretion, and HBV-DNA levels in the culture fluid and cells were analyzed. The resistance of HBV-DNA containing EVs (HBV-EVs) to neutralizing antibody was analyzed by neutralizing test with anti-HBs antibody. To analyze the EVs and virions, we used stimulated emission depletion microscopy (STED microscopy).

Results: Purified EVs from HBV-infected PXB-cells were shown to contain HBV-DNA. Specimens recovered from immune- precipitation with antibodies to EV marker molecules were enriched for HBV-DNA compared with control samples. In addition, GW4869 treatment resulted in decreased HBV-DNA levels in the EV fraction from HBV-infected PXB-cells. These data showed that the HBV genomic DNA containing EVs are produced in HBV-infected PXB-cells. Furthermore, EVs from HBV-infected PXB-cells were shown to be capable of transmitting HBV DNA to naïve PXB-cells, and these HBV-DNA- transmitting EVs were resistant to antibody neutralization. STED microscopy showed that HBV-EV contained HBc antigen, but not HBs antigen, the target of neutralizing antibodies.

Conclusion: These findings suggest another infectious process mediated by EVs and HBV infectious EV may contribute to evade host immune response.

Keywords: Extracellular vesicle, HBV

17th International Congress of Virology 101

Abstract Submission

Hepatitis viruses II

OR231

PREFERENTIAL RECOGNITION OF HBV-DNA INTEGRATED TARGET BY HBV-SPECIFIC CD8 T CELLS

A. A. Khakpoor 1,*, Y. Ni 2, Z. Z. HO 3, V. Oei 1, J. X. Chow 1, N. Yang 4, C. Tham Yan Lin 1, A. T. Tan 1, S. Urban 2, A. Bertoletti 1

1Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore, 2Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany, 3Lion TCR Private Limited, 4Singapore Institute for Clinical Sciences, A*Star, Singapore, Singapore

Objectives: HBV-infected hepatocytes present in patients with chronic hepatitis B make up a complex mosaic of cells expressing different level of HBV antigens (core or envelope). This heterogeneity is the likely results of the presence of hepatocytes infected for different time and hepatocytes carrying full or partial HBV-DNA integration. We hypothesized that HBV-specific CD8 T cells could differentially recognize these targets. Aim of the study was to develop an experimental system that mimic the intrahepatic HBV-expressing hepatocytes heterogeneity and analyze the ability of HBV-specific CD8 T cells to recognize them

Methods: HepG2 NTCP were infected with HBV. Quantity of viral antigens (mRNA and protein level) as well as MHC class -I / PDL-1 were tested at 18 hrs,1,3,7,21, 28, 37days post infection (p.i.) using nanostring or flow cytometry. Similar quantification was performed in HepG2.2.1.5 (HepG2 with full HBV-DNA integration) or HepG2-preS1-GFP (HepG2 with full HBs DNA integration). Targets were co-cultured with core or envelope-specific CD8T cells with or without cytokines (IFN-gamma and IFN- alpha). Ability of IFN-gamma production or degranulation by CD8T cells was quantified.

Results: HepG2 cells expressing viral antigens from integrated HBV-DNA either HepG2.2.1.5 or HepG2-PreS1-GFP activate efficiently either core or envelope-specific (or both) CD8T cells (approximately 30-60% CD8Tcells). In contrast the ability of HBV infected cells to trigger HBV-specific CD8 T cells fluctuate over time and was in general less efficient. Remarkably, 28 days after infection, HBV-infected HepG2-NTCP were not able to activate envelope-specific CD8T cells, despite HBsAg high level expression. In addition, while cytokines (IFN-gamma, IFN-alpha) didn’t boost the presentation of core and envelope in HepG2.2.1.5, HBV infected HepG2-NTCP required IFN-gamma to efficiently present envelope epitope to CD8T cells. Interestingly, this increased efficiency of epitope presentation was associated with a parallel increased PD- L1 expression

Conclusion: Cells with HBV-DNA integration demonstrate constant and more efficient ability to trigger HBV-specific CD8 T cells than HBV infected ones. This suggest that in a chronically HBV infected liver, HBV-specific CD8 T cells would preferentially recognize hepatocytes with HBV-DNA integration than HBV-infected hepatocytes. Hepatocytes with HBV- DNA integration could act as a decoy for anti-viral T cell immunity and as such further restrain the antiviral efficacy of HBV- specific T cells.

Keywords: CD8 T cell, DNA integration, HBV

17th International Congress of Virology 102

Abstract Submission

Hepatitis viruses II

OR232

IMPAIRMENT OF INTERFERON-BETA RESPONSE BY HEPATITIS B VIRUS IN TUPAIA BELANGERI

M. E. H. Kayesh 1,*, S. Ezzikouri 2, H. Chi 3, T. Sanada 4, N. Yamamoto 4, B. Kitab 3, S. Murakami 5, Y. Tanaka 5, M. Kohara 4, K. Tsukiyama-Kohara 6

1Joint Faculty of Veterinary Medicine, Yamaguchi university (Kagoshima University) (Japan), Kagoshima, Japan, 2Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco, 3Transboundary Animal Diseases Centre, Kagoshima University, Kagoshima, 4Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, 5Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, 6Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan

Objectives: To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, this model is very costly and difficult to use because of ethical and animal welfare issues. We aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and to characterize its intrahepatic innate immune response.

Methods: First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes and HBV-A2 propagation level was found to be higher than that of HBV-C. Moreover, we found that HBV-A2 established persistent infection in some tupaias. Then we aimed to characterize the intrahepatic innate immune response upon HBV-A2 infection in this model at 28 days post-infection (dpi) and at 31 weeks post-infection (wpi). First, six adult tupaias were infected with HBV-A2 (strains JP1 and JP4) and sacrificed at 28 dpi for characterization. Next, nine adult tupaias were infected with HBV- A2 (JP1, JP2, and JP4), and sacrificed after 31 weeks for characterization. HBV-DNA quantification was performed using qPCR, based on TaqMan chemistry. Cytokines and toll-like receptors (TLRs) mRNA expression analysis was performed by one step qRT-PCR. Hepatitis B surface antigens (HBsAg) was measured using the two-step sandwich assay principle with a fully automated chemiluminescent enzyme immunoassay system.

Results: At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1, in which the expression of tumor necrosis factor (TNF)-a was significantly upregulated. Expression of TLR1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. At 31 wpi, serum HBsAg levels were detected and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in two tupaias. Suppression of TLR9 was observed in all tupaias except one, which showed a significant increase in TNF-α expression. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection.

Conclusion: Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β, which might have contributed to the establishment of persistent HBV infection.

Keywords: HBV, IFN-beta, tupaia

17th International Congress of Virology 103

Abstract Submission

Negative strand RNA Viruses

OR233

FIGHTING EBOLA: AN ALTERNATIVE SUBUNIT VACCINE STRATEGY

K. Chappell*, D. Watterson 1, D. Muller 2, A. Depelsenaire 2, A. Young 1, G. Marsh 3, M. Kendall 2, P. Young 1

1School of Chemistry and Molecular Biosciences, 2Australian Institute of Biotechnology and Nanotechnology, The University of Queensland, St Lucia, 3Australian Animal Health Laboratory, CSIRO, Geelong, Australia

Objectives: The 2014/2015 Ebola epidemic in West Africa claimed over eleven thousand lives and highlighted our under preparedness to counter emerging viral epidemics. A lead live replicating recombinant vaccine (rVSV-ZEBOV) whose deployment was expedited during the 2014/2015 Ebola epidemic was shown to be efficacious but questions have been raised with regards to its safety. To provide a safer alternative, we set out to develop a subunit vaccine candidate based on a soluble version of the recombinant Ebola glycoprotein (GP) that is stabilized in its pre-fusion conformation and has the potentially cytotoxic mucin-like and transmembrane domains removed.

Methods: BALB/c mice were immunized via NanopatchTM (NP) microneedle delivery or intradermal injection. We then assessed the antibody response elicited against the live Ebola virus (Zaire strain) using facilities at CSIRO’s Australian Animal Health Laboratories in Geelong (AAHL).

Results: The stabilized Ebola GP was recognized by a panel of highly neutralizing antibodies, indicating both the GP1/2 and glycan cap domains are available and are presented in the desired conformation. Mouse sera showed promising plaque reduction neutralization titres following vaccinations (PRNT50 = ~1/50 sera dilution). We also showed that this candidate vaccine is thermostable, retaining significant antigenicity after extended incubation at 37°C.

Conclusion: Immunization with the stabilized Ebola GP elicited a strong neutralizing immune response, indicative of protection. Long term stability indicates this vaccination strategy may not require cold chain transport. Furthermore, the removal of known cytotoxic domains and the absence of replicative elements ensures that this vaccine will have a safer profile than live recombinant vaccines. Taken together, our results suggest that this vaccine may represent a safer alternative strategy to combat future Ebola outbreaks.

Keywords: Ebola virus, Glycoprotein, Vaccine

17th International Congress of Virology 104

Abstract Submission

Negative strand RNA Viruses

OR234

MRNA CAPPING MACHINERY OF VESICULAR STOMATITIS VIRUS

M. Ogino 1, J. Neubauer 1, T. Ogino*

1Department of Molecular Biology and Microbiology, CASE WESTERN RESERVE UNIVERSITY SCHOOL OF MEDICINE, Cleveland, United States

Objectives: GDP polyribonucleotidyltransferase (PRNTase, EC 2.7.7.88) is an unconventional mRNA capping enzyme that is present as an enzymatic domain in multifunctional RNA-dependent RNA polymerase (RdRp) L proteins of nonsegmented negative strand (NNS) RNA viruses belonging to the Rhabdoviridae family, such as vesicular stomatitis virus (VSV) and rabies virus. The mechanism of mRNA capping with PRNTase is distinct from that with eukaryotic mRNA capping enzyme, guanylyltransferase (GTase, EC 2.7.7.50). It has been shown that PRNTase transfers 5′-phospho-RNA (pRNA) from 5′- triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA (L-pRNA) intermediate to generate the cap structure (GpppA) on RNA, whereas eukaryotic GTase transfers GMP from GTP to 5′-diphospho-RNA via a covalent enzyme-GMP intermediate. The objectives of this work were to reveal the unique enzymatic characteristics of the PRNTase domain in the VSV L protein and to elucidate the mechanisms of co-transcriptional formation of the fully-methylated cap structure (m7GpppAm) with the L protein.

Methods: Using our in vitro oligo-RNA capping systems, including the L-pRNA intermediate formation and pRNA transfer assays with the recombinant VSV L protein, we examined the effects of mutations in the PRNTase domain on the enzymatic reaction and analyzed the acceptor and donor substrate specificities of the PRNTase domain. Using our in vitro VSV transcription system, we investigated the timing of pre-mRNA capping and methylation with a transcribing L protein.

Results: The active site of PRNTase is constituted by five amino acid sequence motifs that are conserved among NNS RNA viruses belonging to the order Mononegavirales (e.g., measles, Nipah, Ebola). These motifs were essential for the PRNTase reaction in the step of the intermediate formation as well as VSV gene expression and propagation in host cells. The PRNTase domain was found to recognize either the ribose-2′- or 3′-hydroxyl group and the guanine-C-2-amino group, but not C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the pRNA transfer reaction. We found that the PRNTase reaction is reversible and both the forward and reverse reactions are completely abolished by ribose-2′-O-methylation of the first nucleotide of the substrate RNAs. The PRNTase domain in a transcribing L protein capped nascent pre-mRNAs of <20 nucleotides in length, whereas the methyltransferase (MTase) domain methylated the cap structure of pre-mRNAs ranging in length from 80 to 150 nucleotides at the 2′-O position followed by guanine-N-7 position.

Conclusion: The VSV PRNTase domain has a unique active site and substrate specificities that are distinct from those of eukaryotic GTase. Since ribose-2′-O-methylation inhibits the forward and reverse PRNTase reactions and is required for efficient guanine-7-methylation of the cap structure, we conclude that the 5′-end modifications proceed in the following order: (1) polyribonucleotidylation of GDP → (2) 2′-O-methylation → (3) guanine-N-7-methylation. These reactions with the PRNTase and MTase domains of the L protein sequentially occur on pre-mRNAs with different chain lengths during mRNA chain elongation catalyzed by the RdRp domain on the same polypeptide.

Keywords: GDP polyribonucleotidyltransferase, mRNA capping, vesicular stomatitis virus

17th International Congress of Virology 105

Abstract Submission

Negative strand RNA Viruses

OR235

FERRET MODELS OF DISEASE ASSOCIATED WITH FILOVIRUS INFECTIONS

G. Marsh*

Objectives: Current animal models for filoviruses consist of either non-human primates that can be infected with un-adapted human isolates of virus or rodent models with viruses that have been adapted following multiple -passages in order to induce disease. The objectives of these studies were to develop ferret models of filovirus disease in which disease results from infection with un-adapted virus isolates.

Methods: Ferrets were exposed to a range of infectious doses of various filoviruses (Ebola, Sudan and Marburg viruses) via the oronasal route. Ferrets were then monitored for the onset of clinical disease and were euthanased when animal reached pre-determined humane endpoints. Terminal samples were collected for virus titration, clinical and anatomical pathology. A serial sacrifice study was also performed with ferrets challenged with Ebola virus investigating disease progression and clinical signs.

Results: Following exposure of ferrets to low doses of various filoviruses, ferrets all developed disease and reached humane endpoints, a surrogate of lethal disease, within 4-7 days following challenge. High levels of viraemia were observed in animals at late stages of disease with systemic spread of virus. Clinical pathological changes were observed in animals suggesting disseminated intravascular coagulation occurred in animals.

Conclusion: The ferret has significant advantages over other rodent models of disease for Ebola virus, the greatest of which is that they are susceptible to wild-type human isolates of virus without the requirement for adaptation. Ferrets studies are also more cost effective than non-human primate studies, and it is easier to accommodate their species welfare requirements. Clinical signs and blood parameter changes observed in ferrets were similar to those recorded in non-human primates and humans infected with Ebola virus. The ferret may therefore provide a small non-primate mammal of Ebola virus disease suitable for testing of vaccines and therapeutics.

Keywords: animal model, disease, Ebola virus

17th International Congress of Virology 106

Speaker Abstract Submission

Negative strand RNA Viruses

STRUCTURAL ELUCIDATION OF THE RABIES VIRUS P PROTEIN-STAT1 INTERFACE: THE MOLECULAR BASIS FOR IMMUNE EVASION

A. Hossain 1, J. Zhan 1, K. Lieu, T. Ose 2, N. Ito 3, P. Gooley 1, G. W. Moseley 1,* 1Dept. of Biochemistry & Molecular Biology, The University of Melbourne, Parkville, Australia, 2Hokkaido University, Sapporo, 3Gifu University, Gifu, Japan

Objectives: Negative-sense RNA viruses such as rabies virus (RABV), Nipah virus and Ebola virus have limited genome capacity resulting in the expression of only a small number of proteins. Nevertheless, such viruses arrest potent control over infected cells and the immune system, and are often highly lethal, with rabies having an almost 100% case-fatality rate. Key to this is the expression of multifunctional proteins that have evolved immune-antagonistic functions additional to conserved ‘core’ functions in genome replication and virion structure. Among these, RABV phospho (P) protein performs roles as the essential polymerase cofactor and the viral interferon (IFN) antagonist. For the latter, P interacts directly with host immune signalling factors including STAT1, resulting in potent shut down of antiviral pathways. Recently, we showed that preventing P-STAT1 interaction does not impact RABV viability in vitro, but prevents viral immune evasion, ablating pathogenicity in animals following intracerebral infection. These data clearly identify the P-STAT1 interface as a potential therapeutic target. However, the structure and molecular details of the interface are unresolved, with no direct structural data available. This leaves major gaps in knowledge on fundamental processes in rabies pathogenesis, and the potential for targeting of P protein-STAT1 interactions to prevent lethal outcomes. We thus sought to elucidate the structural basis of this interaction.

Methods: To determine the molecular structure and function of the P protein-STAT interface, we have used structural and biophysical methods including nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR), together with cell biology and molecular virology approaches, including reporter gene/functional genomic analysis of antiviral immune signaling, live cell imaging approaches, and co-immunoprecipitation/proteomic assays. Results: We report here the first direct structural data on the molecular interface of P protein with STAT1, including detailed molecular mapping of novel sites and residues forming the interacting surfaces. Using mutagenesis guided by the structural data, we demonstrate that the surfaces form the functional interaction in vivo, and are thus critical to immune evasion function. These data have enabled us to generate the first structural model for the P protein-STAT1 complex. Conclusion: Our new findings are revealing the specific molecular mechanisms used by RABV to shut down host antiviral immunity, as well as the molecular strategies by which RABV P protein coordinates its multiple interactions essential to viral replication and immune evasion. Our ongoing research seeks to exploit this knowledge to develop novel vaccine strains and compounds able to inhibit these critical pathogenic interactions.

Keywords: Immune evasion, Interferon, Rabies

17th International Congress of Virology 107

Speaker Abstract Submission

Bridging Session – Microbes and Climate Change

Dengue and Zika transmission: environmental and human interaction

Ling Chen Guangzhou Institute of Biomedicine and Health, China

Dengue and Zika, transmitted by Aedes mosquitoes, have become two of the most important mosquito-borne diseases. With increasing globalisation, urban development with high population density and climate change, the two diseases are set to worsen as they increase in transmission intensity and invade new territories. A trans-disciplinary approach is needed to tackle the complex epidemiology of the diseases, which have inflicted heavy burdens on economies and societies. This presentation will discuss the impact of environmental and human interaction on the diseases, and the possibilities and limitations of vector control and vaccination in controlling the diseases. It will also demonstrate the utility of data science, virology, molecular epidemiology, entomology, climate science and social science in supporting prevention and control programmes.

17th International Congress of Virology 108

20 July 2017 Oral Presentation

17th International Congress of Virology 109

Speaker Abstract Submission

Innate defence against viruses

DUSP4 regulates MAPK and RIG-I signalling in innate immune response to virus infection

Y. Zhang*, H. Jiao 1 1Microbiology & Immunology, National University of Singapore, Singapore, Singapore

Objectives: MAPK phosphatase (MKPs), also known as dual-specificity phosphatases (DUSPs), are cysteine-based protein tyrosine phosphatases that dephosphorylate phosphotyrosine, phosphothreonine, and phosphoserine residues in their substrates. They are originally identified as the major negative regulators of MAP kinases (MAPKs). Our recent study on DUSP10/MKP5 demonstrated that this molecule plays an important role in immune response to virus infection. DUSP4/MKP2 is another DUSP/MKP family member whose function in immune response to virus infection is unclear. This study aims to elucidate the regulatory function of DUSP4 in RIG-I-mediated signaling and in innate immune response to virus infection.

Methods: Wildtype (WT) and DUSP4 knockout (KO) mice and cells were used to examine its function in innate immune response. Bone marrow-derived macrophages (BMDMs) and dendrtic cells (BMDCs) were prepared to examine the regulation of DUSP4 in innate immune cells to RIG-I stimulation and influenza virus infection. WT and DUSP4 KO mice were infected with PR8 (H1N1) influenza virus to study the in vivo function of DUSP4 in immune response to influenza virus.

Results: DUSP4 KO BMDMs and BMDCs have enhaced cytokine expression in response to RIG-I stimulation and PR8 infection. Increased activation of MAPKs and IRF3 was observed in KO cells compared to that in WT cells. The KO mice developed less severe disease in response to PR8 infection, which was associated with increased type I IFN expression in the lung compared to WT mice. Conclusion: Our studies on DUSP4 demonstrate a novel function of this molecules in immune response to virus infection and may be further explored for therapeutic purposes.

Keywords: DUSP, MAPK, RIG-I

17th International Congress of Virology 110

Abstract Submission

Innate defence against viruses

OR236

VACCINIA VIRUS PROTEIN C4 IS AN INHIBITOR OF DNA-PK-MEDIATED DNA SENSING

S. R. Scutts 1,*, S. W. J. Ember 1, H. Ren 1, D. L. Veyer 1, R. P. Sumner 1, C. Ye 1, G. L. Smith 1

1Department of Pathology, University of Cambridge, Cambridge, United Kingdom

Objectives: DNA-dependent protein kinase (DNA-PK) acts as a pattern recognition receptor that binds cytosolic DNA, such as the genome of vaccinia virus (VACV), leading to an IRF3-mediated innate immune response. VACV protein C16 inhibits activation of this receptor by interacting with the Ku heterodimer and diminishing the ability of DNA-PK to bind DNA. Here, we show that VACV protein C4, which shares 43% amino acid identity with C16, also binds to Ku and can diminish activation from this receptor.

Methods: HEK 293T cells were transfected with pcDNA T/O expression plasmids encoding TAP-tagged proteins and immunoprecipitations were performed using FLAG or Streptavidin beads. For DNA pulldown assay, HeLa cells were transfected with biotinylated double stranded oligonucleotide DNA one hour prior to cell lysis. For ELISAs, MEFs were transfected and levels of CXCL10 and IL-6 in cell supernatants were determined. In vivo work utilised BALB/c mice for intranasal inoculation or C57BL/6 mice for intradermal inoculation with sucrose purified virus and weight loss, sign of illness or lesion size were measured at various time points.

Results: Data presented demonstrate that the C-terminal region of C4 mediates binding to Ku and that three conserved residues; C174, Y175 and C176, are required for this interaction. Functionally, C4 blocks the binding of Ku to DNA leading to reduced production of cytokine and chemokines. In vivo, C4 and C16 share redundancy in that single deletion viruses display identical virulence to wild-type Western Reserve virus in the intradermal model of infection, whereas a double deletion virus has reduced virulence.

Conclusion: Taken together, this investigation reveals that VACV has evolved two proteins to inhibit DNA-PK, emphasising the importance of this newly discovered PRR in the response to infection.

Keywords: DNA sensing, Immune evasion, Vaccinia virus

17th International Congress of Virology 111

Abstract Submission

Innate defence against viruses

OR237

ASTROCYTIC RETICULAR NETWORK FUNCTIONS IN THE BRAIN AS A CONDUIT SYSTEM FOR VIRAL ENTRY AND CYTOKINE PRODUCTION

R. Watanabe*

Objectives: Mu-3 virus (Mu-3), a neuropathogenic strain of the mouse hepatitis virus (MHV) JHM viral strain (JHMV) induces the apoptosis of pyramidal neurons in CA2 and CA3 subregions of the hippocampus at 4 days post-inoculation (dpi). The maternal clone of MU-3, srr7 virus (srr7) has been isolated from extremely neurovirulent cl-2 virus (cl-2). In spite of differences in the virulence and neuropathological findings, these viruses exhibit a similar pattern of viral spread during the initial phase of infection. Between 12 and 24 hpi, viral antigens are detected only in the meninges, followed by viral spread into the ventricular wall at 24 to 48 hpi before invasion into the brain parenchyma. Therefore, it was indicated that the viruses use a passage between the meninges and ventricular wall as an entry route into the brain parenchyma. In addition, several kinds of cytokines were produced deep in the brain parenchyma before viral invasion. Thus, the pathways for viral invasion and information similar.

Methods: BALB/c mice were housed in a specific pathogen-free animal facility, and maintained according to the guidelines set by the ethical committee of our university. For the experiment, mice were transferred to a biosafety level 3 (BSL-3) laboratory after obtaining permission from the committee of our university. Six-seven-week-old mice were used for all experiments. For detecting fibroblastic reticular fibers (FRfibs), the expression of their components reported in the lymph node, such as Erasmus University Rotterdam-thymic reticulum antibody 7 (ER-TR7)-reactive ER-TR7-antigen (ERag), collagen, and laminin were investigated. The production of cytokines were studied in the areas where viral antigens were not detected.

Results: [Detection of the pathway] At 48 hpi, the passage for the viral entry was found to be constructed by the extracellular matrix (ECM). The ECM includes an ERag-positive fibers (ERfibs) associated with laminin and collagen. [Cells that produce ERag] Astrocytes produced ERag in addition to collagen and laminin after infection.[Cytokines and ERfibs] The infected cells in the ventricle and ventricular wall during the early phase of infection did not produce IFNb, but deep in the brain parenchymal area where no viral antigens were detected, IFNb-positive cells were found. A fine astrocytic network was found to surround the IFNb-producing. Fibrous structures of ERfibs were closely associated with astrocytic fibers. IL-10: Because immunosuppressive state similar to endotoxin tolerance (ET) was induced after infection with our viruses, and the study failed to detect proinflammatory cytokines in the CA2 and CA3 subregions in the hippocampus, we studied the production of anti-inflammatory cytokines to clarify the mechanisms of indirect effects of viral infection. At 3 dpi, viral antigens are not found in the hippocampus. Nevertheless, the production of IL-10 was detected in the neurons in the CA2 and CA3 subregions.

Conclusion: Highly neurovirulent viruses invade and spread in the brain using the host reactions triggered by infection, which includes immunosuppression, and the production of unique extracellular matrices, which form an astrocytic reticular network (ARN) after infection, and are used as a conduit system similar to FRN reported in the lymph nodes.

Keywords: astrocyte, cytokine, reticular network

17th International Congress of Virology 112

Abstract Submission

Innate defence against viruses

OR238

THE ROLE OF MAST CELLS IN JAPANESE ENCEPHALITIS VIRUS PENETRATION OF THE BLOOD-BRAIN BARRIER

J. Hsieh*, A. St. John 1

1Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore

Objectives: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Mechanisms for JEV penetration of the blood brain barrier (BBB) remain poorly understood. We previously showed that a similar flavivirus, dengue virus, activates mast cells and leads to disruption of blood vessels. The aim of this study is to characterize the mast cell’s response to JEV and its role in JEV-induced break down of the BBB.

Methods: Human ROSA mast cells and mouse bone marrow-derived mast cells were exposed to JEV in vitro and degranulation was measured by beta-hexosaminidase assay. In vivo, wild-type and mast cell-deficient (Sash) mice were infected with JEV via the intraperitoneal (I.P.) route. Serum concentrations of the mast cell specific protease, MCPT1, were measured as a marker for mast cell activation, using ELISA. To quantify BBB compromise, Evans blue dye was administered I.P. and leakage into the brain was quantified by colorimetric measurement. Contributions of mast cells to the clearance of JEV infection in the periphery and the central nervous system (CNS) were assessed using real-time polymerase chain reaction. The neuroseverity score and grip strength meter were used to measure neurologic deficits.

Results: JEV increased beta-hexosaminidase release by both human ROSA mast cells (P <0.05) and mouse bone marrow mast cells (P <0.05) starting at a multiplicity of infection of 1, indicating that JEV induces mast cell degranulation. The virus also increased serum MCPT1 concentration in JEV-infected wild-type mice (P <0.05, n=8). In vivo, JEV was able to promote Evans blue dye leakage across the BBB (P <0.05, n=10), enhanced JEV virus titer in the CNS (P <0.05, n=6), worsened neuroseverity score (P <0.05, n=12), and weakened grip strength (P <0.005, n=12) in wild-type mice. These effects were absent in mast cell-deficient sash mice.

Conclusion: Our findings point to mast cells playing a counter-protective role during JEV infection, inducing BBB breakdown, increasing CNS viral titers, and worsening neurological deficit. We have implicated mast cells as a previously unidentified component of BBB breakdown in JEV infection.

Keywords: Blood-brain barrier, Japanese encephalitis virus, Mast cells

17th International Congress of Virology 113

Speaker Abstract Submission

Viral disease

THE ROLE OF CHONDROCYTES IN ALPHAVIRAL INFLAMMATORY DISEASE

L. Herrero*, H. Liu 1, A. Supramaniam 1, W. S. Lee 1, P. Coles 1, P. Rudd 1 1Institute for Glycomics, Griffith University, Southport, Australia

Objectives: Arthropod-borne arthritogenic alphaviruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) cause large epidemics of severe musculoskeletal disease and have been progressively expanding their global distribution emerging in new regions of the world. It has been well-established that alphaviruses cause severe arthralgia/arthritis in the joints that can be both acute and chronic. The synovial space of joints is glycan rich, containing high levels of glycosaminoglycan (GAGs) frequently linked to protein backbones forming proteoglycan structures. Chondrocytes are the major cell type producing the proteoglycan rich articular cartilage matrix of joint. To date, there have been no studies to specifically elucidate the impact of alphaviruses on cartilage and cartilage producing chondrocytes. Therefore this study aims to characterise the complex interactions between alphaviruses and host cartilage components (chondrocytes and glycans) in disease pathogenesis

Methods: We examined the role of chondrocytes in alphavirus infection using in vitro, ex vivo and in vivo cells and tissues followed by molecular, imaging, bioplex, histology and glycan array assays. The combination of cells and techniques provided the ability to assess alphaviral infection of chondrocytes in an isolated manner and in the context of the cartilage matrix in vivo thereby assessing the role of chondrocytes in both alphavirus infection and pathogenesis

Results: Results show that alphaviruses including CHIKV, RRV and Barmah Forest virus (BFV) can replicate in chondrocytes in cell lines and in primary human cells. This infection resulted in the production of critical soluble factors and degrading enzymes known to contribute to cartilage damage. Results correlated to in vivo where murine chondrocytes were found to be a site of alphavirus infection with RRV-RNA and antigen detected in chondrocytes of the cartilage. Furthermore this infection was found to produce effects, which caused in damage to the murine articular cartilage thereby contributing to disease pathogenesis.

Conclusion: These studies indicate that chondrocytes are a potential target for alphaviral infection and replication during natural infection, contributing to the disease pathogenesis. Detailed understanding of the role of chondrocytes in alphaviral pathogenesis and the specific role of glycans could ultimately lead to the development of safer and improved antiviral therapies against viral pathogens and the arthritic diseases they cause.

Keywords: Alphavirus, cartilage, pathogenesis

17th International Congress of Virology 114

Abstract Submission

Viral disease

OR239

A MURINE MODEL OF ENTEROVIRUS 71 (EV71) INFECTION, DISEASE, AND PATHOLOGY EXHIBITING NEUROGENIC PULMONARY EDEMA

C. B. L. Victorio 1,*, Y. Xu 1, Q. Ng, B. H. Chua 2, S. Alonso 3, V. Chow 3, K. B. Chua 1

1Chua Kaw Bing Lab, Temasek Lifesciences Laboratory, Singapore, Singapore, 2Curtin University, Perth, Australia, 3Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: To develop a murine model of enterovirus A71 (EV-A71) infection that exhibits the full spectrum of clinical diseases observed in human cases of EV-A71 disease.

Methods: One-week-old BALB/c mice were inoculated with a mouse-cell-adapted EV-A71 strain (EV71:TLLmv) via either 6 intraperitoneal (IP) or intramuscular (IM) injection with 10 CCID50 virus. The animals were observed daily for general signs of disease—as well as signs of neurological deficits—over a 28-day observation period. Moribund mice were sacrificed, and gross pathological analysis was performed. Relevant tissues were collected and fixed for subsequent histopathological analyses. Immunohistochemical staining for EV-A71 antigens was also performed to determine tissue sites of virus replication in the animals. To measure catecholamine levels in sera, cardiac puncture was performed in moribund animals following euthanasia. Sera were collected and analysed by ELISA.

Results: Mice inoculated with MCA-EV-A71 strains exhibited disease in a dose-dependent, age-dependent, and inoculation route-dependent manner. Inoculated mice could be classified into four distinct groups based on clinical signs manifested at the time of euthanasia—Class III (survivors) include animals that did not exhibit disease throughout the observation period; Class II include mice that exhibited mild disease that mainly presented wasting and persistent limb paralysis; Class I include mice that exhibited severe disease with signs of neurological deficits within 1 week post-inoculation with the virus. A few animals in this group (Class IA) also exhibited respiratory distress. Some mice also exhibited ataxia, tremors, and signs of autonomic nervous system dysfunction, such as tachycardia and tachypnea. Others also exhibited skin lesions in the feet, reminiscent of HFMD (Hand, Foot, and Mouth Disease) manifestations in humans. Gross pathology of Class IA mice exhibited uncollapsed lungs with focal areas of haemorrhaging. Histopathological analyses revealed signs consistent with pulmonary edema (PE). The absence of distinct pathology in the cardiac tissues ruled out cardiogenic PE, while the absence of viral antigens in the lung tissues rule out direct tissue injury caused by viral replication as a cause of PE. Instead, high levels of serum catecholamine point to a neurogenic origin of PE. Histopathological analysis of brain and spinal cord tissues reveal extensive tissue lesions and viral replication in certain regions, especially in those previously known as associated with triggering NPE in humans, thus confirming the neurogenic origin of PE exhibited by Class IA mice. IHC staining also confirmed that the virus replicated only in CNS tissues of mice inoculated with EV71:TLLmv.

Conclusion: This model of mouse infection with EV71:TLLmv mouse-cell-adapted strain exhibits a wide spectrum of diseases—similar to what is observed in human cases of EV-A71 infection, including neurogenic pulmonary edema. Thus, this model exhibits face validity. Histopathological analyses also revealed that damage due to viral replication was localized to the CNS tissues, consistent with findings in human post-mortem analyses. Thus, this model also exhibits construct validity. This infection model represents the first clinically valid animal model of EV-A71 infection, which could be further employed in studies aimed at understanding the neuro-pathogenesis of EV-A71, especially in the context of NPE induction

Keywords: enterovirus A71, Mouse Model, neurogenic pulmonary edema

17th International Congress of Virology 115

Abstract Submission

Viral disease

OR240

AN IN VITRO HUMAN NASAL EPITHELIAL MODEL FOR THE UNIVERSAL SURVEILLANCE AND CHARACTERIZATION OF RESPIRATORY VIRUS INFECTIONS

K. S. Tan 1, Y. Yan 1, H. H. Ong 1, T. N. Huong 2, H. W. Choi 3, T. Tran 4, R. Sugrue 2, G. Smith 5, V. T. K. Chow 6, D. Y. Wang 1,*

1Department of Otolaryngology, National University of Singapore, 2School of Biological Sciences, Nanyang Technological University, 3Saw Swee Hock School of Public Health, 4Department of Physiology, National University of Singapore, 5Emerging Infectious Diseases Programme, Duke-NUS Medical School, 6Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: Respiratory virus infections pose a constant health threat to the population due to their high transmissibility. Currently, studies on these viruses are highly segregated due to different in vitro systems and hosts required to conduct such studies. Moreover, the nature of viruses requiring a host to propagate poses challenges in the selection of host cells to generate sufficient virus for research and vaccine development purposes. This was apparent during the identification and isolation of the SARS coronavirus. Therefore, in order to respond to such threats, the developement of a universal infection model is necessary to facilitate the universal surveillance, identification, diagnosis, and treatment of these pathogens.

Methods: Our lab previously established an in vitro model of differentiated human epithelial cell (hNECs) using stem cells harvested from donor nasal tissues. The cells are fully differentiated in vitro to give rise to a fully functioning, multi-layered nasal epithelium mimicking the arrangement and cell types of the human nasal epithelium. Using this model, the hNECs were infected separately with different respiratory viruses, namely, Influenza A virus (IAV), Rhinovirus (RV), Respiratory Syncytial Virus (RSV) and Parainfluenza virus (PIV). The hNECs infected with the respective viruses were analysed for their ability to sustain the virus infection and to mount an appropriate immune responses mirroring the nasal epithelium.

Results: Using this model, we have successfully showed the ability of the hNECs to sustain the infection of all respiratory viruses tested (IAV, RV, RSV and PIV). The hNECs also showed that the innate immune response mounted were comparable to other infection models. Furthermore, each tested respiratory virus displayed their own distinct viral replication kinetics, host cell type preference and responses, thus mirroring their unique nature and pathogenicity. These findings indicate that this model truly mimics the human nasal epithelium, with varying responses to different respiratory viruses, similar to natural infections in a human host.

Conclusion: Overall, the hNECs model provides a unified platform for the study of respiratory viruses that aids in our preparedness against respiratory virus outbreaks and facilitates more comprehensive meta-analysis between different respiratory virus infections by employing a universal platform of infection.

Keywords: In vitro model, Nasal epithelium, Respiratory virus infection

17th International Congress of Virology 116

Abstract Submission

Viral disease

OR241

IDENTIFICATION OF RISK FACTORS FOR ROTAVIRUS CIRCULATION IN PIGS FROM PHILIPPINE BACKYARD FARMS

M. C. Otero 1,*, L. A. Murao 1, P. Alviola IV 2, N. Oponda 3, F. Lagat, Y. S. L. Ladera 1, P. G. Gilles 2

1Department of Biological Sciences and Environmental Studies, 2School of Management, 3Department of Mathematics, Physics and Computer Science, University of the Philippines Mindanao, Davao City, Philippines

Objectives: Group A Rotaviruses (GARV) are important pathogens for livestock because they cause severe diarrhea in young mammals and birds. This may result in economic losses due to death of animals, poor growth performance, and added costs for treatment and care. Backyard swine farms in the Philippines constitute over 60 percent of the local swine industry but are plagued by outdated management practices and poor animal health services, thereby promoting viral spread. Subclinical infection of GARV in pigs is also common yet largely undetected, and fecal shedding from asymptomatic pigs is a potential source of new infections or outbreaks. In this study, fecal shedding of GARV was monitored in backyard farms in Davao City, Philippines, in order to determine its association with gastroenteritis and identify risk factors for viral spread.

Methods: A database of 831 backyard swine farms in Davao City was generated in coordination with 20 barangays from four districts. A total of 100 farms were selected using stratified random sampling for fecal sampling and survey on farm practices, household socio-demographics, and geographic information.

Results: The incidence of diarrhea in these farms during the sampling period was low (5 percent). However, around 12 percent of the farms tested positive for GARV based on nested reverse-transcription PCR of the VP6 gene. Probit and logit statistical analysis revealed that the presence of drainage decreases the risk of GARV fecal shedding, while there was an increased risk when the water is sourced from faucets linked to local water reservoirs or from deep wells.

Conclusion: Hence, proper management of water in backyard farms appears to be crucial for control of rotavirus circulation in pigs in Davao City. This is the first report of porcine GARV surveillance in the Philippines.

Keywords: farm practices, porcine rotavirus, surveillance

17th International Congress of Virology 117

Abstract Submission

Virus surveillance, epidemiology & Public Health

OR242

PREVALENCE OF ANTIBODIES TO ZIKA VIRUS IN MOTHERS FROM HAWAII WHO DELIVERED BABIES WITH AND WITHOUT MICROCEPHALY BETWEEN 2009-2012

M. Kumar 1,*, L. Ching 1, J. Astern 1, E. Lim 1, A. Stokes 1, M. Melish 1, V. Nerurkar 1

1University of Hawaii, Honolulu, United States

Objectives: Zika virus (ZIKV) is an emerging mosquito-borne pathogen. ZIKV infection is linked to the development of severe fetal abnormalities that include spontaneous abortion, stillbirth, hydranencephaly, and microcephaly. ZIKV outbreaks have been recorded in the United States. We recently demonstrated the first congenital ZIKV infection in the United States confirmed by high ZIKV IgM antibody titers in serum and cerebrospinal fluid. In this case, a ZIKV-infected mother delivered a baby with microcephaly. In a quest to find a link between ZIKV infection and babies born with microcephaly, we investigated archived blood samples from mothers who gave birth in Hawaii between 2009 and 2012 to babies with microcephaly.

Methods: In this study, we investigated archived blood samples from six mothers who gave birth to babies with microcephaly and 12 mothers who gave birth to healthy babies in Hawaii between 2009 and 2012. We tested maternal blood for the presence of ZIKV IgM and IgG antibodies using commercially available human ZIKV IgM and IgG ELISA kits.

Results: Blood from one mother who delivered babies with microcephaly tested positive for ZIKV IgM antibody (16.6%) and blood from three mothers tested positive for ZIKV IgG antibody (50%). ZIKV IgG antibody was detected in one of 12 (8.3%) mothers who delivered healthy babies and all 12 mothers were negative for ZIKV IgM antibodies. ZIKV showed a trend toward significance with microcephaly. ZIKV IgG antibody positive mothers were more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgG antibodies (Odds ratio [OR]=11.0, 95% confidence interval [CI]=0.8-147.9, p = 0.083). Similarly, ZIKV IgM antibody positive mothers were also more likely to deliver babies with microcephaly than mothers who were negative for ZIKV IgM antibody (OR=6.8, 95% CI=0.2-195.1).

Conclusion: These data provide further evidence of a link between ZIKV infection and microcephaly and suggests presence of ZIKV positive cases and associated microcephaly in the United States as early as 2009.

Keywords: Zika virus, Microcephaly, Antibodies

17th International Congress of Virology 118

Speaker Abstract Submission

Virus surveillance, epidemiology & Public Health

GLOBAL COMPARATIVE EPIDEMIOLOGY OF RESPIRATORY VIRUSES 2010-2015

J. Tang 1,*, J.-M. Heraud 2, T. Hachette 3, M. Waris 4, L. Jennings 5, S. Bialasiewicz 6, E. Koay 7, P. Chan 8, H. Zaraket 9, M. Koopmans 10 1Clinical Microbiology/Virology, University Hospitals of Leicester NHS Trust, Leicester, United Kingdom, 2Virologie Unit, National Influenza Center, Antananarivo, Madagascar, 3Pathology and Laboratory Medicine, Dalhousie University, Halifax, Canada, 4Virology, University of Turku, Turku, Finland, 5Virology, Canterbury Health Laboratories, Christchurch, New Zealand, 6Centre for Children's Health Research, University of Queensland, Brisbane, Australia, 7Molecular Diagnosis Centre, National University Hospital, Singapore, Singapore, 8Microbiology, The Chinese University of Hong Kong, Hong Kong, Hong Kong, 9Faculty of Medicine, American University of Beirut, Beirut, Lebanon, 10Viroscience, Erasmus MC, Rotterdam, Netherlands

Objectives: To set up/maintain a global network of laboratories routinely testing for influenza and other respiratory viruses; to collate and present the contemporaneous, cross-sectional global incidence of respiratory RNA virus infection during 2010-2015, as caused by human influenza, respiratory syncytial, parainfluenza, corona- and rhino- viruses; to understand the patterns of infection across different global populations more clearly; to make and support a case for increased investment into the development of antiviral drugs and vaccines against these non-influenza respiratory RNA viruses.

Methods: Over an 18-month period, multiple laboratories across the world were contacted via email/Skype/personal contact at conferences, etc. to request participation in creating a network of laboratories (INSPIRE: International Network for the Sequencing of resPIRratory vIrusEs: www.global-inspire.org). Anonymised RNA respiratory virus incidence data for 2010-2015 (where routine diagnostic laboratory testing was performed), for each of: influenza A, B; respiratory syncytial viruses (RSV A/B); parainfluenza viruses (PIV types 1-4), human metapneumoviruses (HMPV), coronaviruses (CoVs: OC43, 229E, NL63, HKU1), and rhinoviruses (RVs) was requested from each participating site. Data collected consisted of: the proportion of positives among total specimens tested for each virus, as well as individual age and sex of the patients where anonymised line lists were permitted. Each contributing laboratory sought their own local IRB approval for sharing such anonymised data within the INSPIRE network.

Results: The data received back from the INSPIRE network was quite heterogeneous, as some of the contributing sites national reference laboratories covering the entire population of their country for these tests, whereas others were single- site hospital diagnostic laboratories, covering local patient populations. In addition, the testing methods (viral culture vs. immunofluorescence vs. PCR) and kits used differed between the different sites, as did the patient populations tested (i.e. inpatient vs. outpatients, paediatric vs. adult, acute clinical diagnostic vs public health surveillance – and/or mixtures of all of these). As expected there were distinct peaks of viral incidence for influenza and RSV in temperate countries. Within these temperate climate populations, the Northern and Southern hemisphere peaks of influenza and RSV incidence were frame-shifted by approximately 6 months, coinciding with their respective winter and summer seasons. There was a more mixed, pattern of viral infections that could be detectable throughout the year for the more tropical/subtropical countries. For PIV (including individual types when tested) there was no discernible generalizable pattern of incidence across all sites, though some countries (Mongolia, Japan and The Netherlands) showed higher peaks of incidence during this study period. There were insufficient numbers of sites routinely testing for HMPV, CoVs and RVs to allow a comparative plot of the incidence of these viruses. Conclusion: Using a total data collection approach, we have described the contemporaneous annual incidence patterns of these respiratory viruses, across the world. This highlights their global healthcare burden and demonstrates the urgent need for the development of interventions against these viruses, such as new antiviral drugs and vaccines.

Keywords: Influenza virus, Paramyxovirus, Respiratory tract infections

17th International Congress of Virology 119

Abstract Submission

Virus surveillance, epidemiology & Public Health

OR243

EXPERIENCE OF VIRAL INFECTIONS CAN CHANGE VULNERABILITY OF HOST AGAINST SUBSEQUENT INFECTIONS: PEDIATRIC ACUTE RESPIRATORY INFECTION COHORT STUDY

Y. Furuse 1,*, R. Tamaki 1, M. Saito-Obata 2, V. Tallo 3, S. Lupisan 3, H. Oshitani 1

1Tohoku University Graduate School of Medicine, Sendai, Japan, 2RITM-Tohoku Collaborating Research Center on Emerging and Reemerging Infectious Diseases, 3Research Institute for Tropical Medicine, Muntinlupa, Philippines

Objectives: Acute respiratory infection (ARI) is still of great concern in public health and significance of viral etiology in severe acute respiratory infections has been acknowledged. It is likely that children suffer from multiple ARIs which must be caused by a variety of viruses in their early life. However, interaction among different respiratory viruses has not been fully discussed. In the present study, we studied if experience of viral infections can change vulnerability of host against subsequent ARIs.

Methods: A prospective cohort study for ARI was conducted in the Philippines, between March 2014 and June 2016. Children aged less than 5 years were recruited. Respiratory symptoms (cough and difficult breathing) were recorded every day. When children visited health facilities there with respiratory symptoms, nasopharyngeal swabs were collected for viral detection by PCR and sequencing and severity of the disease was assessed. We tested association between viral etiology and severity of the present ARI. We also investigated if experience of viral infection can be a risk to acquire subsequent ARIs after the viral infection.

Results: Total of 3,858 children was enrolled and 16,844 ARIs were recorded. Nasopharyngeal swabs were taken from 2,800 ARIs. Among them, 1,193 ARIs were classified as severe ARI by physical examination. Most commonly detected virus was Rhinovirus (RhV, 724) followed by Respiratory syncytial virus (RSV, 376), Parainfluenza virus (PIV, 254), Influenza virus (IFV, 206), Human metapneumovirus (hMPV, 105), Adenovirus (AdV, 67), and Enterovirus D68 (EV-D68, 41). After adjustment for gender, age, nutrition status, and socio-economic status, EV-D68 (adjusted odds ratio, 2.0) and RSV (adjusted odds ratio, 2.4) are found to be associated with severe ARI.

Children who experienced infection with IFV-A, PIV-4, RhV-A, and RhV-C suffered from statistically significant large number of severe ARIs after the infection compared with matched controls. Hazard ratios to acquire subsequent severe ARI after viral infection compared with matched controls were IFV-A (hazard ratio, 2.0), PIV-4 (hazard ratio, 7.2), RhV-A (hazard ratio, 1.6), and RhV-C (hazard ratio, 1.8). Infection with RhV-C increased risk to acquire not only severe but also all subsequent ARI (hazard ratio, 1.4). Hazard ratios for subsequent ARI positive for any respiratory viruses and ARI negative for any of them were larger than 1 among children who experienced infection with RhV-A or RhV-C. In contrast, children who experienced infection with RSV were less susceptible to all ARI thereafter (hazard ratio, 0.83) mainly due to protective effect against infection with the homologous virus (hazard ratio, 0.25).

Conclusion: In this study, we revealed that viral etiology plays a significant role in severity of the present ARI and, more importantly, viral infection affects vulnerability of host against subsequent ARIs.

Keywords: children respiratory infection, Respiratory virus infection, Risk factors

17th International Congress of Virology 120

Abstract Submission

Virus surveillance, epidemiology & Public Health

OR244

A STUDY ON THE ORIGIN OF BOVINE ROTAVIRUS STRAINS DETECTED FROM THE CHILDREN OF THE NASU DISTRICT, TOCHIGI, JAPAN

K. Numazaki*

Objectives: Serious gastrointestinal infection of rotavirus (RV) is usually prevalent during winter months and often seen in infants and young children. While at least 11 G genotypes have been isolated from humans, G1, G2, G3, G4, and emerging G9 are major genotypes of human RV. Although G6 human rotavirus is quite rare, it is the major type among rotaviruses from cattle. In a study of Japanese cows, 59.1% of isolates belonged to G6. Usually, bovine G6 strains were combined with P[5], P[1], and P[11] worldwide. Studies on genotypes of prevalent RV strains are quite important for preventing infection, developing vaccines, and its evaluation. We investigated the characteristics of RV infections of the Nasu district of Tochigi, Japan and to compare to the other region before introduction of rotavirus vaccines.

Methods: We examined the clinical findings in 147 patients who attended to the Department of Pediatrics at International University of Health and Welfare Hospital in Nasu-shiobara City, Tochigi, Japan during the time of April 1, 2008 to March 31, 2010. We analyzed the clinical findings of the 37 patients with a fecal sample positive for RV antigen on VP6, NSP4 gene RT-PCR genotyping. Viral genotypes were determined using rotavirus-positive samples from 27 of these 37 patients.

Results: In 37 cases in VP6, NSP4 gene-based RT-PCR 24 samples were positive and the genotypes were determined as G1P [8] in 5 samples, G3P [8] in 5 samples, G9P [8] in 3 samples, and G6P [9] in 2 samples. Of particular note, we detected G6P [9] which were extremely rare in human beings but common in cattle. KF 17 strain of a patient showed G6 type of bovine strains and P [9] was admitted to reconcile with human strains. Per match ratio in each gene of VP6, VP1, VP2, VP3, and NSP4 showed a high match rate (84.1-86.4%) with DS-1 type. Per match ratio in each gene of VP4, NSP1, NSP3, NSP4 and NSP5 also showed a high match rate (94.2-96.9%) with AU-1 type. Bovine type strain gene segments become reassortant major two genes in these stocks.

Conclusion: In the phylogenetic analysis the gene of a strain KF17 of G6P [9] was located in a human lineage including other human G6 strains. Similarly, all other genes of the strain except for the NSP3 gene were relatively closely related to at least one of the human G6 RVs reported in Europe and the U.S. In a study in Miyagi Prefecture G6P [9] (M72S11) per sample was found from a 2-year-old toddler in 2011. Also G6P [9] have been detected from more than 3 cats of Mie Prefecture. These findings suggest that human G6 RVs which had occurred by reassortment between human and bovine RVs are distributed worldwide, despite low prevalence. G6 RV may occur independently in different areas through reassortment among local strains.

Keywords: None

17th International Congress of Virology 121

Speaker Abstract Submission

Viruses and Cancer

Oncolytic measles virotherapy: preclinical and clinical insights

Kah-Whye Peng, Ph.D. Department of Molecular Medicine, Mayo Clinic, Rochester Minnesota 55905

The live attenuated Edmonston strain of measles virus is a replication competent virus that is being developed for cancer therapy. The virus is engineered to express the human sodium iodide symporter (NIS) to enable noninvasiave longitudinal imaging of the pharmacokinetics of viral replication in the test subject over time. This MV-NIS virus is being investigated as an investigational drug in ovarian cancer, myeloma, head and neck cancer, mesothelioma, breast cancer, malignant peripheral nerve sheath tumor, delivered by intratumoral, intraperitoneal, intrapleural and intravenous routes of administration at Mayo Clinic. We demonstrated for the first time, that intravenously administered measles virus can induce complete regression of disseminated myeloma disease. In addition, we also recently completed a phase 1 clinical protocol whereby mesenchymal stem cells were used as Trojan horses to deliver the oncolytic measles virus to patients with recurrent ovarian cancer as a way to improve virus delivery and to circumvent neutralizing antiviral antibodies. In this presentation, we will elaborate on the preclinical and clinical data obtained so far, and possible mechanism(s) on why some patients might have durable complete responses to systemic measles virotherapy.

17th International Congress of Virology 122

Abstract Submission

Viruses and Cancer

OR245

EPSTEIN-BARR VIRUS INFECTION IS RELATED TO THE DEVELOPMENT AND LOCAL RECURRENCE OF LARYNGEAL CANCER IN TAIWANESE PATIENTS

L.-A. Lee 1,*, C.-G. Huang 2, T.-J. Fang 1, H.-Y. Li 1, T.-C. Chen 3

1Department of Otorhinolaryngology-Head and Neck Surgery, 2Department of Laboratory Medicine, 3Department of Pathology, Linkou-Chang Gung Memorial Hospital, Taoyuan City, Taiwan

Objectives: Epstein-Barr virus (EBV) is an oncogenic virus causing nasopharyngeal cancer and Burkitt's lymphoma. The role of EBV in laryngeal cancer remains ambiguous. This study aimed to examine the effect of EBV infection on disease development and local control among patients with primary laryngeal cancer after standard treatment.

Methods: This study was a prospective case-controlled study matched by age and sex. We recruited 27 patients with newly diagnosed, primary laryngeal cancer and 27 patients with benign laryngeal lesion in the discovery cohort. Surgical specimens were examined by real-time PCR to detect EBV BamH1 gene. Epstein-Barr encoding region (EBER) RNA expression in tissue microarray specimens was detected by in situ hybridization. Tumor markers (c-Myc, EGFR, Bcl-2, p53, p16, pRb, E-cadherin) and host mucosal immune responses (MHC class I, β2–macroglobulin, MHC class II, CD1d, CD3, CD4, CD161) in tissue microarray specimens were determined by immunohistochemistry. Patients with primary laryngeal cancer were followed between surgery and death or up to 24 months. The primary endpoints were the risk of local recurrence and the time to local recurrence. Local recurrence models were validated in an independent validation cohort (n = 15).

Results: We observed a significantly higher BamHI-W DNA positive rate in the primary laryngeal cancer group compared with the benign laryngeal lesion group (48% vs. 18%, P = 0.042; unadjusted odds ratio [OR]: 4.09, 95% CI: 1.19–13.95; P = 0.025, simple logistic regression). Multivariate analysis using a logistic regression model identified BamHI-W DNA (OR, 10.45; 95% CI, 1.53–71.18; P = 0.017), cigarette smoking (OR, 19.55; 95% CI, 1.49–256.69; P = 0.024), Reflux Symptom Index ≥ 13 (OR, 0.01; 95% CI, 0–0.81; P = 0.039), c-Myc (OR, 0.01; 95% CI, 0–0.39; P = 0.016), Bcl-2 (OR, 1.04; 95% CI, 1.01–1.08; P = 0.012), and β2–macroglobulin (OR, 1.04; 95% CI, 1.01–1.08; P = 0.018) were important risk factors for laryngeal cancer. The 2-year local recurrence rate was 32% (n = 7, 95% CI: 11%–54%). We further found a combination of ‘EBV BamHI-W DNA positivity plus EBER nucleus positive index ≥ 0.04%’ was a predictor for 2-year local recurrence (‘positive’ vs. ‘negative’: 75% vs. 19%; P = 0.045, log-rank test). Multivariate analysis using a Cox regression model identified ‘EBV BamHI-W DNA positivity plus EBER nucleus positive index ≥ 0.04%’ (HR, 8.55; 95% CI, 1.26–58.16; P = 0.028)’ and ‘betel nut chewing (HR, 9.22; 95% CI, 1.27–66.82; P = 0.028)’ could significantly predicted 2-year local recurrence after adjustment with ‘P16 (HR, 0.97; 95% CI, 0.94–1.00; P = 0.090)’. Accordingly, we identified 2 distinct risk categories with 2- year local recurrence rates ranging from 11% (≤1 risk factor; n = 20) to 100% (>1 risk factors; n = 7) in the discovery cohort. The validity of the risk stratification was internally confirmed in the validation cohort (P = 0.019).

Conclusion: EBV infections in Taiwanese patients with laryngeal cancer are common and clinically relevant to local recurrence. If independently validated, our composite prognostic stratification comprising EBV infection may be useful for allocating laryngeal cancer patients to risk-adapted therapies.

Keywords: Carcinogenesis, Epstein-Barr virus, Laryngeal cancer

17th International Congress of Virology 123

Abstract Submission

Viruses and Cancer

OR247

ONCOLYTIC VIROTHERAPY POTENTIATED BY PHOSPHATASE INHIBITOR

M. Selman 1 2,*, A. Chen 1, F. Tzelepis 1, J.-S. Diallo 1 2

1Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, 2Faculty of medicine, University of Ottawa, Ottawa, Canada

Objectives: Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics, based on their selective replication and lysis of tumour cells, without causing damage to normal cells. Numerous studies have shown that viral oncolysis, spread and overall efficacy is improved using pharmacological compounds that manipulate the cellular innate anti-viral immune response. A number of viruses have evolved various strategies to antagonize the antiviral activity. For instance, some viral proteins target phosphatases to evade innate immune recognition. Hence, we tested a chemical panel of phosphatase inhibitors (PI) to identify novel compounds that could enhance OV activity.

Methods: A phosphatase inhibitor panel was screened for their ability to enhance of oncolytic rhabdovirus VSVΔ51 in 786- 0 cells, a human renal carcinoma cell line which is highly resistant to infection with VSVΔ51. Promising compounds were tested for cancer specific enhancement of VSVΔ51 in ex vivo tumours and normal organ samples. The mechanism of action was examined by microarray analysis. In vivo anti-tumour efficacy of the compounds alone and in combination with VSVΔ51 was tested in resistant mouse tumour models was also tested.

Results: We identified phosphatase inhibitors compounds that could enhance OV infection in vitro and ex vivo, in resistant tumour cell lines. Furthermore, one phosphatase inhibitor tested increased anti-tumour efficacy in combination with OV in several syngenic tumour models, leading to durable responses in models otherwise refractory to OV and drug alone. Microarray analyses suggest this potentiation occurs by inhibition of the type I interferon signaling pathways and through enhanced immune-stimulation in addition to improved oncolysis.

Conclusion: We show that phosphatase inhibitors compounds can maximize anticancer immunity, and the ability to enhance the growth and spread of oncolytic rhabdoviruses, in addition to OV mediated viral lysis in cancer cells leading to increase efficacy of OV treatment in in vivo models.

Keywords: Interferon signalling, oncolytic virus, Rhabdoviruses

17th International Congress of Virology 124

Abstract Submission

Antiviral immunity

OR248

PD-1+ CD8+ T CELLS PREDICT HEPATITIS B VIRUS CONTROL UPON ANTI-VIRAL THERAPY DISCONTINUATION

L. Rivino 1,*, N. Le Bert 2, U. Gill 3, K. Kunasegaran 1, Y. Cheng 4, D. Tan 2, E. Becht 4, E. Newell 4, P. Kennedy 3, A. Bertoletti 1

1Emerging Infectious Diseases, Duke-NUS Medical School, 2Singapore Institute for Clinical Sciences, A*STAR, Singapore, Singapore, 3Hepatology, Barts and The London School of Medicine & Dentistry, London, United Kingdom, 4Singapore Immunology Network, A*STAR, Singapore, Singapore

Objectives: The clinical management of chronic hepatitis B (CHB) relies exclusively on virological parameters that alone cannot determine in which patients nuclos(t)ide analogue (NUC) treatment can be safely discontinued, without the development of significant hepatic flares (HF). HBV-specific T cell responses are associated with viral control, however current therapies are limited in their ability to restore the functional HBV-specific repertoire and consequently NUC therapy is lifelong in the majority. The aim of this study is to identify immunological biomarkers to predict viral control in CHB patients upon therapy discontinuation.

Methods: PBMC were analysed at 4-weekly intervals prior to and after NUC discontinuation in 19 HBeAg negative, virally suppressed patients. The expression of 41 markers for phenotypic and functional characteristics of T, B & NK cells were studied by CyTOF and HBV-specific T cell responses were assessed by flow cytometry and ELISPOT. NanoString Technology was used to analyse transcriptional regulation of over 600 immune based genes in sorted CD8+ T cells.

Results: Following NUC discontinuation, patients differentiated into 2 groups based on the presence/absence of HF. Using CyTOF, patients with and without HF demonstrated similar immune signatures of lymphocyte subsets. However, patients that controlled HBV in the absence of HF displayed higher frequencies of HBV-polymerase/core specific T cells and increased expression of the inhibitory/co-stimulatory genes PD-1, TIM-3, EGR2, KLRC1 and SLAMF1 on total CD8+ T cells. Notably, PD-1+ CD8+ T cells display a tendency towards higher cytokine producing capabilities in patients without HF, and functional HBV-specific T cells were enriched in the PD-1+ fraction of T cells.

Conclusion: Our data suggests that circulating HBV-polymerase/core specific T cells and PD-1 expression on total CD8+ T cells may represent novel biomarkers to identify patients where NUC therapy can be safely discontinued, without the development of HF. These results further underline the importance of HBV-specific T cells in viral control and the potential to stop NUC therapy in eAg negative CHB patients. We hypothesize that expression of inhibitory co-stimulatory molecules on T cells might be beneficial in a situation of chronic antigenic stimulation, since it might allow PD-1+ virus-specific T cells to persist and exert a partially protective role.

Keywords: Anti-viral therapy, Hepatitis B virus, T cells

17th International Congress of Virology 125

Abstract Submission

Antiviral immunity

OR249

Characterisation of RIG-I-like receptor agonists for dengue virus therapy

V. Ho*, H. Y. Yong 1, L. Dahai 2, K. Fink 3

1Lee Kong Chian School of Medicine (NTU), Nanyang Technological University (NTU), 2Lee Kong Chian School of Medicine (NTU), Nanyang Technological University , 3Singapore Immunology Network (A*STAR), A-STAR, Singapore, Singapore

Objectives: To characterize a RIG-I-like receptor agonist and it's potential application in the area of dengue virus therapy

Methods: - In vitro synthesis of 5’-ppp dsRNA.

- RNA screening with type I interferon bioassay using HEK-293T MX1P-luc cells.

- qPCR for type I interferon production by transfected A549 cells.

- Bioassay for type I interferon production by transfected A549 cells.

- qPCR and bioassay for type I interferon production by transfected U937-DC-SIGN cells.

- DENV-2 infection and flow cytometry analysis on transfected U937-DC-SIGN and A549 cells.

- Isolation of Primary Human Skin DCs

- DENV-2 infection and flow cytometry analysis on transfected Primary Human Skin DCs

- LL171 bioassay for type I interferon production by mice treated with dsRNA.

- Type I interferon bioassay for RLR-expressing HEK-293T cells transfected with 5’-ppp dsRNA.

- Type I interferon bioassay for RIG-I-KO U937-DC cells transfected with 5’-ppp dsRNA.

Results: Using the smallest dsRNA ligand that can activate RIG‐I signaling (OHYr23), we demonstrated that this molecule is capable of priming human epithelial A549 cells and human monocytic U937 cells into an anti‐viral state through type I interferon signaling activation. Modifications to dsRNA sequences through the addition of mismatches in various locations of the stem structure resulted in differences in activation of type I interferon signaling, leading to the identification of OHYr05, a dsRNA ligand more potent than OHYr23 in activating type I interferon response in cell lines as well as inducing these cells into an anti‐viral state. Both OHYr05 and OHYr23 has also been shown to potently inhibit DENV replication in primary human dendritic cells isolated from human skin samples (skin DCs). Both 5’‐ppp dsRNA ligands could induce type I interferon production in mice when injected together with a cationic polymer. Conclusion: Results suggest that OHYr23 and OHYr05 can activate anti‐viral responses and possibly confer short‐term protection (as anti‐viral) against DENV. Future work will involve investigating the signaling pathway involved in this immune activation, as well as potential for these 5’-ppp ligands to be used as vaccine adjuvants.

Keywords: Dengue, Immunology, RIG-I

17th International Congress of Virology 126

Abstract Submission

Antiviral immunity

OR250

NOVEL CONSENSUS DNA VACCINE INDUCES PROTECTIVE IMMUNITY AGAINST ZIKA VIRUS

K. Muthumani*, S. B. Kudchodkar 1, E. L. Reuschel 1, B. D. Griffin, H. Choi 1, K. A. Kraynyak 2, J. N. Maslow, Y. K. Park, L. Humeau 2, N. Y. Sardesai 2, J. J. Kim 2, G. P. Kobinger 3, D. B. Weiner 1

1Vaccine Center, The Wistar Institute, Philadelphia, PA, USA, 2Public Health Agency of Canada, Winnipeg, Canada, 3Inovio Pharmaceuticals, Plymouth Meeting, PA, USA, 4Gene One Life Science Inc., Seoul, South Korea, 5Immunology, Centre de recherche en infectiologie de l'Université Laval, Québec City, Canada

Objectives: Significant concern has been raised over the past two years from the increased global spread of the mosquito- borne flavivirus, Zika (ZIKV). Accompanying this spread has been an increase in cases of the devastating birth defect microcephaly as well as cases of Guillain-Barre syndrome in adults in many affected countries. Additional concerns have been raised by recent findings indicating that ZIKV can also be sexually transmitted which increases the exposure risk for a virus without a vaccine or current therapy.

Methods: Here we describe the development of a novel synthetic DNA consensus antigen-based vaccine targeting the pre- membrane + envelope proteins of ZIKV

Results: Following construct expression confirmation, we found that mice and non-human primates generated ZIKV- specific cellular and humoral immune responses, including neutralization antibody responses, following electroporation- enhanced delivery of the vaccine. Immunization of mice lacking receptors for interferon (IFN)-α/β immunization with this DNA vaccine provided 100% protection against weight loss, death, and pathology in brain tissue following ZIKV challenge. Additionally, vaccinated rhesus macaques were protected and failed to demonstrate any clinical signs of diseases subsequent viral challenge.

Conclusion: This initial study in a pathogenic mouse and in NHP challenge model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control in humans. This represents the first ZIKV vaccine approved for human trials.

Keywords: DNA vaccine, Zika virus

17th International Congress of Virology 127

Speaker Abstract Submission

Antiviral immunity

GLYCOSYLATION OF C-TERMINAL TAIL OF IMMUNOGLOBULIN A CONFERS DIRECT ANTIVIRAL ACTIVITY AGAINST ENVELOPED SIALIC ACID-BINDING VIRUSES

M. Maurer 1, L. Meyer 2, M. Bianchi 3, H. Turner 3, V. Orlowski 2, N. Le 4, A. Wyrzucki 2, A. Ward 5, M. Crispin 4, L. Hangartner* 1Brain Research Institute, 2Institute of Medical Virology, University of Zurich, Zurich, Switzerland, 3Immunology and Microbial Science, The Scripps Research Institute, La Jolla, United States, 4Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 5Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States

Objectives: When the impact of the antibody isotype on the in vitro neutralizing activity of human heterosubtypic antibodies against influenza A viruses was investigated, it was found that heterosubtypic antibodies neutralize influenza A virus up to 1000-fold more potently when expressed as IgA antibodies compared to IgG1. The responsible molecular mechanism was therefore determined, and its applicability to other viruses investigated.

Methods: IgA1/IgG1 chimeric and glycosylation knock-out variants of IgA1 antibodies were used to determine the origin of the more potent neutralization. IgA1 antibodies were then expressed in glycosylation deficient cell lines, or treated with glycosylases, to determine the responsible molecular moiety. The findings were then applied to other antibodies and the other sialic acid binding viruses, such as Newcastle disease virus.

Results: It was found that sialilation of the N-linked glycan in the C-terminal tail of IgA molecules directly interferes with receptor binding of influenza A viruses. When the glycosylation of this site was analyzed in detail, it was found that not only alpha 2,6'-linked but also 2,3'-linked sialic acid can be found in IgA purified from human saliva. The inhibitory activity of the C-terminal sialic acid does not require Fab-mediated binding, as non-specific IgA antibodies also inhibit infection of sialic-acid binding viruses at higher concentrations. Conclusion: The direct antiviral activity of the C-terminal tail of IgA may constitute an innate defense mechanism against sialic acid binding viruses, and perhaps other pathogens. It may also be harnessed to potentiate the efficacy of antibodies used for passive immunization.

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Keywords: heterosubtypic antibodies, IgA, Influenza A virus

17th International Congress of Virology 128

Speaker Abstract Submission

RNA-protein interactions

IN-GEL PROBING TO EXAMINE RNA STRUCTURES WITHIN HIV RNA-PROTEIN COMPLEXES OF VARYING STOICHIOMETRY

Z. Long 1, J. Zeng 1, A. Lever 1 2, J. Kenyon 1 3 4,* 1Medicine, University of Cambridge, Cambridge, United Kingdom, 2Medicine, 3Microbiology and Immunology, National University of Singapore, Singapore, Singapore, 4Homerton College, University of Cambridge, Cambridge, United Kingdom

Objectives: To develop a technique to examine structural changes occurring in individual RNA species within RNA- protein complexes of varying stoichiometry. To use this technique to better understand HIV-1 packaging.

Methods: High-throughput SHAPE (selective 2’OH acylation analysed by primer extension) has revolutionized RNA secondary structure probing. SHAPE reagents acylate the 2’OH according to the flexibility of the backbone: where nucleotides are Watson-Crick paired the backbone is rigid and refractory to acylation; where nucleotides are single- stranded the backbone binds the SHAPE reagents readily. Primer extension assays are then used to map the relative amounts of SHAPE reagent bound at each nucleotide position along the backbone and this information is fed into modeling algorithms, alongside free energy parameters, to produce structural models. Capillary sequencing using fluorophore-labelled primers is used to examine the primer extension assays and make the technique high-throughput. In 2013 we published a novel technique based upon high-thoughput SHAPE: “in-gel SHAPE” (NAR 2013, Oct;41(18):e174). This technique separates different RNA conformers within a mixed sample using native PAGE, and then probes their structures whilst still inside the gel matrix, enabling analysis of each conformer individually- something that was not previously possible. RNAs are recovered from the gel using electroelution and are then used in primer extension assays, as in the high-throughput SHAPE technique. We used in-gel SHAPE to examine the structures of the HIV-1 (human immunodeficiency virus type-1) packaging signal RNA in its monomeric and dimeric forms and to show that a structural switch occurred between the two. Now, we have developed this technique further to examine the structures of RNAs in RNA-protein complexes of varying stoichiometry.

Results: Using HIV-1 packaging signal RNA- Gag protein interactions as a model we show that we can follow the RNA structural changes occurring as RNAs interact with each other and with successive protein molecules. When binding to monomeric packaging signal RNA, the Gag protein begins to push the structure towards that of the 'hemi dimer'. When binding to dimeric packaging signal RNA, Gag binding continues to stabilise its structure. The nucleocapsid domain of Gag in isolation does not show these effects. Conclusion: In-gel SHAPE can be used to follow RNA structural changes that occur when RNAs interact with each other and with proteins, gaining valuable insights into molecular rearrangements occuring during the HIV-1 lifecycle.

Keywords: HIV-1, packaging, RNA-protein interactions

17th International Congress of Virology 129

Abstract Submission

RNA-protein interactions

OR251

STRUCTURAL BASIS OF VIRAL RNA-DEPENDENT RNA POLYMERASE CATALYSIS AND TRANSLOCATION

B. Shu 1, P. Gong*

1N/A, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

Objectives: RNA viruses encode a unique class of RNA-dependent RNA polymerases (RdRPs) to carry out their fully RNA- based genome replication and transcription. Although the chemical nature of nucleotide addition is essentially shared by all nucleic acid polymerases, the structural and mechanistic details taken by each polymerase class differ to various extents. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain based active site closure mechanism, and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. In this study, we seek to identify novel NAC states using crysllographic methods.

Methods: Using the RdRP from another human enterovirus, enterovirus 71 (EV71), we obtained a crystal form of RdRP elongtion complex (EC) that allows multiple NACs to occur in the crystal lattice. A time-resolved strategy and different NTP combinations were used in subsequent EC crystal soaking experiemnts.

Results: Here we report seven crystal structures of enterovirus 71 RdRP elongation complex at 2.5-2.8 Å resolution. In these structures the polymerases are poised at various distinct stages to reveal mechanistic details of initial NTP binding, key amino acid side chain conformational switches during active site closure, and in particular the post-catalysis movement of the RNA duplex on the way to vacate the active site for the next NAC.

Conclusion: Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs as compared to other processive polymerases.

Keywords: crystal structure, nucleotide additino cycle, RNA-dependent RNA polymerase

17th International Congress of Virology 130

Abstract Submission

RNA-protein interactions

OR252

IDENTIFICATION OF POTATO VIRUS A COAT PROTEIN AS AN IMPORTANT REGULATOR OF VIRAL TRANSLATION AND REPLICATION

K. Mäkinen*, A. Lõhmus 1, J. Besong-Ndika 1, A. Hafren 1, K. Ivanov 1

1Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland

Objectives: In addition to encapsidation of the viral genome and its protection against degradation, many non-structural functions have been attributed to viral coat proteins (CPs). Potyviruses are a large family of economically important positive- stranded plant RNA viruses. In the current study we sought to understand the molecular mechanisms underlying the CP- mediated regulation of translation and replication of potato virus A (PVA; a potyvirus) and the interplay between CP, host proteins and viral RNA in these processes.

Methods: The infectious cDNA of PVA allows readily insertions into several positions within the genome without compromising the infectivity. This property was instrumental for our in vivo studies. We quantitated viral gene expression and translation by virus-derived Renilla luciferace and viral RNA by qRT-PCR. We purified viral ribonucleoprotein (RNP)- complexes via affinity tags fused to viral proteins and subjected them to mass spectrometry identification. Many advanced techniques in biochemistry, molecular biology and cell biology were used to understand the molecular mechanism how CP exerts its function on PVA gene expression.

Results: Our results suggest that PVA CP inhibits viral RNA translation through CP translated from viral RNA in cis. The action of two host chaperons, heat shock protein 70 and CP-interacting protein (CPIP, a HSP40 family member), was found to relieve the translation inhibition likely by detaching CP from viral RNA and targeting it to degradation. The RNA-binding property of PVA CP and its capacity to inhibit translation were both found to be regulated by host kinase CK2-mediated CP phosphorylation. We found that both the functional chaperon system and CP phosphorylation were essential for PVA replication. High CP concentration in cells was found to overpower the function of CPIP and stop PVA gene expression.

Conclusion: We have shown that PVA CP and the host proteins CK2, HSP70 and CPIP are essential for PVA replication and that CP inhibits translation. We propose that the transient translational block formed by the non-phosphorylated CP is essential for replication complex formation and contributes to the switch of PVA RNA as a template for translation to one for replication. We further propose that later in the infection, when CP inhibits viral RNA translation through co-translational interactions between excess CP accumulated in trans and CP translated from viral RNA in cis, viral RNA could be sequestered from translation and specifically selected for encapsidation.

Keywords: coat protein, Potyvirus, viral translation and replication

17th International Congress of Virology 131

Abstract Submission

RNA-protein interactions

OR253

INVESTIGATING THE MECHANISM OF AUF1 NEGATIVE REGULATION OF PICORNAVIRUS INFECTIONS

W. Ullmer*, B. Semler 1

1Microbiology and Molecular Genetics, University of California Irvine, Irvine, United States

Objectives: Picornaviruses such as poliovirus, coxsackievirus B3 (CVB3), and encephalomyocarditis virus (EMCV) are (+) ssRNA viruses that replicate in the cytoplasm of infected cells through the actions of both viral and host proteins. Replication of these viruses requires nuclear proteins that are re-localized to the cytoplasm during infection following cleavage of nucleoporins by viral proteinases. Mis-localization of host proteins that have a negative impact on virus replication is an unintended consequence of the disruption of nucleocytoplasmic trafficking. The host mRNA decay protein, AUF1, is an example of a negative regulator that is re-localized during infection by several picornaviruses. Identified from an RNA affinity screen as binding to the 5’ noncoding region (NCR) of poliovirus RNA, it was later discovered that AUF1 knockdown or knockout in human or mouse cells results in increased replication of the picornaviruses poliovirus, CVB3, enterovirus 71, and rhinovirus 1a. Evidence that AUF1 restricts picornavirus replication through inhibition of viral translation or by promoting decay of viral RNA has been previously reported. Our work seeks to clarify the mechanism by which AUF1 acts as a negative regulator of poliovirus and CVB3 infection.

Methods: Single cycle growth analyses for poliovirus, CVB3, and EMCV were performed in HEK-293 cells stably expressing tetracycline-inducible control or AUF1-targeting shRNAs. Viral translation was measured using recombinant poliovirus or CVB3 encoding Renilla luciferase immediately following the 5’ NCR. Translation driven by the viral internal ribosome entry site (IRES) in the 5’ NCR was measured using reporter RNAs encoding the poliovirus or CVB3 5’ NCR upstream of firefly luciferase.

Results: We discovered that AUF1 negatively regulates replication of EMCV in addition to poliovirus and CVB3. While AUF1 is known to promote the decay of mRNAs following binding to the 3’ NCR, we found that replication of a mutant poliovirus lacking its 3’ NCR increased in AUF1 knockdown cells similar to wild type virus, indicating that AUF1 does not negatively regulate poliovirus replication through the 3’ NCR. Instead, we found that AUF1 knockdown resulted in increased translation of full-length poliovirus and CVB3 RNA, as well as increased translation of a poliovirus or CVB3 5’ NCR reporter RNA.

Conclusion: These data suggest that AUF1 negatively regulates poliovirus and CVB3 replication through inhibition of viral IRES-driven translation and not through destabilization of viral RNA.

Keywords: AUF1, picornavirus, poliovirus

17th International Congress of Virology 132

Speaker Abstract Submission

Viruses and microRNA

ELEVATED EXPRESSION OF THE MIR-125B-5P TARGETING TO MULTIPLE SIGNALING PATHWAYS CONTRIBUTES TO JAPANESE ENCEPHALITIS VIRUS PERSISTENT INFECTION

R.-Y. Chang 1,*, R. Y.-L. Wang 2, C.-W. Huang 1, K.-N. Tsai 1, Y.-S. Chen 1 1Department of Life Science, National Dong Hwa University, Shoufeng, 2Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

Objectives: MicroRNAs (miRNAs) have participated versatile roles in multiple biological processes. How miRNAs may have involved in the establishment of persistent infection of flaviviruses remains mostly unknown. This study aimed at identification and characterization of miRNAs and miRNA-mediated signaling in the establishment of persistent infection of Japanese encephalitis virus (JEV).

Methods: We implemented NanoString nCounter technology to obtain the miRNA expression profile in comparison between the acute and persistent infection of JEV. The miR-125b-5p was identified and further confirmed by Northern blot analysis and RT-qPCR. Function of the elevated expression was characterized.

Results: The expression level of miR-125b-5p was significantly increased (approximate 8 folds) in persistent infected cells. As soon as the cells established persistent infection, the significant high expression of miR-125b-5p was readily observed. Transfecting of miR-125b-5p at low dosage (< 5 nM) slightly increased cell viability, while high concentration at 25 nM significantly reduced cell viability indicting that miR-125b-5p has dose effect. Transfecting of miR-125b-5p at 0.5 nM did not rescue virus-induced cell death but significantly reduced virus titers. Six potential targets of miR-125b-5p were predicted using bioinformatics tools. These targets were down regulated in persistently infected cells and were direct targets of miR-125b-5p as evidenced by luciferase reporter assay. Conclusion: Our results demonstrated that miR-125b-5p up regulated in persistently infected cells and inhibited virus replication presumably by targeting to several host regulators in signal transduction pathways that could lead to persistent infection. This is the first report that characterized potential roles of miRNAs in the establishment of JEV persistent infection.

Keywords: miR-125b-5p, persistent infection

17th International Congress of Virology 133

Abstract Submission

Viruses and microRNA

OR254

HOST MICRORNA MIR-181D PLAYS A NEGATIVE REGULATORY ROLE IN THE ENTEROVIRUS 71 REPLICATION BY TARGETING FKBP1A

W.-F. Tang*, J.-T. Horng

Objectives: Enterovirus A 71 (EV A71) is a member of the genus Enterovirus within the family Picornaviridae. EV A71 infection causes hand-, foot, and- mouth diseases and is occasionally associated with acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. The mechanism underlying EV A71 replication in the cell is still largely unknown, and few studies have addressed the relationship between cellular miRNAs and EV A71 infection. We are interested in the response of miRNA to EV A71 infection and the biological function of their target genes in EV A71 replication.

Methods: We employed microRNA array analysis to understand the roles of cellular microRNAs during EV A71 infection. The differentially expressed miRNAs were verified by qPCR. To investigate the role of these miRNAs in viral replication, miRNA mimics or inhibitors were transfected into RD cells, and the effect on the synthesis of viral proteins, viral RNA, and viral titers was studied. To identify the host cellular factors involved in EVA71replication, we used online prediction databases such as TargetScan (http://www.targetscan.org/) and miRanda (http://www.microrna.org/microrna/home.do) combined with literature searches to identify potential candidate target genes. A 3’UTR reporter assay was performed to confirm candidate gene authenticity. To investigate the importance of the candidate genes during EVA71replication, siRNA or overexpression plasmid was introduced into RD cells and the virus replication was analyzed.

Results: We observed 5 downregulated miRNAs and 2 upregulated miRNAs in response to EV A71 infection at 10 h p.i. We identified a cellular miRNA, miR-181d, the expression of which was downregulated by EV A71 infection in a time- dependent manner. In miR-181d mimic-transfected cells, we found that the levels of the viral protein, RNA, virus progeny, and EV A71 replicon were substantially decreased, and knockdown of miR-181d increased virus replication. FKBP1A is a miR-181d target gene. The viral replication increased in FKBP1A-knockdown cells and decreased in FKBP1A- overexpressed cells, indicating that the presence of FKBP1A was unfavorable for EV71 replication. The role of FKBP1A in EV A71 replication was further analyzed, and it was found that the TGF-beta signaling pathway was activated in response to EV A71 infection and the p-SMAD 2/3 levels were increased in FKBP12-knockdown cells. The phosphorylated form of SMAD 2/3 was partially colocalized with the replication complex.

Conclusion: 1. Virus controls the expression of miRNA to help virus replication.

2. FKBP1A is a miR-181d target gene.

3. FKBP1A might regulate EV A71 replication by modulating the TGF-beta signaling cascade.

Keywords: enterovirus A71, FKBP1A, miR-181d

17th International Congress of Virology 134

Late breaking abstract

Viruses and microRNA

OR255

ROLES OF MIR-122 IN HEPATITIS B VIRUS (HBV) REPLICATION

T. Tan May Chin*

Objectives: MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression post-transcriptionally. MiRNAs have been shown to affect viral infection and replication including that of hepatitis B virus (HBV). It is estimated that approximately 240 million people are chronically infected with HBV worldwide and are at enhanced risk of developing hepatocellular carcinoma (HCC). miR-122, a liver-specific miRNA, has been reported to regulate HBV replication either directly or indirectly. This talk aims to delineate the role of miR-122 in HBV replication and reactivation.

Methods: Prediction of putative binding sites for miR-122 on HBV genomes was carried out using RNAHybrid and the sites were further verified experimentally using the relevant luciferase report constructs. Effects of overexpression of miR-122 on HBV replication was examined using 2.2.15 cells. Analysis of the miR-122 binding sites was also carried out using HBV sequences from chronic hepatitis B e-negative patients.

Results: Possible binding sites of miR-122 on HBV genomes in addition to the reported sites were predicted using RNAHybrid. Over-expression of miR-122 significantly down-regulated extracellular HBV DNA levels in 2.2.15 cells. Moreover, the interaction of miR-122 with the HBV transcripts at the predicted target binding sites was observed by dual luciferase reporter assay. Analysis of HBV sequences of chronic hepatitis B patients (HBe negative) with and without reactivation at the predicted sites suggests that in some patients, specific point mutations could weaken the miR-122 interaction with HBV viral transcripts and thus may enhance HBV replication during reactivation.

Conlusion: Overall, the results suggest that miR-122 may inhibit HBV replication by binding directly to the HBV transcripts, and weaker binding of miR-122 with mutated HBV sequences at two of these sites may enhance viral replication during reactivation. The various other means that the virus uses to counter the high expression of miR-122 in hepatocytes will also be discussed.

Keywords: None

17th International Congress of Virology 135

Abstract Submission

Viruses and microRNA

OR256

A PRIMARY TONSILLAR CRYPT CELL LINE FOR IN VITRO MODELLING OF ENTEROVIRUS INFECTION AND PATHOGENESIS

N. Min*

Objectives: Human enterovirus type 71 (EV71) along with Coxsackievirus A16 (CA16) and Coxsackievirus A6 (CA6) are well characterized major causative agents of hand-foot-mouth disease (HFMD), belonging to human Enterovirus Species A of the family Picornaviridae. Generally, HFMD causes mild clinical manifestations such as fever and blisters in mouth, on palms or on sole of the feet; occasionally, fatal cases were also reported presenting meningitis and polio-like flaccid paralysis. Currently, little information is known on pathogenesis of EV71, CA16 and CA6 in human. However, squamous epithelium lining of tonsillar crypt cell was recently being identified as possible reservoir during EV71 infection supporting active viral replication and faecal-oral spreading. Here, we explored the mechanism of infection in human primary tonsillar crypt cell (HtEpic) with EV71, CA16 and CA6 viruses together with in vitro role of miRNA during infection.

Methods: In this study, we performed in vitro characterization of the HtEpic cell line during EV71 and CA16 infection. Active replication of the virus was confirmed by growth kinetic studies followed by plaque assay. RNA and protein accumulation were also observed across multiple time points via real-time polymerase chain reaction, western blotting and indirect immunofluorescence assay (IFA). Moreover, we also conducted next generation sequencing (NGS) of HtEpic miRNome during EV71 and CA6 infection for 6 and 12-hour post infection (HPI) time points.

Results: Generally, viral titer in HtEpic cell line peaked at 24 HPI for EV71 (2.1x106 PFU/ml) and CA16 (1.7x106 PFU/ml) viruses where CA6 (1.3 x104 PFU/ml) peaked at 12 HPI. Our real-time PCR unveiled active RNA strand synthesis for all three viruses across 48-hour time point. VP3 protein cleaved from the polyprotein were also detected in increasing manner from 12 to 24 HPI. Additionally, through IFA, punctate signal of enterovirus dsRNA was observed at the same time points. Furthermore, NGS profiling of miRNome during infection revealed miRNA involving in an array of various cellular processes being dysregulated.

Conclusion: We established for the first time, a primary cell line model that shows susceptibility to EV71, CA16 and CA6 infections. The optimal cell line may serve as a valuable tool in better understanding enterovirus mechanism of infection. Furthermore, the primary cell line could facilitate drug screening experiments as well as host factor interaction studies as proven by our miRNome screen.

Keywords: Enterovirus, miRNA, Primary Cell

17th International Congress of Virology 136

Abstract Submission

HIV and Retroviruses

OR257

ALIX AND THE GENOMIC RNA PACKAGING SIGNAL OF HIV-1 COOPERATE TO OPTIMIZE VIRUS MATURATION AND RELEASE

A. Lever*, C. Hellmund 1, B. Meng 1

1Medicine, University of Cambridge, CAmbridge, United Kingdom

Objectives: During and after budding of HIV-1 the structural polyprotein Gag undergoes cleavage by the viral protease in a precisely coordinated manner leading to maturation of the viral core. Mutation of an RNA stem loop (SL1) in the 5 untranslated region of the genome involved in dimerisation and packaging causes a delay in the final step of proteolytic processing of Gag. We sought to understand the connection between the RNA processes of genome encapsidation and the maturation of the viral particle

Methods: We analysed mutants in SL1 and the neighbouring packaging motif SL3 to see if they all caused defects in Gag processing and whether this related to genomic RNA dimerisation as has been shown in HIV 2. We investigated the putative linkage between the ESCRT related protein ALIX, which has been shown to be important in the viral budding process, and the Gag protein which has been suggested to involve RNA. We investigated how previously published mutants in Gag, that compensate for SL1 mutation related defects in Gag processing, influence viral budding.

Results: We found that both SL1 and SL3 mutants cause budding defects typical of 'late domain' defects seen when domains of Gag interacting with ESCRT proteins are mutated. in HIV 1 Gag processing was not linked to genome dimerisation. We found that an SL1 mutation reduces the ability of ALIX to rescue the budding defect seen when interaction of Gag with the ESCRT protein TSG101 was disrupted. The previously described comepnsatory mutants in Gag were found to cause a general acceleration of Gag processing but retain the requirement for intact late domains.

Conclusion: These data suggest tha the viral genomic RNA (gRNA) is involved in regulating virus budding and maturation. We propose a model whereby gRNA bridges Gag and ALIX, acting as a checkpoint to initiate processing and release of viable virions containing the viral genome

Keywords: ALIX, HIV, Packaging signal

17th International Congress of Virology 137

Abstract Submission

HIV and Retroviruses

OR258

CONFORMATIONAL STATES OF HIV-1 GP41 REVEALED BY CYSTEINE SCANNING MUTAGENESIS

A. Khasawneh 1 2 3,*, C.-S. Lay 3 4, A. Laumaea 3 5, H. Drummer 2 3 5, P. Poumbourios 2 3

1Department of Basic medical sciences, Faculty of medicine, Hashemite University, Al Zarqa, Jordan, 2Department of Microbiology, Monash University, Clayton VIC 3168, 3Virus Fusion Lab, Burnet Institute, Prahran VIC 3004, 4Department of Biochemistry and Molecular Biology, Monash University, Clayton VIC 3168, 5Department of Microbiology and Immunology, The University of Melbourne, VIC 3010, Australia

Objectives: The binding by HIV-1 gp120 to CD4 and a chemokine receptor (CCR5 or CXCR4) activates the membrane fusion glycoprotein, gp41. The membrane fusion function of gp41 involves the initial refolding of its ectodomain into a prehairpin intermediate, which is believed to largely comprise an extended trimeric coiled coil with terminal hydrophobic sequences that tether the cell and viral membranes. Further refolding produces the trimer of hairpins, which apposes the membrane-interactive termini leading to virus-cell membrane fusion and nucleocapsid penetration. In this study, we examined gp41 conformational states using cysteine-scanning mutagenesis.

Methods: Cysteine residues were substituted into central a and d positions of the predicted central coiled coil domain of the prefusion conformation of gp41 (i.e. prior to activation by gp120-receptor interactions) and recombinant models of the prehairpin intermediate and trimer of hairpins based on maltose binding protein-gp41 chimeras expressed in E. coli. The presence of coiled coil structure was inferred from the ability of Cys substitution mutants to form interprotomer disulfide bonds. Disulfide bond formation was monitored by SDS-PAGE in the presence and absence of reducing agents.

Results: We found that the introduction of a Cys residue at position 538 in the N-terminal polar segment of gp41 led to efficient interprotomer disulfide formation in the recombinant models of the prehairpin intermediate and trimer of hairpins but not in the context of prefusion gp41. By contrast, mutations at other positions did not lead to disulfide formation. The N- terminal polar segment of gp41 mediates functionally important hydrophobic packing interactions with the C-terminal membrane proximal ectodomain region (MPER) of gp41 in the trimer of hairpins conformation. By using mutant recombinant proteins, we found that the T538C disulfide formed independently of polar segment-MPER interactions, indicating that the coiled coil extends into the polar segment in an MPER-independent manner.

Conclusion: We infer a model for the conformational states of gp41 that are associated with membrane fusion function whereby the coiled coil is propagated in an N-terminal direction to encompass the polar segment in the prehairpin intermediate. These changes would propel the fusion peptide, which is N-terminal to the polar segment, towards the target cell membrane for insertion. The extended coiled coil structure provides a packing surface for the C-terminal helical region and adjacent MPER, forming the trimer of hairpins. These studies extend our understanding of the structural changes in gp41 during fusion and reveal new targets for antiviral development.

Keywords: Cysteine replacement mutagenesis , HIV-1 gp41, Prefusion transmembrane glycoprotein

17th International Congress of Virology 138

Abstract Submission

HIV and Retroviruses

OR259

OPTIMAL HIV-1 UNCOATING, REVERSE TRANSCRIPTION AND REPLICATION REQUIRES INTERACTION BETWEEN THE STRICTLY CONSERVED E300 HIV-1 RT RESIDUE AND CELLULAR EEF1A

D. Rawle 1 2,*, D. Li 2, J. Swedberg 3, D. Soares 4, D. Harrich 2

1School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, 2Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, 3Institute for Molecular Biosciences, University of Queensland, St Lucia, Australia, 4MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

Objectives: HIV-1 is an enveloped virus containing a viral core structure coated with capsid protein, which “uncoats” in the cytoplasm by a poorly understood mechanism. The core contains the viral genomic RNA and viral enzymes required to synthesise viral DNA by reverse transcription and integrate it in the host chromosomes, however cellular factors are also required. We have previously reported that cellular eukaryotic elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 RT and this is crucial for late stage HIV-1 reverse transcription. The objectives of this study were to better define how HIV-1 RT is interacting with eEF1A by identifying surface exposed RT residues that facilitate the direct interaction with eEF1A, and determine whether this interaction was important for optimal HIV-1 uncoating kinetics and replication.

Methods: Mutant recombinant HIV-1 RT was analysed by co-immunoprecipitation and NanoBRET (Promega) to identify surface exposed RT mutations that are important for interaction with eEF1A. These mutations were inserted into virus and characterised for in vitro RT enzymatic activity by standard colorimetric assay, and reverse transcription kinetics in cells by nevirapine addition assay. Capsid uncoating kinetics were tracked using a cyclosporine A washout assay, and virus replication was monitored by measuring HIV-1 CAp24 by ELISA in the supernatant of infected human T cells.

Results: We identified the first surface exposed residue of HIV-1 RT that is important for interaction with cellular eEF1A, the strictly conserved E300 residue in an acidic patch of the thumb domains alpha-J helix. Using complementary assays the E300R mutation resulted in reduced RT interaction with eEF1A, without affecting in vitro RT catalytic activity. We show that using HIV-1 encoding an RT protein with reduced interaction with eEF1A (E300R), uncoating and reverse transcription are delayed and less efficient, and a strong defect in replication in human CD4+ T cells was observed.

Conclusion: The interaction of the HIV-1 RT thumb domain acidic patch with cellular eEF1A is important for optimal HIV-1 uncoating, reverse transcription and replication in CD4+ T cells. Furthermore, eEF1A has several surface exposed basic patches that could support interaction with RT through hydrogen bonding or a salt bridge. This RT acidic patch may be an attractive and completely novel drug target for development of a new class of RT:eEF1A interaction inhibiting antivirals to target early replication stages of HIV-1.

Keywords: eEF1A, HIV-1, Uncoating

17th International Congress of Virology 139

Abstract Submission

HIV and Retroviruses

OR260

INHIBITORY ACTIVITY OF A NEW ALKALOID, GLOBOSPIRAMINE, IN LATENTLY HIV INFECTED CELLS

M. S. De Jesus 1,*, A. P. Macabeo 1, J. D. Ramos 1, T. Okamoto 2

1College of Science and the Graduate School, University of Santo Tomas, Manila, Philippines, 2Department of Cellular Molecular Biology, Nagoya City University, Nagoya, Japan

Objectives: There is a growing number of HIV exhibiting resistance to some of the commonly used drugs for antiretroviral therapy. Screening natural products is one way of identifying candidate anti-HIV drugs that will address this concern of drug resistance. Preferably, the mode of action should suppress HIV transcription if not eliminate the virus. In this study, three indole alkaloids isolated from an endemic Philippine plant, Voacanga globosa, was investigated for its capability to suppress HIV transcription using simple experimental assays for screening these compounds.

Methods: The purified compounds, globospiramine, vobtusine and deoxyvobtusine, were subjected to WST-1 cytotoxicity assay and cytotoxic concentration at 50 % (CC50) was determined. Compounds at different concentrations were then introduced to latently HIV infected OM10.1 and J-Lat cells in the presence of 1-5ng/mL of TNF (tumor necrosis factor). After 3 days of incubation, supernatants were harvested and tested for the HIV p24 antigen ELISA assay. Compounds that showed inhibitory action were introduced to 293 cells in the presence of 5 ng/mL for transfection-luciferase assay using specific promoter plasmids: HIV LTR-luc, NFkB-luc and mutant kB-luc. Flavopiridol was used as positive control.

Results: Globospiramine at 500nM (IC50) inhibited TNF-induced HIV replication as measured in HIV p24 antigen levels in ELISA. Based on the cytotoxic and inhibitory concentration at 50%, globospiramine exhibited a therapeutic efficiency of 3.18 in OM10.1 and J-Lat cells. Based on the transfection-luciferase assay, globospiramine was found to suppress HIV transcription by inhibiting activation of HIV-LTR by cellular transcription factors including NFkB. Other compounds did not show inhibitory action.

Conclusion: Among the 3 compounds, only globospiramine, a new spirobisindole alkaloid, showed the potential to suppress HIV transcription but with some cytotoxicity. With further studies on this alkaloid, this is a good candidate for novel anti-HIV drug.

Keywords: alkaloid, anti-HIV, inhibitory

17th International Congress of Virology 140

Virology

Poster Presentation

17th International Congress of Virology 141

100 Years of Bacteriologic

17th International Congress of Virology 142

Abstract Submission

100 Years of Bacteriologic PO600

A NEW PHIKMVVIRUS INFECTING RALSTONIA SPP. DISPLAYS EPS-DEGRADING PROPERTIES A. D. S. Xavier 1, F. P. Silva 1, P. M. P. Vidigal 2, T. T. M. Lima 1, F. O. Souza 1, P. Alfenas-Zerbini 1,* 1Microbiology, 2Núcleo de Análise de Biomoléculas, UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, Brazil

Objectives: Bacterial wilt caused by Ralstonia spp., a soil-borne Gram-negative bacteria, is considered one of the most important plant diseases in tropical and subtropical regions of the world. A large number of bacteriophages capable of lysing or physiologically reprograming cells of Ralstonia spp. have been reported in Asia. Despite the potential use of these organisms in the management of bacterial wilt, information on viruses that infect Ralstonia spp. is nonexistent in the Americas. The objectives of this study were to isolate and characterize Brazilian native viruses capable of infecting Ralstonia spp.

Methods: Soil samples were collected in tomato fields in areas with incidence of bacterial wilt, specifically from rhizospheric soil of symptomatic and asymptomatic plants were used as source material and screened for the presence of bacterial virus by standard soft agar overlay method using R. solanacearum RSB1 as host. Single lysis plaques were picked, propagated and purified. Morphological characterization of the viral particle was performed by transmission elctron microscopy. The total nucleic acid was isolated from purified virions by phenol-chloroform extraction and the genome was sequenced. Open reading frames (ORFs) in the viral genome were annotated and analyzed using GeneMark.HMM, PRODIGAL and ORF Finder. Phylogenetic trees were constructed using Bayesian inference. The host specificity was evaluated against a collection of isolates of Ralstonia spp. and other bacterial species by plaque assay using the standard soft agar overlay method.

Results: Soil samples were collected in tomato fields in areas with incidence of bacterial wilt, specifically from rhizospheric soil of symptomatic and asymptomatic plants were used as source material and screened for the presence of bacterial virus by standard soft agar overlay method using R. solanacearum RSB1 as host. Single lysis plaques were picked, propagated and purified. Morphological characterization of the viral particle was performed by transmission elctron microscopy. The total nucleic acid was isolated from purified virions by phenol-chloroform extraction and the genome was sequenced. Open reading frames (ORFs) in the viral genome were annotated and analyzed using GeneMark.HMM, PRODIGAL and ORF Finder. Phylogenetic trees were constructed using Bayesian inference. The host specificity was evaluated against a collection of isolates of Ralstonia spp. and other bacterial species by plaque assay using the standard soft agar overlay method.

Conclusion: We describe RVphiAP1, a novel species in the genus Phikmvvirusi infecting Ralstonia spp. Its lytic arsenal reveals a robust mechanism of pathogenesis that highlights the biotechnological potential of viral peptides for the management of bacterial wilt.

Keywords: bacterial virus, Cell lysis, plant pathogenic bacteria

17th International Congress of Virology 143

Abstract Submission

100 Years of Bacteriologic PO601

ANALYSIS OF ADSORPTION OF PHAGES, S13' AND S24-1, BELONGING TO THE FAMILY PODOVIRIDAE GENUS P68VIRUS, USING STAPHYLOCOCCUS AUREUS STRAINS WITH DIFFERENT GLYCOSIDIC LINKAGE PATTERNS OF WALL TEICHOIC ACIDS

J. Uchiyama 1,*, M. Taniguchi 1, K. Kurokawa 2, I. Takemura-Uchiyama 1, T. Ujihara 3, H. Shimakura 1, Y. Sakaguchi 4, H. Murakami 1, M. Sakaguchi 1, S. Matsuzaki 3 1School of Veterinary Medicine, Azabu University, Kanagawa, 2Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki, 3Kochi University, Kochi, 4Kitasato University School of Medicine, Kanagawa, Japan

Objectives: One of the important therapeutic phage groups against Staphylococcus aureus infections is family Podoviridae genus P68virus. α- and/or β-O-glycosidic linkages of wall teichoic acids (WTAs) on the cell walls are considered to be important for adsorption of this phage group. We have reported the members of the P68virus genus, phages S13’ and S24- 1, which the genes for receptor-binding proteins (RBPs) are slightly varied on their genomes. In this study, the associations of genetic variation and phage adsorption were studied using phages S13’ and S24-1.

Methods: Genetically-modified S. aureus RN4220 clones installed with WTA glycosylation modification were prepared. Adsorption of phages S13’ and S24-1 belonging to this viral group were examined using the bacterial strains. Binding activity of the recombinant RBPs manufactured in Escherichia coli was examined by Western blot, using the bacterial strains. The phylogenetic analysis of the protein was performed.

Results: First, we found that β-O-N-acetylglucosamine of WTAs was essential for infection and adsorption of phage S13’, while the N-acetylglucosamine regardless of α- and β-O-glycosidic linkages of the WTAs were essential for infection and adsorption of phage S24-1. Next, examining the binding activities of their RBPs to cell walls with different glycosidic pattern of the WTA, the results were coincident with the experimental results of phage infection and adsorption. Moreover, the RBPs among Podoviridae and Siphoviridae families of S. aureus phages were phylogenetically examined. Considering the protein sequence similarity of RBPs and evolution of the Siphoviridae family by modular evolution theory and recombination, the plausible hypothesis of horizontal gene transfer of the RBP gene from the Podoviridae to Siphoviridae families was proposed.

Conclusion: Because S. aureus strains have different WTA glycosidic linkage patterns, we believe that this study will contribute the development of the therapeutic phage with improved adsorption based on the P68virus genus in the future.

Keywords: Adsorption, Bacteriophage, Staphylococcus aureus

17th International Congress of Virology 144

Abstract Submission

100 Years of Bacteriologic PO602

Diversity of phages in an anaerobic sludge digester

J. Ni 1,*, K. Kubota 1, S. Kazama 2, Y.-Y. Li 1 1Civil and Environmental Engineering, 2NICHe, TOHOKU UNIVERSITY, Sendai, Japan

Objectives: Phage is a generic term for viruses that infect bacteria, and it can be divided into ssDNA, dsDNA, ssRNA and dsRNA phages depending on the genome structure. It is said that many DNA phages may affect prokaryotes and also can affect the microbial structure. Anaerobic sludge digester is widely used for energy-creation and sludge-reduction. The microbial community structure in anaerobic sludge digester has been well studied, but little is known about phages there. In this study, we tried to elucidate phage diversity in anaerobic sludge digester with a metagenomic approach.

Methods: Samples were taken from an anaerobic sludge digester in a sewage treatment plant in June (DS1606) and July (DS1607), 2016. SCOD, SS, VSS etc. of the samples were measured. For prokaryotic community analysis, the V4 region of 16S rRNA gene was amplified with the primer set of 515F-806R. Sequencing of the amplified products was conducted using MiSeq platform and the obtained sequence data was analyzed by QIIME with the Greengene database. For metagenomic analysis of phages, the samples were initially diluted 10 times with the SM buffer. The diluted solution was centrifuged, and then the supernatant was filtered through a 0.2 μm filter. The filtrated water was subjected to ultrafiltration with a 30 kDa filter. Thereafter, pellet was recovered by ultracentrifugation. Nucleic acids were extracted from the recovered pellet using the NucliSENS. RNA library was prepared using SeqPlex RNA Amplification kit and True-seq DNA PCR-free LT Sample Prep Kit B.

Results: The characteristics of the samples were as follows: SS of DS1606 was 15.1 g/L and VSS was 11.7 g/L; the soluble COD of DS1606 and DS1607 samples were 627 and 606.52 mg/L, respectively; and TOC were 165 and 201 mg/L, respectively. Prokaryotic communities of the DS1606 and DS1607 samples were similar. Four phyla with the highest relative abundance were Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes. Relative abundance of Archaea, mostly belonging to Euryarchaeota, were 1.38% (DS1606) and 1.31% (DS1607). The results were similar with the previous studies, which indicates that the anaerobic sludge digester was in good operation. The sequencing reads number of DS1606 sample with 150-250 bases over Q25 was 1,984,414, in which only 68 sequence reads were close to known phages, and 7,563 sequence reads were assigned as virus. The remaining 1,976,783 reads had low sequence identity to the phages currently being known. Among the known phages, the Coliphage, Bacteriophage S13 and Propionibacterium phage had the relative abundances of 22.1%, 10.3% and 8.8%, respectively. As for the DS1607 sample, the filter-passed sequencing read number was 1,684,844 in total, 253 sequence reads of which were similar to known phages, 706 sequence reads were close to virus, and the remaining 1,683,055 reads had low sequence identity to known phages. Among the known phages, Tetrasphaera phage TJE1, Coliphage, Salicola phage, Cellulophaga phage and Bacteriophage S13 had relative abundances of 18.6%, 15.8%, 6.7%, 6.7% and 5.1%, respectively. Eight known phages were detected in both DS1606 and DS1607 samples.

Conclusion: Small portion of the total sequence reads was assigned as known phages, and most of the sequence reads showed low identity with known phage, indicating the most of phages in the anaerobic sludge digester are unknown. To clarify phage diversity and their roles, it is necessary to analyze unassigned sequence reads as well as isolation of phages there.

Keywords: anaerobic sludge digester, metagenomics analysis, phage diversity 17th International Congress of Virology 145

Abstract Submission

100 Years of Bacteriologic PO603

GENETIC DISSECTION OF A SINGLE REPEAT OF THE TAIL TAPE MEASURE PROTEIN OF THE LACTOCOCCAL PHAGE TP901-1

M. Alqarni*, J. Mahony 1, S. Spinelli 2, C. Cambillau 2, D. Van Sinderen 1 1Microbiology, UNIVERSITY COLLEGE CORK, Cork, Ireland, 2Architecture et Fonction des Macromolecules Biologiques (AFMB), Marseille, France

Objectives: The overall objective is to study the effect of deleting specific amino acids within one of the 29 repeats (comprised of 11 or 18 aa) of the TP901-1 tail Tape Measure Protein TMP. employing the ssDNA recombineering approach. 16 in-frame deletion mutants were generated in order to assess the effect of the deletions on (1) the phage's infectivity on its host strain Lactococcus lactis subsp. cremoris 3107 and (2) the phage morphology/assembly.

Methods: Recombineering mutagenesis was applied to generate desirable mutations in the TP901-1 tail Tape Measure Protein TMP. Phage analysis varied between plaque assays and lysogenic conversion assay to asses phage infectivity. Regarding phage morphology electron microscopy was used to study the effect of mutations on the TP901-1 tail Tape Measure Protein TMP.

Results: Dissection of a single repeat: To understand the importance of (i) individual amino acids and(ii) the length/periodicity of a repeat element, a single repeat was genetically dissected by sequentially deleting each of the individual amino acids sequentially from the fourth repeat in combination with repeats 1-3 (for ease of detection of the mutants). This particular repeat was selected as it was known that deletion (del)1-3 displays similar plaque forming and lysogeny characteristics to that of the wild typephage, and because deletion of larger DNA regions (rather than individual codons) facilitatedthe detection of desired mutants based on size differences of the generatedtmpTP901-1deletions. Additionally, the effect of removing a half repeat (i.e. in the wild type background)was assessed by generating the mutant del3-3.5 in which the first five amino acids of the fourth repeat were deleted as well as the third repeat, which is known not to affect TP901-1infectivity. Thus, 11 mutants were constructed carrying incremental deletions and named del1-3.1 to del1- 4 (=del1-3.11), and each mutant was characterised with respect to lysogeny and plaque forming ability. All mutants lysogenised the host at apparently equal efficiency;however, only mutants del1-3.3, del1-3.7 and del1-3.10 were capable of forming plaques (or atleast countable plaques).

Conclusion: The application of the recombineering system to the mutational analysis of the bacteriophage TP901-1 TMP has facilitated the development of 16 mutations within the TMP-encoding gene. The mutants have been assessed for their infective capability and impact on phage morphogenesis. It is evident that certain amino acids within a single repeat are essential to the correct functioning and stability of the phage structure, while others create minimal impact. This study suggests that the impact of the mutations may be more consistent with effect of changing the periodicity of the tail tube helices rather than requiring specific amino acid residues. Due to the change within the TMP structure caused by deletions, a number of the non-plaquing mutants of TP901-1 were challenging in terms of obtaining images of fully intact phages when subjected to purification for electron microscopy. The effect of pH, temperature and osmotic stressors on the mutants generated have been examined to define environmental factors that may impact on phage stability based on lysogenic assays its was clear that mutants sustained their ability to inject there DNA which showed intact phages, the cause of this apparent instability remains unclear. Image:

17th International Congress of Virology 146

Keywords: Bacteriophage , Lactococcus lactis , TP901-1

17th International Congress of Virology 147

Abstract Submission

100 Years of Bacteriologic PO604

PRESENTATION OF THE FIRST PHAGE THAT INFECTS THE LD28 CLADE, AN ABUNDANT FRESHWATER METHYLOTROPHIC BACTERIAL GROUP

K. Moon 1,*, I. Kang 2, S. Kim 2, S.-J. Kim 1, J.-C. Cho 2 1School of Biological Sciences, Seoul National University, Seoul, 2Department of Biological Sciences, Inha University, Incheon, Korea, Republic Of

Objectives: Bacteriophages (phages) are the most abundant biological entities on earth and known to be present as 10- times more abundant than their host bacteria. In aquatic environments, their abundances are calculated as ~106 particles/ml of seawater and ~107 particles/ml of lake water. Most importantly, bacteriophages participate in the nutrient cycling in aquatic environments. Not only that they interfere with bacteria-mediated various nutrient cycles by predation, but they also contribute in increase of the dissolved carbon source through lysis of bacterial cells. Despite of their extensive population, bacteriophages infecting major groups of freshwater heterotrophic bacteria have been rarely isolated, leaving large unexplainable area in freshwater viral metagenome studies. To our knowledge, no phage has been identified and isolated that infects major freshwater heterotrophic bacterial groups, such as acI, acIV, LD12, Limnohabitans, Polynucleobacter, and LD28. In this study, we attempted to isolate bacteriophages infecting the LD28 clade, one of the major freshwater bacterial groups and perform in-depth genome study of the isolated bacteriophage for better understanding of freshwater microbial ecology.

Methods: In April 2014, surface water of Lake Soyang, located in South Korea, was collected. The water sample was initially filtered through a 0.2-μm PES membrane filter to remove bacterial sized particles, followed by addition of methanol and vitamin mixture. An LD28 strain, IMCC19250, isolated from the Lake Soyang was added to the lake water sample from the above to promote the growth of bacteriophage that infects the LD28 strain. After success enrichment and isolation of the bacteriophage that showed lysis of the LD28 strain, the viral particles were collected to perform whole genome sequencing. To observe genome distribution in lake waters, competitive binning of the LD28 bacteriophage against Lake Soyang virome was performed using DIAMOND.

Results: From the surface layer of the Lake Soyang, a bacteriophage infecting an LD28 strain was successfully isolated and named P19250A. Morphological and genomic analyses revealed that P19250A is a lytic siphovirus with a ~38.6-kb genome. In the binning analyses of freshwater viromes, P19250A appeared to be the most highly-assigned freshwater phage in several viromes, including five viromes from Lake Soyang and Lough Neagh, UK, and the proportion of P19250A- assigned reads fluctuated following the abundance of LD28 clade in Lake Soyang.

Conclusion: These results showed that P19250A would be an essential resource for analyses of freshwater viromes, and also suggest that phages of other abundant freshwater bacteria are needed to be isolated for better understanding on freshwater viruses.

Keywords: Bacteriophage, Freshwater, Methylotroph

17th International Congress of Virology 148

Antiviral Immunity

17th International Congress of Virology 149

Abstract Submission

Antiviral immunity

PO605

25-HYDROXYCHOLESTEROL INHIBITS GLYCOPROTEIN MATURATION IN LASSA, EBOLA AND NIPAH VIRUSES.

P. Shrivastava-Ranjan*, M. K. Lo 1, E. Bergeron 1, A. Chakrabarti 1, M. Flint 1, C. Albarino 1, S. T. Nichol 1, C. F. Spiropoulou 1

1Viral Special Pathogens Branch, Centre for Disease Control and Prevention, Atlanta, United States

Objectives: The interferon-stimulated gene cholesterol 25-hydroxylase encodes an enzyme that catalyzes the production of 25-hydroxycholesterol. 25-hydroxycholesterol is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here we determine the mechanism of action of cholesterol 25-hydroxylase and 25-hydroxycholesterol in inhibition of Lassa, Ebola and Nipah viruses.

Methods: 1. We investigated the expression levels of Nipah G, Ebola GP1 and Lassa GP1 in cells either treated with 25- hydroxycholesterol or overexpressing cholesterol 25-hydroxylase by Western Blot.

2. We used quantitative reverse-transcription PCR (qRT-PCR) to determine viral RNA copy numbers in supernatants of infected cells.

3. We determined virus titer in Lassa, Nipah and Ebola virus infected cells treated with 25-hydroxycholesterol or overexpresssing cholesterol 25-hydroxylase.

Results: 1. Overexpression of cholesterol 25-hydroxylase or treatment with 25-hydroxycholesterol decreased glycoprotein N-glycan maturation and reduced production of infectious Lassa virus.

2. Depletion of endogenous cholesterol 25-hydroxylase using siRNA enahnced level of fully glycosylated Lassa GP1 and increased infectious virus production.

2. Preliminary investigation of the attachment glycoproteins of Nipah and Ebola viruses led to similar observations of decreased glycoprotein maturation and virus infectivity.

Conclusion: Our findings identify a novel role for cholesterol 25-hydroxylase in controlling viral propagation, and indicate that either modulation of cholesterol 25-hydroxylase expression or direct administration of 25-hydroxycholesterol could potentially be useful as a broad-spectrum antiviral therapy.

Keywords: Nipah, Ebola, Lassa, Cholesterol 25-hydroxylase

17th International Congress of Virology 150

Abstract Submission

Antiviral immunity

PO606

CHARACTERIZATION OF A NOVEL INHIBITOR OF THE INTERFERON-REGULATORY FACTOR 3-DEPENDENT INNATE ANTIVIRAL RESPONSE

R. Ambrose 1,*, J. Liu 2, C. Rootes 1, T. Adams 2, A. Bean 1, C. Stewart 1

1Health and Biosecurity, CSIRO, Geelong, 2Manufacturing, CSIRO, Parkville, Australia

Objectives: The host recognition of intracellular viral RNA and subsequent induction of cytokine signalling is tightly regulated at the cellular level and is a target for manipulation by viruses and therapeutics alike. In this study we have defined the function of a poorly-understood human protein that promotes infection by several highly pathogenic RNA viruses, in the context of antiviral signalling.

Methods: We utilised siRNA knockdown and overexpression plasmids of this protein during virus infection or stimulation with the viral RNA mimic poly(I:C). Using quantitative PCR, immunoblotting and protein interaction assays we interrogated the dsRNA-induced signalling pathways culminating in interferon α/β transcription. We also performed bioinformatics analyses on the amino acid sequence to study its conservation across species and identify putative functional domains.

Results: Bioinformatic analyses showed that the novel protein is highly evolutionarily conserved. Overexpression of this protein suppressed the transcription of interferons (IFN)-α/β and the pro-inflammatory tumor necrosis factor (TNF)-α in response to the double-stranded RNA mimic poly(I:C). However, transcriptional activity of interferon regulatory factor 3 (IRF3) was affected without impairing its activation or nuclear translocation. As the uncharacterised protein localised to the nucleus and was upregulated in response to poly(I:C), we proposed that it may act as part of a negative feedback loop to turn off IFN production. However, unlike most IRF3 inhibitors, this protein did not induce IRF3 degradation or impair IRF3- DNA binding.

Conclusion: We have defined the function of a highly conserved human protein, with no functionally characterised domains, in antiviral immunity. The protein inhibits IRF3-dependent antiviral cytokine transcription via a mechanism hitherto undescribed for any protein, and may represent a novel class of immune regulator with implications for antiviral immunity, autoimmune disorders and cancer.

Keywords: Interferon signalling, IRF3 regulation

17th International Congress of Virology 151

Abstract Submission

Antiviral immunity

PO607

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells

Y.-C. Chao 1,*

1INSTITUTE OF MOLECULAR BIOLOGY, ACADEMIA SINICA, Taipei, Taiwan

Objectives: Although baculovirus has been used as a safe and convenient gene delivery vector in non-host mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of host negative regulators is necessary to improve baculovirus-based expression systems.

Methods: Here, we performed high throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line.

Results: Among the genes screened, suppression of receptor interaction protein kinase 1 (RIP1, alias RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further studies revealed that baculovirus transduction instead plasmid DNA transfection results RIP-1-mediated inhibition of foreign gene expression.

Conclusion: This study has initiated a rather complete survey to demonstrate most, if not all, of human cell immune response against a “naïve virus”. Our findings revealed that knockdown of RIP1- a common mediator upstream of TBK1 in TLR-, RIG-I- and cytosolic-DNA sensing receptor signaling pathways inhibits the downstream signaling cascade, which then subsequently blocks expression of interferons and cytokines. In addition, this study has made baculovirus a more efficient gene transfer vector for at least several of the most frequently used mammalian cell systems, including A549, Vero E6, and U2OS cells.

Keywords: baculovirus, mammalian cells, antiviral.

17th International Congress of Virology 152

Abstract Submission

Antiviral immunity

PO608

IFNλ is a potent non-inflammatory therapeutic for treatment of IAV infection

S. Davidson 1, H. H. Gad 2,*, T. McCrabe 1, S. Crotta 1, R. Hartmann 2, A. Wack 1

1Mill Hill Laboratory, The Francis Crick Institute, London, United Kingdom, 2Department of Molecular Biology and Genetics, Aarhus University, Århus C, Denmark

Objectives: Influenza A virus (IAV) is a common human pathogen that causes three to five million cases of severe illness and up to 500,000 deaths every year. Both type I and III IFNs are involved in the immune response towards IAV infection and can restrict viral replication in the airway epithelia. However, excessive type I IFN levels has also been shown to lead to uncontrolled inflammation as it acts on immune cells and can thus contribute to disease. In contrast, type III IFN has a much more limited effect on immune cells and we therefore wanted to test if type III IFN could potentially be used as a therapeutic against IAV infection.

Methods: Murine IFNλ2 was expressed in E. coli and purified from inclusion bodies under denaturizing conditions by metal- ion affinity chromatography. The protein was renatured in vitro and further purified by cation-exchange and size-exclusion chromatography to obtain a highly pure and active protein. The recombinant IFNλ2 as well as commercially available IFNα4 was used to treat congenic B6.A2G-Mx1 mice either before or during infection with IAV while assessing their antiviral and proinflammatory effect.

Results: We have established a simple and efficient expression system to produce highly pure and active murine type III IFN and used it to assess the effects of treatment with exogenous type I and III IFNs during the course of IAV infection in vivo in mice. Both type I and III IFNs could restrict viral replication when administered during IAV infection but whereas type III IFN protected the mice from disease and increased their survival, type I IFN exacerbated disease and caused higher mortality. This may be explained by the fact that type I IFN treatment had a proinflammatory effect on immune cells in contrast to type III IFN and thus led to increased inflammation and lung damage.

Conclusion: Here, we demonstrate a simple and efficient method for producing highly pure and active type III IFN, which may be a potent non-inflammatory therapeutic for treatment of IAV infection.

Keywords: Influenza virus, Innate immunity, Interferon

17th International Congress of Virology 153

Abstract Submission

Antiviral immunity

PO609

PROFILING OF THE DOMINANT NEUTRALIZING EPITOPES OF HUMAN ANTIBODIES AGAINST ADENOVIRUS TYPE 14 AND 55

Y. Feng, X. Sun, Q. Wang, C. Li, L. Feng*, L. Chen*

Objectives: The emergence of highly virulent adenovirus type 14 (Ad14) and 55 (Ad55) has exerted substantial threats for public health globally. However, the dominant neutralizing epitopes targeted by human antibodies, which are important for the design of preventive vaccines, remain unknown. In this study, we clarified the epitopes of the neutralizing antibodies (nAbs) elicited in individuals infected by these two adenoviruses.

Methods: We generated series of chimeric Ad14 reporter viruses each harboring a single or the entire hyper-variable-region (HVR) from Ad55 hexon, and detected the neutralizing capacity of serums from Ad14 or Ad55 seropositive individuals. The epitope recognizing profiles of mice anti-Ad14 or Ad55 nAbs were also studied.

Results: Hexon-specific nAbs from different donors possessed widely diverse recognition profiles, but were mainly directed to HVR 2, 5, and 7. Immunization-induced mice nAbs, however, recognized dramatically different epitopes in comparison with human nAbs, which were localized in HVR1, 2, 7 of Ad14 and HVR1, 2 of Ad55, respectively. We also analyzed human antibody responses to the other two capsid proteins, penton and fiber. Although penton-binding antibodies were frequently detected, residual, if any, penton-specific nAbs were generated in these donors. Contrarily, fiber-specific nAbs were observed in almost all Ad14 and Ad55 double-positive serums. Subtraction analysis with fiber knob revealed that fiber- specific nAbs contributed substantially for the cross-reactivity. Additionally, immunization with fiber knobs generated robust nAbs which neutralized both Ad14 and Ad55, implying that fiber may be an ideal antigen for a bivalent vaccine.

Conclusion: These results not only added to our knowledge about human nAb responses against Ad14 and Ad55 infection, but may have significant implications in vaccine development.

Keywords: Epitopes, Human Adenovirus , Neutralizing antibodies

17th International Congress of Virology 154

Abstract Submission

Antiviral immunity

PO610

SCHLAFEN 14 (SLFN14) IS A NOVEL ANTIVIRAL FACTOR INVOLVED IN THE CONTROL OF VIRAL REPLICATION.

R.-K. Seong*, S.-W. Seo 1, J.-A. Kim 1, S.-K. Park 1, M. Kumar 2, O. S. Shin 1

1Biomedical Science, Korea University, Seoul, Korea, Republic Of, 2Tropical Medicine, Medical Microbiology and Pharmacology, Honolulu, HI, United States

Objectives: Schlafen (SLFN) proteins have been reported to play an important function in the control of cell proliferation, differentiation and the induction of immune responses. The antiviral roles of SLFN during viral infection have not been well explored.

Methods: To examine the role of SLFN during viral infection, RAW264.7 mouse macrophage, human THP-1 or A549 cells were infected with either wild-type PR8 or delNS1/PR8 influenza A virus. The induction of SLFN expression following influenza A virus infection was measured by quantitative PCR, western blotting analysis and immunofluorescence assay. The effect of SLFN over-expression or knock-down on viral replication was measured and plaque assay was conducted to determine the viral replication.

Results: In this study, we sought to determine antiviral activities of putative RNA-helicase domain containing SLFN14 against viral infection. First, murine SLFN14 expression was specifically induced by TLR3-mediated mitogen-activated protein kinase (MAPK) pathways and type I Interferon (IFN) signaling pathways in RAW264.7 mouse macrophages. Furthermore, overexpression of SLFN14 plasmid DNA in A549 cells led to the reduction of plaque formation and inhibition of viral NP expression, whereas knockdown of SLFN14 in RAW 264.7 cells resulted in enhanced viral titer. Meanwhile, SLFN13 knockdown in A549 cells resulted in enhanced viral replication for both influenza A and B virus.

Conclusion: In conclusion, our data suggest that SLFN14 is a novel antiviral factor for influenza virus.

Keywords: Schlafen 14, RNA helicase, Antiviral factor

17th International Congress of Virology 155

Abstract Submission

Antiviral immunity

PO611

SENDAI VIRUS V PROTEIN PLAYS AN ANTIAPOPTOTIC ROLE DURING INFECTION

T. Irie 1,*, R. Kawabata 1, A. Yoshida 2, I. Okamoto 1, K. Oda 1, T. Sakaguchi 1

1Department of Virology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan, 2Virginia- Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, United States

Objectives: The apoptotic response to viral infections plays opposing roles in viral pathogenesis and replication in the infected host: a host antiviral defense for viral clearance and a progressive replication of viral infection. Sendai virus (SeV), a prototypic member of the family Paramyxoviridae that causes fatal pneumonia in mice, has been reported to induce apoptosis in infected cells by a relatively slow process. SeV-induced apoptosis has been reported to be mediated by the IRF3/Bax apoptotic pathway. Recently, we have demonstrated that one of the SeV accessory proteins, V, inhibits activation of IRF3 by a physical interaction to prevent production of interferon-beta (Irie et al., J Virol 2012). In this study, we tried to reveal a novel antiapoptotic function of the V protein.

Methods: To achieve this goal, we characterized a V-deficient SeV recombinant, V(-), in terms of apoptosis induction. Inhibitory effects of Caspase inhibitors, Bcl-xL, which functions as a dominant-negative version of Bax, as well as the WT as well as an IRF3-interaction-deficient mutant of V proteins.

Results: The V(-) virus induced cell death with typical apoptotic markers, such as chromatin condensation and DNA fragmentation, much more quickly and severely than did the wild-type virus. This apoptosis was shown to be dependent on the V-IRF3 interaction, and prevented in the presence of a pan-caspase inhibitor as well as by over-expression of Bcl-xL.

Conclusion: The results obtained here indicate that the V protein may prevent SeV-induced, IRF3/Bax-mediated apoptosis by interacting with IRF3. The V(-) virus has been shown to be dramatically attenuated in mice, despite its normal growth in cell cultures. In addition, we previously reported that a quick clearance of the V(-) virus in lungs of infected mice is lost in IRF3-KO mice (Kiyotani et al., Virology 2007). Taken together, the SeV V-IRF3 interaction may play an important role in viral pathogenesis.

Keywords: Apoptosis, Innate immunity, Paramyxovirus

17th International Congress of Virology 156

Abstract Submission

Antiviral immunity

PO612

STING IS INVOLVED IN ANTIVIRAL IMMUNE RESPONSE AGAINST VZV INFECTION IN HUMAN DERMAL CELLS

J.-A. Kim 1,*, S.-K. Park 1, S.-W. Seo 1, C.-H. Lee 2, O. S. Shin 1

1Brain Korea 21 Plus for Biomedical science, College of Medicine, Korea University, Seoul, 2Department of Microbiology, Chungbuk National University, Cheongju, Korea, Republic Of

Objectives: Varicella Zoster Virus (VZV) is a human-restricted a-herpesvirus that exhibits tropism for the skin. The VZV host receptors and downstream signaling pathways responsible for the antiviral innate immune response in the skin are not completely understood.

Methods: To investigate the antiviral role of STING in human dermal cells, shRNA-mediated STING knock-down cells were infected with varicella-zoster virus. We performed plaque assay to measure viral titers. Immunoblotting and real-time PCR were conducted to measure viral and host protein and mRNA expression levels., respectively. Additionally, to obtain information on differential and dynamic transcriptome response to VZV infection, we performed RNA-sequencing analysis.

Results: Stimulator of interferon genes (STING) mediates an important host defense against VZV infection in dermal cells including human dermal fibroblasts (HDFs) and HaCaT keratinocytes. Inhibition of STING using small interfering RNA (siRNA) or short hairpin RNA (shRNA)-mediated gene disruption resulted in enhanced viral replication but diminished IRF3 phosphorylation and induction of IFNs and pro-inflammatory cytokines. Pre-treatment with STING agonists resulted in reduced VZV glycoprotein E (gE) expression and the inhibition of plaque formation. Additionally, using RNA-sequencing to analyze dual host and VZV transcriptomes, we identified several novel host genes significantly induced by VZV infection.

Conclusion: Our data provide a new insight into STING-mediated induction of type I IFNs and subsequent antiviral signaling pathways that regulate VZV replication in human dermal cells.

Keywords: stimulator of interferon gene, varicella-zoster virus

17th International Congress of Virology 157

Abstract Submission

Antiviral immunity

PO613

USP18 interacts with HBV Polymerase protein and surface antigen protein to promote virus secretion

M. Yao*

Objectives: Hepatitis B virus(HBV) infection is a global epidemic, with 240 million people chronically infected with HBV, which poses a great threat to the public health. Interferon-alpha remains one of the best treatment options for HBV, however almost half of the patients do not respond. Our previous studies and others demonstrated that increased hepatic baseline (pretreatment) expression levels of a subset of ISGs including ISG15 and USP18 were associated with treatment nonresponse to type I IFN-based therapy in HBV patients. The underlying mechanism remains unclear. In this study, we explore the effect of USP18 on HBV production, focusing on the interactions between USP18 and HBV proteins to further dissect the role of USP18 in HBV infection and response to IFN treatment

Methods: Over-expression of USP18 or USP18-C64S inactive mutant was achieved by tranfection of the plasmids into HepG2.2.15 cells. HBV DNA within the cells and in the culture supernatant were quantified by Real-time PCR. In order to identify which HBV proteins interact with USP18, plasmids encoding HBV four open reading frames (ORF:P gene, S gene, C gene and X gene) were constructed with routine molecular cloning techniques. The expression of HBV proteins (polymerase/P, surface antigen protein/S, core protein/C , X protein/X) were confirmed by Western Blot(WB). Co- immunoprecipitation (CO-IP) assay were employed to identify the interactions between USP18 and HBV proteins.

Results: Over-expression of USP18 wild-type or C64S inactive mutant significantly increased the levels of secreted HBV DNA in the culture supernatants although no effect on the replication of HBV DNA in the cells. In addition, Co-IP assay identified that USP18 interacted with HBV Polymerase protein and surface antigen protein.

Conclusion: We concluded that USP18 stimulated HBV secretion independent of its enzymatic activity possibly through interaction with HBV P and/or S proteins. This study will shed light on how HBV exploits the host innate immune response to benefit its own secretion, which may reveal new promising candidates for further antiviral therapy development.

Keywords: None

17th International Congress of Virology 158

Late breaking abstract

Antiviral immunity

PO875

Protective capacity of human antibody response during acute dengue infection

J. Xu*, C.-I. Wang 1, K. Fink 1

1SINGAPORE IMMUNOLOGY NETWORK, Singapore, Singapore

Objectives: Dengue is the leading arthropod-borne viral disease that puts half of the world’s population at risk of infection. Vaccine development has been impeded by the lack of defined protective immune correlates following natural dengue infection. We seek to understand the human antibody response following natural dengue infection.

Methods: Dengue-specific antibodies were isolated from the plasmablasts of two naturally infected Dengue patients by single B cell cloning. Antibody function and binding characteristics were determined by plaque reduction neutralization and competition ELISA respectively. Epitope mapping studies were conducted by alanine scanning mutagenesis. AG129 mice were used as animal models for in-vivo studies.

Results: A panel of 18 dengue-reactive, cross-reactive but weakly neutralizing antibodies were categorized into 4 groups based on binding characteristics. While fusion loop specific antibodies dominated the antibody response, we identified smaller groups of antibodies that recognize previously undescribed cryptic epitopes in domain II of the Envelope protein. Although weakly neutralizing, these antibodies were most efficacious in promoting survival of infected mice. Finally, we describe the discovery of a unique antibody that recognizes a quaternary structure epitope and possesses potent neutralization across all four dengue virus serotypes in-vitro and in-vivo.

Conlusion: The differential ability of antibodies to decrease viremia and promote survival suggests new-insights that might help to shape the correlates of protection.

Keywords: antibody response, Dengue

17th International Congress of Virology 159

Antivirals

17th International Congress of Virology 160

Abstract Submission

Antivirals

PO614

A novel fast and robust methodology for screening of anti-Chikungunya virus compounds

R. Bonotto*, B. Pascoalino 1, L. Holanda Gondim de Freitas-Junior 1

1Grupo de ação rápida de doenças emergentes, Butatan, São Paulo, Brazil

Objectives: Chikungunya is arthpord borne virus that belongs to Togaviridae family, responsible for chikungunya fever (CHIKV) in humans. It is estimated that about one billion people live in risk areas around the globe, especially in the tropics, The acute symptoms of the disease include high fever, headache, nausea, vomiting, conjunctivitis, and the disease envolves to severe joints pain, eventually and finally to the arthigenic syndrome that can last from weeks to years. The recently reported outbreaks in Brazil 2015 for exemple, shows the demand for the development of prevention or treatment approaches, since no antiviral or vaccine are available,therefore the treatment consists only on prophylaxis and drug to release the symptoms. In the recent years several studies aiming the development of antiviral molecules pointed cloroquine, arbidol with blocking activity of early steps virus in vitro. Another compounds ribaverin, 6-azaraudine and more recently faviparavir, showed activity impairing replication. Although those compounds demonstrated efficacy against the virus in vitro assays, they revealed unsefe in vivo tests. Currently High-throughput screening is the most employed methodology by the pharmaceutical industry for the triage of molecules against several pathogens. The clear advantagens of this technology are the possibility of screening a large amount of compounds with agility, reliability and robustness. In the present work, our group developement a cell-based high-throughput screening system, capable of detecting active molecules against chikungunya virus(chikv) infecting the human hepatoma Huh-7 cells using a cell viability output.

Methods: This novel technology is based in measurement of flourescence intensity generated by the labeling of the host cell nuclei with DAPI, after 72 hours of infection with CHIKV and treatment with diverse compounds to verify its ability protect the Huh-7 cells from lysis due to the viral infection.

Results: This methodology was validate using the interferon alpha2A as reference drug and sucessfully identified active molecules against CHIKV.

Conclusion: Thereby DAPI viability assay showed a robust and reliable assay to screening a range of compounds and indentified antiviral molecules agaisnt chikungunya virus

Keywords: Antivirals, Chikungunya virus, drug discovery

17th International Congress of Virology 161

Abstract Submission

Antivirals

PO615

Antioxidant activity and Anti-inflammatory effects of water extracts from the organic Flos Lonicerae Japonicae (FLJ) in Virus-infected RAW264.7 Cells

H.-W. Lin, C.-W. Liu, Y.-Y. Chang, H.-L. Chiou*

Objectives: Flos Lonicerae Japonicae (FLJ),a traditional Chinese medicine, has been used for treating various diseases. This study aimed to investigate the antioxidant and anti-virus inflammatory capacity and underlying molecular mechanisms of water extracts from FLJ of various stages including green bud stage (GB), white bud stage (WB), silver flower stage (SF), and gold flower stage (GF).

Methods: Antioxidant activities of water extracts obtained from dried FLJ of various stages were measured based on DPPH radical scavenging, and their relationships with total phenolics were investigated. The impact of extracts on expressions of NO, iNOS, COX-2 and inflammatory cytokines (IL-6, TNF-a, Rantes and MCP-1) in pseudorabies virus (PRV)-infected RAW264.7 cells were studied by western blotting. In addition, the effect of FLJ on virus replication was investigated by plaque assay.

Results: Our results showed WB of FLJ had the highest DPPH free radical scavenging activities and total phenolic contents, compared with that of other stages. Further experiments demonstrated that PRV-induced NF-κB activation is regulated through inhibiting STAT1 and STAT3 phosphorylation in response to WB of FLJ. Additionally, WB of FLJ caused HO-1 induction via upregulating pNrf2; both of which involved in the secretion of proinflammatory mediators. Taken together, our data indicate that WB of FLJ diminished expressions of proinflammatory mediators (NO, IL-6, TNF-a, Rantes and MCP-1) and their regulatory genes, iNOS and COX-2, in PRV-infected RAW264.7 cells by inhibiting STAT1/3 dependent NF-κB activation and inducing Nrf2-mediated HO-1 expression. Additionally, results of plaque assay demonstrated that WB of FLJ did inhibit viral replication.

Conclusion: Results from this study revealed that water extract of WB of FLJ could act as potential antiviral agent based on their antioxidant and anti-virus inflammatory capacity.

Keywords: anti-inflammation, Flos Lonicerae Japonicae, pseudorabies virus

17th International Congress of Virology 162

Abstract Submission

Antivirals

PO616

AR-12 SUPPRESSES DENGUE VIRUS REPLICATION BY DOWN-REGULATION OF PI3K/AKT AND GRP78

C.-P. Chang 1,*, H.-H. Chen 1, C.-C. Chen 2, Y.-S. Lin 1, C.-F. Lin 3, C.-L. Chen 4

1Department of Microbiology & Immunology, National Cheng Kung University, Tainan, 2Department of Pathology, Chia-Yi Christian Hospital, Chia-Yi , 3Graduate Institute of Medical Sciences, 4Translational Research Center, Taipei Medical University, Taipei, Taiwan

Objectives: Dengue is the most prevalent mosquito-borne viral disease in the world and causes at least 390 million people infections per year, yet there are no approved effective antiviral drugs to treat dengue. It has been reported that activating PI3K/AKT signaling pathway and up-regulating GRP78 are crucial for supporting DENV replication. Targeting these pathways may server as a promising anti-DENV treatment. AR-12, a derivative of celecoxib without inhibiting activity on cyclooxygenase, can abolish PI3K/AKT signaling and suppress GRP78 levels, and has been recently reported to block the replication of several hemorrhagic fever viruses. However, the efficacy of AR-12 in treating DENV infection is still unclear.

Methods: Here, we evaluated DENV replication by detecting the expression of non-structure proteins and infectious viral particles in different cell lines including A549, Huh-7, U937 and Meg-01, in the presence of AR-12. Furthermore, we used a DENV infection mice model, in which the newborn mice were administered with DENV and AR-12 via intracerebral and intraperitoneal injection, to examine the anti-DENV activity of AR-12 in vivo.

Results: We provided evidences to show that four serotypes of DENV replication are all significantly reduced in AR-12 treated cells. Treatment of AR-12 does not inhibit DENV binding to cells or up-regulate type-I interferon, but reduces the expression of pAKT and GRP78 in DENV infected cells. Rescue of GRP78 is able to attenuate anti-DENV effect of AR-12. Using a DENV-infected suckling mice model, we further demonstrate that treatment of AR-12 before or after DENV infection abolishes virus replication and mice mortality.

Conclusion: In conclusion, we uncover the potential efficacy of AR-12 as a novel drug for treating dengue.

Keywords: antivirus, AR-12, dengue

17th International Congress of Virology 163

Abstract Submission

Antivirals

PO617

Broadly anti-influenza agents developed by viral polymerse genes

H. Zhao*, K. To

Objectives: Currently, more and more drug-resistant viral strains emerge after the antiviral drug application for treatment. Thus, it is urgently needed to develop broadly antiviral agents with prophylactic and therapeutic effects against influenza A virus with low possibility to cause drug resistance.

Methods: PCR and cloning were used to construct plasmids expressing shortened viral polymerase genes. Antiviral efficacy of shortened viral polymerase genes against influenza A virus in cells was evaluated by plasmid transfection, real time PCR and plaque assay. The antiviral mechanism of shortened viral polymerase genes was identified by real time PCR, plaque assay and HA assay. The antiviral activity of shortened viral polymerase genes in vivo was detected by animal challenge with lethal dose of influenza A virus.

Results: Shortened viral polymerase genes could significantly inhibit different subtypes of influenza A virus replication in cells. Shortened viral polymerase genes could inhibit 10~1000-fold viral replication in cells. The antiviral mechanism of shortened viral polymerase genes was attributed to completely inhibit normal viral package to generate defective virus, which could further inhibit normal viral replication in multiple life cycles. The in vivo antiviral activity showed that shorten viral polymerase genes could protect more than 80% mouse survivals for prophylactic treatment and also protect about 50% mouse survivals for therapeutic treatment.

Conclusion: Shortened viral polymerase genes could provide broadly and potently anti-influenza virus activity in vitro and in vivo. The shorted viral polymerase genes may be developed to be an antiviral with broadly anti-influenza activity and low possibility to cause drug resistance.

Keywords: antiviral, Influenza A virus

17th International Congress of Virology 164

Abstract Submission

Antivirals

PO618

Broadly antiviral peptide against respiratory viruses in vitro and in vivo

H. Zhao*, X. ZHAO

Objectives: Defensins are the front line of host innate immunity and have broad spectrum antiviral effects. However, there are limited data to show if these defensins have good antiviral activity in vivo and what the antiviral mechanism is. Thus, this study aims to investigate widespread antiviral activity of defensin peptide in vitro & in vivo and illustrate the antiviral mechanism.

Methods: Antiviral peptide library derived from mouse beta defensin-4 was synthesized by company. Recombinant beta defensin was expressed by E. coli. Antiviral activity in vitro was assayed by plaque assay, qPCR. Antiviral activity in vivo was detected by animal challenge with lethal dose of A(H1N1)pdm09 and H7N9 virus. The antiviral mechanism was identified by western blot, ELISA, confocal microscope and qPCR.

Results: Peptide P9 could significantly inhibit H1N1, H5N1, H7N9, SARS-CoV and MERS-CoV replication in vitro. Also, one dose of P9 showed potently prophylactic protection on mice with 100% and 50% survivals against H1N1 and H7N9 virus, respectively. The antiviral mechanism of P9 was attributed to the binding of P9 to viral glycoproteins and the enriched basic amino acids of P9 which could prevent endosomal pH decrease to inhibit viral replication.

Conclusion: We identify a new peptide which shows broad-spectrum antiviral activity against respiratory viruses (H1N1, H5N1, H7N9, SARS-CoV and MERS-CoV) in vitro and has potently antiviral activity in vivo. This peptide binds to viral glycoproteins and subsequently blocks viral replication by inhibiting virus-endosome pH decrease. The broadly antiviral activity of P9 implies that this kind of peptide may be a good candidate to be developed as an antiviral for treating emerging infectious diseases.

Keywords: Antiviral peptides, defensin, Influenza A virus

17th International Congress of Virology 165

Abstract Submission

Antivirals

PO619

Chloroquine: a candidate for broad-acting antiviral against Hand, Foot and Mouth Disease

Y. W. Tan*, J. J. H. Chu 1 2

1Collaborative Translation Unit for HFMD, Institute of Molecular and Cell Biology, 2Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: The Hand, Foot and Mouth Disease is a viral infection common in young children, characterized by the appearance of vesiculopapular rashes on the hands, foot and mouth. In some cases, the afflicted patients develop severe complications with neurological involvement. Currently, there is no effective antiviral available for treatment of HFMD and medical care is limited to supportive symptomatic therapies. In the absence of an effective treatment option, which is especially critical in severe infections, there is a need for research into an effective anti-HFMD therapy.

Methods: We have evaluated the efficacy of chloroquine against Enterovirus 71 (EV71) in Rhabdomyosarcoma (RD) cells using different treatment protocols, as well as a selection of viruses of other serotypes with one treatment protocol. The efficacy of chloroquine against EV71 was also evaluated in EV71- infected 6-day old Balb/c pups.

Results: Results from in vitro studies in cultured cells indicated that chloroquine effectively reduced viral replication in EV71- infected RD cells by treating them before or after virus inoculation or both. In addition, treatment of RD cells with chloroquine both before and after virus entry was able to reduce viral progeny production of Coxsackievirus A6 and A16, and Echovirus 7 hence indicating the broad-spectrum inhibitory effect on other human enteroviruses. Results from animal studies confirmed that the treatment of EV71-infected Balb/c pups improved their survival rates, reduced their clinical signs and lowered their viral loads. Histological analyses of animal tissues revealed a lower extent of pathology observed in the limb muscle tissues, the primary site of disease manifestation for the animal model of choice.

Conclusion: Taken together, the in vivo findings confirm that chloroquine can inhibit EV71 infection in EV71- infected susceptible animals and the antiviral activities observed across the selected serotypes in in vitro studies, similar to what had been observed for EV71, suggest that chloroquine may be a good candidate for a broad-acting anti-HFMD therapy.

Keywords: chloroquine, Enterovirus 71, HFMD

17th International Congress of Virology 166

Abstract Submission

Antivirals

PO620

Clostridium S45-5 isolated from human displays broad spectrum of antiviral activity in vitro and in vivo

J.-S. Lee*

Objectives: Probiotics are likely to have an impact through gut mucosa by inhibiting the growth of pathogenic microorganisms and by enhancing local and systemic immune responses in the host. This study aimed to investigate the potential antiviral activity of Clostridium species using in vitro and in vivo model.

Methods: For this purpose, we tested antiviral properties with 122 strains of Clostridium species isolated from infant fecal samples and selected the Clostridium S45-5. For antiviral effects of Clostridium S45-5, we used Influenza A virus (PR8), Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV) and Herpes Simplex Viruses (HSV). Mechanically, we checked the secretion of type I IFN, pro-inflammatory cytokines and transcriptional activation of antiviral genes after treatment of Clostridium S45-5. Moreover, for the prophylactic effects in mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2 or H7N3), oral administration of Clostridium S45-5 were performed and checked the levels of IL-6 or IFN-β and also IgA secretion in the small intestine and lung.

Results: Pretreatment of an effective dose Clostridium S45-5 significantly reduced the replication of diverse RNA or DNA viruses both in immune (RAW264.7) and epithelial (HEK293T) cells by establishing the antiviral state, by production of Interferons or proinflammatory cytokines. Moreover, oral administration of Clostridium S45-5 for 2 weeks exhibited prophylactic effects in BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2 or H7N3). Clostridium S45-5 inoculated mice displayed reduced lung viral titers and faster weight recovery. Further, Clostridium S45- 5 inoculated mice showed increase levels of IgA, IL-6, IFN-λ and IFN-β secretion in the serum, small intestine and lungs fluid.

Conclusion: These results suggested that Clostridium S45-5 can induce the cellular antiviral state of immune and epithelial cells in vitro and in vivo. Consequently, Clostridium S45-5 could be a potential source of promising probiotic or used to design other antiviral/anti-influenza agents. [This work was supported by the Technology Innovation Program, 10046418 (TGM0721311) of the Ministry of Trade, Industry & Energy (MI, Korea)].

Keywords: antiviral activity, Clostridium, probiotic

17th International Congress of Virology 167

Abstract Submission

Antivirals

PO621

Compound 2d reduces dengue virus replication and dengue virus-induced cytokine production

L.-C. Yen 1,*, Y.-C. Tsai 1

1Biochemistry, National Defence Medical Center, Taipei, Taiwan

Objectives: Dengue virus (DENV) is a member of the Flavivirus genus and includes four serotypes (DENV-1, -2, -3, and - 4). DENV infection can result in dengue fever (DF) to severe and life-threatening dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). However, despite decades of efforts, only one dengue vaccine has been approved in a few countries and there are still no effective antiviral therapy. In previous studies, the compound 2d could be used for the treatment of rheumatoid arthritis due to its potent and long-lasting anti-inflammatory activity. As DENV infection could lead to cycloxygenase-2 (COX-2), prostaglandin E2 (PGE2) and inflammatory cytokines production, in this study, we aim to examine whether the compound 2d could have anti-DENV activity and reduce the cytokine levels induced by DENV.

Methods: Western blot, Immunofluorescence assay, Plaque assay, ELISA and mouse model experiments.

Results: Treatment with the compound 2d could inhibit viral protein expression and viral progeny production in a dose- dependent manner in HEK-293 cells and Raw264.7 cells infected by all four DENV serotypes. Besides, the compound 2d could reduce COX-2, PGE2 and inflammatory cytokine levels of IL-6 and IL-8 in DENV-infected HEK-293 cells and Raw264.7 cells. Administration of DENV-2 infected AG129 mice with the compound 2d at 5mg/kg could prolong survival time of the treated mice compared to the controls.

Conclusion: Our findings imply that the compound 2d could have therapeutic potential by decreasing the massive inflammatory mediators during DENV infection.

Keywords: Cytokine, Dengue virus

17th International Congress of Virology 168

Abstract Submission

Antivirals

PO622

Coptidis Rhizoma extract inhibits replication of respiratory syncytial virus in vitro and in vivo by inducing antiviral state

J.-S. Lee*

Objectives: RSV, a negative-strand non-segmented RNA virus, belongs to the genus Pneumovirus of the family Paramyxoviridae. It is considered to be a leading cause of lower respiratory tract illness in infants and children worldwide. However, there are no commercial RSV vaccines and application of ribavirin which is only approved drug, to manage RSV infection is limited by its side effects. Therefore, novel antiviral agents for RSV with better effects and safety than ribavirin is target for study. In this study, we demonstrated several lines of evidence showing that Coptidis Rhizoma extract (CRE) has the immunemodulatory ability to inhibit RSV replication.

Methods: In this study, we evaluated the antiviral activities of Coptidis Rhizoma extract (CRE) against RSV in human respiratory tract cell line (HEp2) and BALB/c mice. We determined the induction of antiviral, IFN-stimulated genes (ISGs) and the secretion of IFN-β and IL-6 by CRE in vitro. To elucidate the features in antiviral signaling, we also evaluated the effect of CRE on the signaling molecules including IRF-3, TBK1, p65, ERK and p38 in the type I IFN and NF-κB signaling pathways. In mouse model, we tested whether oral administration of CRE is influence on the lung RSV titer and we checked the immune status in blood serum, BALF and SIF of CRE inoculated BALB/c Mice. In HPLC analysis, we found the presence of several compounds in CRE and we confirmed that palmatine was related to the antiviral properties.

Results: An effective dose of CRE significantly reduced the replication of RSV in HEp2 cells and reduces the RSV-induced cell death. This antiviral activity against RSV was through the induction of type 1 interferon-related signaling and the antiviral state in HEp2 cells. More importantly, oral administration of CRE exhibited prophylactic effects in BALB/c mice against RSV. In HPLC analysis, we found the presence of several compounds and confirmed that palmatine was related to the antiviral properties and immune-modulation effect.

Conclusion: Taken together, an extract of Coptidis Rhizoma and its components play roles as immunomodulators and could be a potential source as promising natural antivirals that can confer protection to RSV. These outcomes should encourage further allied studies in other natural products. [This study was supported in part by the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant no. 316043-3)]

Keywords: Coptidis Rhizoma , palmatine, RSV

17th International Congress of Virology 169

Abstract Submission

Antivirals

PO623

Development of a fluorescence resonance energy transfer–based intracellular assay for real-time monitoring dengue virus infections and antiviral potency

S.-H. Kung*, Y.-C. Liang 1

1Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan

Objectives: The dengue viruses (DENV) are mosquito-borne viruses of the family Flaviviridae that comprised 4 serotypes. DENVs are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. However, there is currently no specific antiviral therapy available for the effective clinical treatment of infections by DENVs. For rapid diagnosis and antiviral screening, we developed a real-time assay system that allows monitoring of intracellular DENV protease NS2B/NS3 activity based on fluorescence resonance energy transfer (FRET).

Methods: This was accomplished by stable expression of a fusion substrate construct composed of the GFP2 (green fluorescent protein 2) and DsRed2 (red fluorescent protein 2), with a cleavage motif of the viral protease connected in between. The rationale is that FRET occurs when the two fluorophores are in close proximity while FRET abrogates when the fusion substrate is cleaved in the presence of DENV protease activity.

Results: The FRET biosensor showed a real-time and quantifiable impairment of FRET upon infection by either DENV serotype in a time- and titer-dependent manner. We also showed that the FRET impairments were solely attributed to the catalytic activity of the viral protease. Treatment of the FRET biosensor with a known DENV inhibitor prochlorperazine reversed the virally reduced FRET in a dose-dependent fashion.

Conclusion: The spatiotemporal imaging enabled by FRET can shed light on the protease-substrate behavior in their normal milieu, and the quantitative FRET measurements permitted efficient screening of antivirals against DENV infections.

Keywords: antiviral, Dengue virus, FRET

17th International Congress of Virology 170

Abstract Submission

Antivirals

PO624

DIRECT ANALYSIS OF RNA CAPS USING NON-ION-PAIRING REVERSE-PHASE LC-MS FOR ANTIVIRAL DRUG SCREENING

A. Chew 1,*, P. Dedon 2 3, D. Luo 4, J. Wang 2

1School of Biological Sciences, Nanyang Technological University, 2Infectious Diseases IRG, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore, 3Biological Engineering and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA, United States, 4Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

Objectives: Almost all eukaryotic mRNAs have a 5’ cap structure of either type 1 (m7GpppNm) or type 2 (m7GpppNmNm). In the case of viruses, many have evolved capping or pseudocapping mechanisms to enhance viral replication and avoid innate immune detection. This mechanism is distinct from the host mRNA cap formation, which provides opportunities for vaccine and antiviral development. However, most reported RNA cap formation assays are based on chemical cross-linking, fluorescence polarization, thin-layer chromatography or radiolabeling. These methods are indirect, insensitive, low- throughput and prone to artifacts, which renders them inadequate as a drug-screening platform. Standard liquid chromatography-coupled mass spectrometry (LC-MS) methods for direct nucleotide analysis use ion-pairing (IP) agents, but this dampens MS detection sensitivity and contaminates the LC-MS system. To address these issues, we aimed to develop a novel non-IP reverse-phase LC-MS method for highly sensitive and accurate detection and quantification of RNA caps.

Methods: A triple quadrupole MS with electrospray ionization source was operated in positive mode.

Results: The total duration of a single run amounted to 30 min. Ion suppression, matrix effects, intra- and inter- day variability, response linearity and stability were all assessed for method validation.

Conclusion: We have developed a novel non-IP reverse-phase LC-MS method for highly sensitive and accurate detection and quantification of RNA caps. With further optimization, this method can be adapted to a high-throughput system for antiviral drug development targeting the viral capping process.

Keywords: LC-MS, RNA Cap

17th International Congress of Virology 171

Abstract Submission

Antivirals

PO625

Drug repurposing: gemcitabine and fidaxomicin are potent antivirals against Human Enterovirus 71 infection

J. Sun*

Objectives: Enterovirus 71 (EV71) causes hand, foot and mouth diseases (HFMD) as well as severe neurological complications in young children. Currently, there are no specific drugs and lack of approved vaccines for EV71 infection. Thus, it is urgent to develop antivirals against EV71. This project is to identify effective antiviral compounds against EV71 infection by drug repurposing of a FDA approved drug collection library.

Methods: A high-throughput phenotypic assay screening was performed using an FDA-approved drug library and the selected hits were further evaluated by qRT-PCR, Western Blotting, drug combination and EV71 IRES inhibition assays.

Results: Eighteen hits were identified in the screening. Two of the hits, gemcitabine HCl and fidaxomicin, showed significant inhibition effects against EV71 at non-cytotoxic concentrations. Drug inhibition assay showed that EV71 titer was decreased by 2.5 and 3 log10 pfu/mL by gemcitabine and fidaxomicin respectively. Western Blot study revealed that both gemcitabine and fidaxomicin inhibited the synthesis of structural and non-structural viral proteins during EV71 infection. Gemcitabine and fidaxomicin also inhibited viral RNA synthesis during EV71 infection. Furthermore, time-of-drug-addition and time-of- drug-removal studies suggested that both gemcitabine HCl and fidaxomicin targeted the early stage of EV71 replication. Further studies showed that fidaxomicin targeted viral IRES-dependent translation. In addition, to improve the antiviral efficacy of the compounds, drug combination studies were performed. Gemcitabine HCl and fidaxomicin showed synergistic effects with interferon-b.

Conclusion: Gemcitabine HCl and fidaxomicin were effective antiviral compounds against EV71 replication and they had synergistic effects with interferon-b.

Keywords: EV71 inhibition, fidaxomicin, Gemcitabine HCl

17th International Congress of Virology 172

Abstract Submission

Antivirals PO626

GRIFFITHSIN, A NOVEL INHIBITOR OF HENIPAVIRUS ENTRY AND FUSION M. K. Lo 1,*, B. R. O'Keefe 2, A. Chattopadhyay 3, J. K. Rose 3, A. L. Hotard 1, S. T. Nichol 1, C. F. Spiropoulou 1 1Viral Special Pathogens Branch, US CENTERS FOR DISEASE CONTROL AND PREVENTION, Atlanta, 2National Cancer Institute, National Institutes of Health, Frederick, 3Department of Pathology, Yale University School of Medicine, New Haven, United States

Objectives: Nipah and Hendra viruses are highly pathogenic members of the genus Henipavirus, and cause fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against. Griffithsin (GRFT) is a high- mannose binding lectin that has shown variable broad-spectrum activity against viruses across many virus families, including SARS Coronavirus, HIV-1, and Japanese Encephalitis virus. The objective of this study is to evaluate the therapeutic potential of GRFT and its synthetic derivative tandemers against Nipah and Hendra viruses. Methods: - We utilized a luciferase reporter Nipah virus to evaluate the antiviral activity of wild-type GRFT across multiple cell lines. - We then assayed the ability of GRFT to inhibit virus-induced cytopathic effect for both wild-type Nipah and Hendra viruses - We performed infectious virus yield inhibition assay against Nipah virus as additional confirmation of antiviral activity of GRFT - We performed time-of-addition assays using the reporter Nipah virus - We performed virus neutralization assays using both the reporter Nipah virus as well as a replication-deficient VSV pseudotyped with Nipah virus glycoproteins to determine whether there was dose-dependent neutralization by GRFT - We performed Nipah virus minigenome assays to determine whether GRFT interferes with intracelleular RNA transcription/replication. - We performed immune fluorescence assay to determine ability of GRFT to inhibit cell-to-cell viral spread. - We performed syncytia assays to determine ability of GRFT to block Nipah F and G glycoprotein mediated cell-to-cell fusion.

Results: 1. GRFT inhibits reporter Nipah virus replication in a dose-dependent manner across multiple cell lines 2. GRFT inhibits wild-type Nipah and Hendra virus-induced cyopathic effect in a dose-dependent manner 3. At high concentrations, GRFT decreases viral infectious yield by roughly 100-fold (2 Log TCID50) 4. Our time-of-addition assays indicated that 50% effective inhibition concentrations steadily increased as we increased the delay between infection and GRFT treatment. 5. Our neutralization assays indicated that GRFT neutralizes Nipah reporter virus and replication-deficient Nipah F/G VSV- pseudotyped virus. 6. GRFT does not inhibit RNA transcription/replication as shown by our minigenome assay. 7. We treated cells with GRFT after infection, and observed a decrease in cell-to-cell spread of viral antigen as indicated by syncytia and viral antigen. 8. We determined that the inhibitory action of GRFT required its natural dimeric state. 9. We evaluated synthetic dimers, trimers, and tetramers of monomeric GRFT, and found that trimeric and tetrameric GRFT enhanced inhibition of reporter and wild-type Henipaviruses. 10. We transfected cells with Nipah F/G glycoproteins, and observed dose-dependent inhibition of syncytia formation. Conclusion: - GRFT is a potent inhibitor of both Henipavirus entry AND cell-to-cell spread. - Given the unique characteristics of GRFT (lack of T-cell mitogenicity), further development is warranted for its potential as a potential antiviral therapeutic against Henipaviruses.

Keywords: Griffithsin, Henipavirus, Nipah virus

17th International Congress of Virology 173

Abstract Submission

Antivirals

PO627

High-Throughput Screening to Identify Inhibitors of BSL-4 Viruses

M. Flint 1,*, P. Chatterjee 1, S. Welch 1, M. Lo 1, L. McMullan 1, E. Bergeron 1, C. Albarino 1, S. Nichol 1, C. Spiropoulou 1

1Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, United States

Objectives: The 2013–2015 Ebola virus epidemic in West Africa highlighted the need for medical countermeasures, both vaccines and antivirals, for viral hemorrhagic fevers. We established methods and assays to permit high-throughput screening of compounds for antiviral activities against multiple biosafety level-4 viruses.

Methods: To facilitate campaigns to identify small molecule inhibitors of these viruses under BSL-4 containment, we generated recombinant viruses expressing fluorescent reporter proteins: Ebola virus, Lassa fever virus, Crimean-Congo hemorrhagic fever virus, Nipah virus, and Rift Valley fever virus. Each of these recombinant viruses undergoes a full replication cycle and is infectious. Compounds with antiviral effects may be detected by reduction of fluorescence in cells treated with the compound and infected with the recombinant viruses. A sixth virus, Alkhurma hemorrhagic fever virus (AHFV), causes a robust cytopathic effect in A549 cells, and an assay based on cell viability was used to test for compounds with anti-AHFV activity.

Results: Each of these assays was optimized for use in 384-well plates, and each demonstrated Z′-factors > 0.6, indicating their suitability for high-throughput screening. Details of assay setup and performance will be described. A test library of FDA-approved compounds was used to establish the reproducibility of these assays, and data on hits from this test library against each of the viruses will be presented. Screening of a diverse library of 50,000 compounds is underway.

Conclusion: Methods and assays to permit high-throughput screening of compounds against multiple BSL-4 viruses were established and used to identify compounds with in vitro antiviral activities.

Keywords: Antivirals, BSL-4, Ebola virus

17th International Congress of Virology 174

Abstract Submission

Antivirals

PO628

INACTIVATION EFFECTS OF CALCIUM HYDROGEN CARBONATE MESOSCOPIC CRYSTALS ON VARIOUS KINDS OF ANIMAL VIRUSES INCLUDING FOOT-AND-MOUTH DISEASE VIRUS.

M. Osaki 1, K. Frusaki 2, R. Onishi 2, T. Onodera 3, H. V. Dang 4, R. Kirisawa 1,*

1Pathobiology, Rakuno Gakuen University, Ebetsu, 2Mineral Activation Technical Research Center, Omuta, 3Research Center for Food Safety, The University of Tokyo, Tokyo, Japan, 4Biochemistry and Immunology, National Institute of Veterinary Research, Hanoi, Viet Nam

Objectives: Virucidal effects of a novel electrically charged disinfectant, CAC-717, on various kinds of viruses were investigated. CAC-717 is an alkaline solution (pH 12.3) and has a self-electromotive force, but it is harmless and not irritant to humans and animals because it contains no chemicals. CAC-717 is produced by applying an electric field to mineral water containing calcium hydrogen carbonate in order to generate mesoscopic crystals.

Methods: Sixteen kinds of animal viruses were used: foot-and-mouth disease virus (FMDV) (A, O and Asia1 types), bovine rhinitis B virus, bovine adenovirus type 7 (BAdV-7), infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus 3, bovine respiratory syncytial virus, bovine viral diarrhea virus (BVDV) (genotypes I and II), equine herpesvirus 1 (EHV-1), equine influenza A virus (H3N8), Aujeszky’s disease virus (ADV), swine influenza A virus (pandemic, H1N1), canine parvovirus 2 (CPV-2), canine distemper virus, canine herpesvirus 1, Newcastle disease virus and bulbul orthoreovirus. Nine volumes of CAC-717 were mixed with one volume of virus and incubated for 1 hour at room temperature. CAC-717 was removed by gel filtration on Sephadex LH-20. Virus titers were determined by titration in appropriate cell cultures. Effects of CAC-717 against viral genomes inside virions were examined by quantifying their genomes by real-time PCR.

Results: Virus titration analyses showed that CAC-717 treatment achieved reductions of 2 log10 to 5.5 log10 and over against all of the viruses tested in this study. The influence of fetal bovine serum (FBS) as organic matter on the antiviral effect of CAC-717 was examined. In the presence of 20% to 50% FBS, the virucidal effect of CAC-717 was abolished. Real- time PCR revealed that among the DNA viruses tested, CAC-717 treatment tended to reduce the amounts of BAdV-7, IBRV and EHV-1 genomes. Genomes of RNA viruses except for BVDV were reduced drastically after CAC-717 treatment. Direct treatment of CAC-717 to ADV and CPV-2 DNAs and BVDV RNA, the quantity of which was not reduced after CAC-717 treatment to virions, destroyed their genomes.

Conclusion: Virucidal effects of CAC-717 on enveloped DNA and RNA viruses and on non-enveloped DNA and RNA viruses were observed. CAC-717 could inactivate all types of animal viruses including FMDV. However, organic matter influenced the virucidal effect of CAC-717. CAC-717 might act on the inside of most RNA viruses and destroy their RNA genomes. The electrical potential generated on the surface of minerals in CAC-717 and its high pH value (pH 12.3) might play key roles in the anti-viral mechanism of CAC-717. CAC-717 may be used effectively and safely as a disinfectant against various kinds of animal viruses.

Keywords: antivirus, disinfectant, mesoscopic crystal

17th International Congress of Virology 175

Abstract Submission

Antivirals

PO629

Inhibition of chikungunya virus (CHIKV) replication by proteasome inhibitors

P. Kaur 1,*, J. J. H. Chu 1

1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, NATIONAL UNIVERSITY OF SINGAPORE, SINGAPORE, Singapore

Objectives: Chikungunya virus (CHIKV) is an arbovirus that has re-emerged as a significant public health threat in the last decade. Despite this, there is currently no antiviral treatment for chikungunya infection. The aim of this study was to study the inhibitory effects of proteasome inhibitors against CHIKV. In addition, the potential mechanism of anti-CHIKV action of bortezomib, and FDA-approved proteasome inhibitor, was also studied.

Methods: In this study, a plaque-assay-based screen of a panel of proteasome inhibitors was performed. Bortezomib was selected for further analysis of its antiviral mechanism. The anti-CHIKV activity of bortezomib was tested in different cell lines, on different CHIKV strains and multiplicity of infection (MOI) to ensure that results were not due to a cell-line specific effect. In order to identify the stage in the CHIKV replication cycle that bortezomib inhibited, time-of-addition and time-of- removal studies were performed. In addition, Western blot and quantitative reverse transcription-PCR (qRT-PCR) analyses were conducted to determine the effect of bortezomib on CHIKV RNA production as well as viral protein expression.

Results: The majority of proteasome inhibitors were found to inhibit CHIKV infection at minimally cytotoxic concentrations, suggesting that the proteasome may play a pro-viral role in CHIKV infection. Bortezomib, a proteasome inhibitor approved for the treatment of multiple myeloma, displayed potent inhibition of CHIKV infection across different CHIKV strains, MOI and cell lines. Bortezomib treatment resulted in an inhibition of 1 to 2 log10 PFU/ml (or 90-99% inhibition) with minimal cytotoxicity. Time-of-addition and time-of-removal data suggested that bortezomib inhibited an early, post-entry stage of the CHIKV replication cycle. Western blot analysis revealed a significant decrease in CHIKV structural protein levels. However, levels of CHIKV nsP4 (RNA-dependent RNA polymerase) was prominently elevated from early timepoints, suggesting the possibility that CHIKV nsP4, like other alphaviruses, is degraded by the proteasome. Despite obvious changes in protein levels, qRT-PCR did not show major changes in viral RNA levels at early timepoints.

Conlusion: This study suggests that proteasome inhibitors like bortezomib may represent a class of compounds with anti- CHIKV activities. One possible way in which bortezomib exerts its inhibitory effects is by preventing proteasomal degradation of CHIKV nsP4 synthesis, as well as reducing levels of viral structural proteins.

Keywords: antiviral, Chikungunya virus, proteasome

17th International Congress of Virology 176

Abstract Submission

Antivirals

PO630

Nanoparticulate v-ATPase blocker exhibits potent antiviral activity against feline peritonitis virus

W.-S. Chang 1,*, W.-L. Wang 1, Z.-S. Fang 1 2, L.-L. Chueh 1, T. Takano 3, T. Hohdatsu 3, C.-M. J. Hu 2 4, H.-W. Chen 1 4

1Department of Veterinary Medicine, National Taiwan University, 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 3School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan, 4Research Center for Nanotechnology and Infectious Diseases, Taipei, Taiwan

Objectives: Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. So far, it remains incurable, and there is no effective vaccine commercially available.The compound diphyllin, a novel vacuolar ATPase blocker, has been shown to inhibit the intracellular endosomal acidification, thus interfering the infection of influenza virus and dengue virus. This study aims to investigate the antiviral activities of diphyllin and nanoparticulate diphyllin against the type II feline infectious peritonitis virus (FIPV) strain NTU156.

Methods: Viral plaque assays and cell cytopathic effect protection assays were performed to investigate the antiviral activity of diphyllin against the local strain of FIPV, NTU156. Acridine orange labeling and immunofluorescence staining were respectively used to examine the inhibition level of endosomal acidification and virus expression of diphyllin on fcwf-4 cells. To investigate the optimal time point for diphyllin treatment, a time-of-addition drug assay was performed. Furthermore, the cooperative antiviral effect was evaluated by combinatorial use of dipyllin and Galanthus nivalis agglutinin (GNA), a pathogen-targeting antiviral drug. In addition, the in vitro antibody dependent enhancement model (ADE) of FIPV infection was established in two different cell lines to mimic clinical manifestation, and the viral inhibitory effect of diphyllin in the context of ADE was also examined. Towards a safer and more efficacious translational use of diphyllin, nanotechnology was applied in this study to prepare a nanoparticulate diphyllin formulation. The properties of diphyllin-loaded nanoparticles were analyzed, and the antiviral effect was investigated.

Results: Diphyllin dose-dependently inhibited endosomal acidification in Fcwf-4 cells. Treatment with diphyllin altered the cellular susceptibility to FIPV and inhibited the downstream virus replication. Furthermore, combinatorial treatment of the host-targeting diphyllin with pathogen-targeting GNA demonstrated enhanced cell protection. In the in vitro model of ADE of FIPV infection, diphyllin also showed a significant antiviral activity against FIPV in Fcwf-4 cells and U937 cells. In addition, nanoparticle-delivered diphyllin further demonstrated a decreased cytotoxicity and enhanced inhibitory effect against FIPV. These results collectively suggest that diphyllin possesses therapeutic potential for the treatment of FIP.

Conclusion: The diphyllin compound demonstrated prominent inhibitory activity against FIPV infection through attenuating cellular organelle acidification, thus blocking virus entry and uncoating. In addition, diphyllin treatment exhibited suppression in viral load in the context of ADE during FIPV infection in vitro. By using a nanoparticle delivery system, diphyllin-loaded nanoparticles showed lower cytotoxicity and enhanced antiviral activity in the nanomolar range. The results generally provide useful information towards discovery and development of new potential therapeutics and superior antiviral strategies against FIP.

Keywords: Feline infectious peritonitis virus, nanoparticles, Vacuolar ATPase inhibitor

17th International Congress of Virology 177

Abstract Submission

Antivirals

PO631

NUCLEOSIDE INHIBITORS OF TICK-BORNE ENCEPHALITIS VIRUS: STRUCTURE-ACTIVITY RELATIONSHIPS AND VIRAL RESISTANCE STUDY

L. Eyer 1,*, R. Nencka 2, D. Ruzek 1

1Virology, Veterinary Research Institute, Brno, 2Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Prague, Czech Republic

Objectives: Tick-borne encephalitis virus (TBEV) represents one of the most serious arboviral neuro-infection in Europe and northern Asia with thousands of TBEV-infected people and many reported deaths annually. The characteristic clinical symptoms of acute TBE range from a mild meningitis to severe meningoencephalitis/myelitis with the risk of temporary or permanent neurologic sequelae after TBE infection. As no specific antiviral therapy is available at present, there is an urgent need for efficient drugs to treat patients with TBEV infection. We report here a structure-activity relationship study based on the anti-TBEV activity/cytotoxicity profiles of 29 chemically modified nucleosides. Moreover, we describe the molecular base of acquired resistance of TBEV to 2´-C-methylated nucleosides and its phenotypical consequences.

Methods: Following methodological approaches were used: in vitro evaluation of antiviral activity and cytotoxicity of nucleoside analogues replication in cell lines of neuronal as well as extraneural origin, in vivo studies of these compounds using a mouse model of TBEV infection, in vitro selection of drug-resistant TBEV mutants and their molecular and phenotypical characterization, advanced computational methods to simulate the interactions of selected nucleosides with TBEV polymerase active site.

Results: Our data demonstrate a relatively stringent structure-activity relationship for modifications at the 2´, 3´, and 4´ nucleoside positions. Whereas nucleoside derivatives with the methylation at the C2´ position or azido modification at the C4´position exerted a strong TBEV inhibition activity (EC50 from 0.3 to 11.1 µM) and low cytotoxicity in vitro, substitutions of the O2´ and O3´ positions led to a complete loss of anti-TBEV activity (EC50 > 50 µM). Moreover, some structural modifications of the heterobase moiety resulted in a high increase of cytotoxicity in vitro. Antiviral activity of 7-deaza-2´-C- methyladenosine was evaluated in vivo using a lethal rodent model of TBEV infection; the dose of 25 mg/kg twice a day (intraperitoneal application) resulted in survival rate of 60%. In order to assess the acquired resistance of TBEV to 2´-C- methyl modified nucleosides, we isolated a drug-resistant TBEV mutant by serial in vitro passage of the Hypr TBEV strain on porcine stable kidney cells under the selective pressure of 7-deaza-2´-C-methyladenosine. Our results strongly indicate that a signature mutation (S603T) within the active site of the viral RNA-dependent RNA-polymerase confers a high-level resistance of tick-borne encephalitis virus to the family of 2´-C-methylated nucleosides, however, it leads to resistance- associated loss of viral fitness in cellular culture, as well as in decreased replication capacity and virulence potency (attenuation) in vivo.

Conclusion: C2´ Methylated or C4´ azido substituted pharmacophores appear to be useful as scientific models, research tools or starting points not only for searching new antiviral drugs against TBEV or other (flavi)viral infections, but also for studies of the resistance development towards nucleoside drugs.

This study was supported by the Czech Science Foundation project GA16-20054S; Czech Agency for Medical Research, Ministry of Health of the Czech Republic (grant No. 16-34238A); and by project LO1218, with financial support from the Ministry of Education, Youth, and Sports of the Czech Republic under the NPU I program.

Keywords: drug resistance, structure-activity relationship, tick-borne encephalitis virus

17th International Congress of Virology 178

Abstract Submission

Antivirals

PO632

Screening and identification of entry inhibitors against Lassa Virus from an FDA-approved drug library

P. Wang 1,*, W. Wang 1

1Chinese Academy of Sciences, Wuhan Institute of Virology, Wuhan, China

Objectives: Lassa virus(LASV) is responsible for outbreak of fatal Lassa hemorrhagic fever in western Africa, where as many as 300,000 infections occur per year. There are no FDA-licensed LASV vaccines and current therapy is limited to the use of ribavirin which has only limited efficacy. Viral entry represents the first step of every viral infection and is a determinant for the host-range and disease potential of a virus.

Methods: Here we performed a high throughput drug screen from an FDA-approved drug library to identify potential entry inhibitors for LASV using pseudotype virus expressing Lassa glycoprotein.

Results: We uncovered a small molecular Lacidipine, which is a L-type voltage-dependent calcium channel blocker, exhibits significant anti-LASV activity with a 50% inhibitory concentration(IC50) of 2.6 μM. Lacidipine also inhibited pseudotypes generated with other arenavirus envelopes including the Old-world group Mopeia and New-world group Guanarito but had no antiviral activity against pseudotypes incorporating the unrelated G protein from vesicular stomatitis virus. Quantitative binding and virus internalization assays reveal that LASV internalization was affected by Lacidipine treatment. Through selection of Lacidipine-resistant virus, we found substitution of threonine at the ectodomain loop of stable signal peptide(T40) with lysine might confer LASV resistance to Lacidipine.

Conclusion: Together, our study identifying an effective small molecular entry inhibitor against Lassa Virus and may provide a novel candidate for further therapy against arenavirus.

Keywords: Glycoprotein, Lacidipine, Lassa virus

17th International Congress of Virology 179

Late breaking abstract

Antivirals

PO633

Structure-based discovery of inhibitors targeting influenza A virus nucleoprotein

J. N. Makau 1,*, K. Watanabe 1, T. Ishikawa 1, S. Mizuta 1, T. Hamada 2, N. Kobayashi 1, N. Nishida 1

1Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, 2Nagasaki Advanced Computing Center, NAGASAKI UNIVERSITY, Nagasaki, Japan

Objectives: The propensity of influenza viruses to develop resistance to current therapeutics calls for continued development of drugs with novel mechanisms of action. Influenza A virus nucleoprotein (NP) is highly conserved and an essential component of the ribonucleoprotein complex for transcription and replication of the virus. In this study, we aimed at finding small molecule inhibitors of NP-NP oligomerization using structure-based drug design approach.

Methods: Our work employed an original structure-based drug discovery algorithm termed Nagasaki University Docking Engine (NUDE) established especially for the Destination for GPU Intensive Machine supercomputer. NUDE was used to select compounds that bind to NP oligomerization pocket from a custom chemical library. Compounds that showed high binding scores during in silico screening were evaluated for antiviral activity in a cell-based assay. The binding affinities between compounds and recombinant NP was then investigated using surface plasmon resonance assay and molecular interactions analyzed by fragment molecular orbital calculations.

Results: We found a compound designated NUD-1 belonging to 4-hydroxyquinolinone family that effectively suppressed the replication of influenza A virus. Analysis of binding between NUD-1 and NP revealed that the compound could bind to NP and inhibit NP-NP oligomerization. Time of addition assay showed that the compound inhibited the mid-stage of virus replication, corresponding to assembly of the NP and other viral proteins. Moreover, NUD-1 exhibited potent activity against various strains of influenza A virus including a clinical isolate of 2009 H1N1 flu pandemic with a 50% inhibitory concentration range of 1.8 – 2.1 µM.

Conlusion: Taken together, our data demonstrate that NUD-1 has a novel mechanism of action and is a potential lead compound for anti-influenza drug development. In addition, NUDE drug discovery aligorithm is a helpful tool for fast identification of important chemical compounds.

Keywords: Influenza A virus, Nucleoprotein, Structure based drug design

17th International Congress of Virology 180

Abstract Submission

Antivirals

PO634

THE EFFECT OF ANTIVIRAL AGENTS ISOLATED FROM FUNGI AGAINST PEPPER MOTTLE VIRUS-VB1/GFP IN PEPPER.

H. M. Lee 1,*, E. G. Song 1, S. M. Ryu 2, K. H. Ryu 1

1Plant virus genbank, Seoul women`s university, 2Biosystems and Biotechnology, Korea University, Seoul, Korea, Republic Of

Objectives: Major crops containing food, vegetable and flowers can cause by plant pathogens: fungi, bacteria, insect, nematodes and viruses. Plant viruses cause an estimated US $60 billons loss in crop yields worldwide each year (McDonald, 2012). Effective pesticides for fungi, bacteria, insect and nematodes were already developed. However, effective antiviral agents were not reported. In this study, we study to find effective antiviral agents for plant virus and to prevent major crops against virus diseases.

Methods: Pepper (Capsicum annuum L.) infected with pepper mottle virus-Vb1/GFP (PepMoV-Vb1/GFP), a green fluorescent protein (GFP) tagged PepMoV, was used in leaf disc method and systemic host method.

1. Leaf disc method

3 Leaf discs produced from pepper infected with PepMoV-Vb1/GFP were put into 1/2 MS media with antiviral agents. The 20 extracts isolated from fungi were evaluated in leaf disc method and observed in visible and ultraviolet (UV) lights for 7 days. The compounds isolated from F1882 EtOAc-extract were also used in leaf disc method.

2. Systemic host method

Inactivation effect

The antiviral agents mixed with PepMoV-Vb1/GFP were incubated for 30 minutes before mechanically inoculated in systemic host plant (pepper).

Protective effect

The antiviral agents mixed with tween 20 were spread on leaves of healthy plants and then PepMoV-Vb1/GFP was mechanically inoculated in the treated leaves after 1 hour.

Results: F1882 EtOAc-extract derived from wood-rotting basidiomycete fungus was observed antiviral activity in leaf disc method. This result was confirmed by RT-PCR and western blot analysis. In systemic host, the compounds isolated from F1882 EtOAc-extract were evaluated using RT-PCR and western blot analysis. Consequently, these compounds showed resistance to PepMoV-Vb1/GFP in leaf disc method and systemic host method.

Conclusion: This study demonstrated that the compounds isolated from fungi were antiviral activity against PepMoV- Vb1/GFP in pepper.

Keywords: leaf disc method, PepMoV-Vb1/GFP, systemic host method

17th International Congress of Virology 181

Abstract Submission

Antivirals

PO635

THE TLR4 ANTAGONIST ERITORAN INHIBITS DENGUE NS1 INDUCED ACTIVATION IN VITRO AND IMPROVES SURVIVAL IN AN IN VIVO MODEL OF DENGUE INFECTION

N. Modhiran 1,*, D. Watterson 1, S. Cheung 1, K. J. Stacey 1, P. Young 1

1School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia

Objectives: Dengue virus (DENV) is the most prevalent arboviral infection, especially in tropical and subtropical regions, affecting millions of people annually. Life-threatening complications of DENV infection include potential hemorrhage and shock. With no specific antiviral treatment currently available for DENV infection there is an ongoing need for drug development. We previously revealed that the highly conserved non-structural protein, NS1 activates cells via Toll-like receptor (TLR) 4, which in vivo may lead to an over-robust inflammatory response that contributes to vascular leak. We further showed that the natural TLR4 antagonist, LPS-RS inhibited both NS1-mediated activation of peripheral blood mononuclear cell (PBMC) and NS1-induced vascular leak in vitro, and reduced dengue virus-induced vascular leakage in a mouse model. This discovery suggested that other TLR4 antagonists could be effective in therapeutic intervention of dengue infection. Considerable efforts have been made over the last 20 years to develop TLR4 antagonists as sepsis inhibitors. In this study we explored the potential of one of the leading sepsis candidates to have gone through clinical trials, Eritoran (Eisai Co., Ltd.), as an inhibitor of NS1-mediated TLR4 activation.

Methods: To investigate the potential of re-purposing sepsis drugs for treatment of dengue infection, the inhibitory effects of Eritoran and LPS-RS on NS1-mediated innate immune responses induced in vitro were assessed in PBMC by measuring IL-6 production. We also assessed the effect of Eritoran on NS1-induced endothelial cell monolayer disruption by measuring trans-endothelial electrical resistance (TEER). In addition, the AG129 mouse model of dengue infection was used to confirm the effect of Eritoran in vivo. The prophylactic and therapeutic effects of Eritoran treatment was assessed in groups of mice administrated both IV and IP. Survival, morbidity and weight loss were observed and serum collected to determine viral load and IL-6 levels.

Results: We demonstrated that Eritoran potently inhibited DENV NS1 stimulated IL-6 production in PBMCs. The drug also inhibited NS1-mediated monolayer disruption in vitro. Intravenous and intraperitoneal injection of Eritoran starting from day 2 post-infection, but not a pre-treatment regimen, improved survival to 60% and 40%, respectively, at day 8 post infection. Viral loads were significantly reduced by all treatments compared to the matched control groups. A non-statistically significant reduction of serum IL-6 from the Eritoran-treated group was observed at day 3 post-infection compared to untreated infected mice.

Conclusion: These results suggest that Eritoran blockade of NS1 induced responses represents a novel therapeutic approach for DENV treatment.

Keywords: Dengue virus, NS1, TLR4

17th International Congress of Virology 182

Abstract Submission

Antivirals

PO636

THERAPEUTIC EFFECTS OF FAVIPIRAVIR IN THE TREATMENT OF LETHAL SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME (SFTS) VIRUS INFECTION

H. Tani 1 2,*, S. Fukushi 2, T. Kurosu 2, A. Uda 3, S. Morikawa 3, T. Komeno 4, N. Nakajima 4, Y. Furuta 4, M. Shimojima 2, M. Saijo 5

1Virology, University of Toyama, Toyama, 2Virology I, 3Veterinary Science, National Institute of Infectious Diseases, Tokyo, 4Research Laboratories, Toyama Chemical Co., Ltd., 5Virology I, National Institute of Infectious Diseases, Toyama, Japan

Objectives: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with a high case fatality rate caused by SFTS virus (SFTSV). Because there are currently no effective therapeutics for SFTS, potent and safe antivirals are needed for the treatment of SFTS. Recently, we reported the efficacy of favipiravir in treating SFTSV infection both in vitro and in vivo. In this study, the therapeutic effects of favipiravir against SFTSV infection after onset of the disease were investigated in a mouse lethal model.

Methods: Mice lacking the type I interferon receptor (IFNAR-/-) were used as an in vivo lethal model for SFTSV infection. IFNAR-/- mice were orally administered twice daily with favipiravir at either dose of 120 and 200 mg/kg/day after challenging with 106 TCID50 of SFTSV (SPL010). Viral RNAs in the blood samples of infected mice were determined by quantitative RT-PCR.

Results: All the mice treated with favipiravir at either dose of 120 and 200 mg/kg/day survived lethal SFTSV infection when the treatment was initiated on or earlier than 3 and 4 days postinfection, respectively. Although the RNA levels increased until administration of favipiravir, they were almost undetectable at 14 days postinfection.

Conclusion: The efficacy of favipiravir at the approval dosage for human on lethal SFTSV infection was demonstrated in IFNAR-/- mice. Furthermore, favipiravir was highly effective against lethal SFTSV infection even when treatments were initiated after the onset of the disease in the mouse lethal model. These results suggest that favipiravir treatment may be one of the choices for the treatment of SFTS patients.

Keywords: Favipiravir, SFTS virus

17th International Congress of Virology 183

Abstract Submission

Antivirals

PO637

VIRUCIDAL ACTIVITIES OF PROTEOGLYCANS PREPARED WITH UMEZU, A SALT-EXTRACT OF JAPANESE APRICOT

K. Ikeda 1, M. Yamaguchi 2, T. Nagao 3, M. Nishide 4, M. Yamaguchi 2, A. H. Koyama 5,*, T. Kuwahara 5

1School of Health and Nursing, Wakayama Medical University, 2Faculty of Education, Wakayama University, Wakayama, 3Faculty of Nursing , Shikoku University, Tokushima, 4Food and Nutrition, Wakayama Shin-Ai Women’s Junior College, Wakayama, 5Molecular Microbiology, Kagawa University Faculty of Medicine, Kita-gun, Japan

Objectives: To develop the safe disinfectant applicable for human mucosal membrane and injured body surface, we have been isolating and characterizing antiviral and virucidal activities of food-derived materials, such as polyphenols, amino acids, fatty acids and their derivatives. In this report, we show the virucidal activity of proteoglycans (PG) from salmon.

Methods: The PG were extracted from salmon cartilage with “umezu”, derived from the salt-extracts of Japanese apricot (Nanko-mume; Prunus mume Sieb et Zucc, a special product of Wakayama) or acetic acid by an ordinary acid-extraction method. The viruses used were poliovirus type 1 Sabin vaccine strain (PV) and feline calicivirus strain F4 (FCV) as non- enveloped viruses and herpes simplex virus type 1 strain F (HSV-1) and influenza viruses A/PR8 (H1N1), A/Aichi/68 (H3N2) and B/Tokyo as enveloped viruses. The virucidal activity was determined by incubating the virus in the buffer containing PG at various concentrations. Immediately after the incubation, the virus preparations were diluted with ice-cold phosphate- buffered saline (PBS) containing 0.1 % BSA for influenza viruses or 0.5 % FBS for other viruses and the number of infectious virus was measured by a plaque assay.

Results: (1) Umezu-extracted PG inactivated influenza viruses concentration-dependently. The most effective inactivation was observed in B/Tokyo virus which lost the infectivity less than one hundredth at the concentration of 2 mg PG/ml in the incubation mixture. The similar inactivation was observed for two subtype of A viruses, showing influenza viruses can be inactivated by PG regardless of its type or subtypes. (2) HSV-1 was also inactivated by PG, but PV-1 and FCV, a non- enveloped viruses, were not inactivated. (3) When PG were extracted with acetic acid, instead of umezu, the PG preparation showed stronger virucidal activity, indicating that the activity does not accompany with a possible contaminated virucidal substance from apricot. (4) PG did not show notable antiviral activities or cytotoxic effects at the concentrations with their virucidal activities. (5) PG decreased the plating efficiency (the E.O.P.) of HSV-1 as expected by its receptor activity for the virus, but it did not affect the E.O.P. of influenza virus or PV. (6) Surprisingly, column-purified PG preparation showed no virucidal activity, indicating that it is not PG molecules to have these virucidal activities, but probably some contaminated substance(s) in the PG preparations.

Conclusion: PG extracted from salmon cartilage with umezu or acetic acid showed remarkable virucidal activity at the concentrations without notable cytotoxic effects. Further characterizations suggested that the activity was associated with possible contaminated substance with high molecular weight, because the column-purified PG lost the activity. These results may indicate a potential usefulness of PG in the health care fields if considered that a disinfectant with low cytotoxicity and nonirritative property may be applicable in vivo against topical infectious diseases.

This work was supported in part by a research grant from JA-Kinan and Tanabe City, Wakayama Japan.

Keywords: None

17th International Congress of Virology 184

Abstract Submission

Antivirals

PO872

MARVAS09 acts as a potent inhibitor of Human Enterovirus 71 replication through suppressing viral IRES in vitro and in vivo – a suitable clinical candidate for treating HEV71 infection

S. Gunaseelan 1,*, J. J. H. Chu 2

1Physiology, 2Microbiology and Immunology, NATIONAL UNIVERSITY OF SINGAPORE, Singapore, Singapore

Objectives: Through eukaryotic studies, 2 translational mechanisms have been described, namely the canonical cap- dependent translation and the IRES-facilitated cap-independent translation. By mapping these translational pathways into a bicistronic construct, potential antivirals that exclusively suppress HEV71 IRES-mediated translation without influencing host-dependent translation can be identified. The most effective drug can then be selected and studied in vitro for its cytotoxicity and inhibitory profiles, spectrum of antiviral activity, antiviral mechanism and drug resistance properties; and can be subjected for evaluation in murine models to reaffirm its efficiency against HEV71 infection.

Methods: To screen a 500-compound flavonoids library, a bicistronic reporter construct consisting of renilla and firefly luciferase genes was developed. After transcription, the renilla and firefly luciferase genes are subsequently translated into their corresponding proteins, in a cap-dependent and HEV71 IRES facilitated cap-independent manner respectively. A bicistronic reporter hairpin was established as a negative control, which terminated cap-independent translation only. Transient transfections of both constructs along with heterogeneous G418 selection were carried out to establish their expression levels overtime. Z-factor tests were also performed to reaffirm their consistency and reliability. Selected hits from screening were further verified with cell viability and viral plaque assays. The most effective antiviral was then subjected to downstream secondary assays such as western blot and qRT-PCR to study its effect on viral protein and RNA synthesis. Alternative HEV71 strains (H, C4 and B5), related Enteroviruses (CA6, CA16 and ECHO7) and non-related viruses (HSV and CHIKV) were also treated with the identified drug to determine its spectrum of activity. HEV71-resistant mutants were obtained via continuous HEV71 culturing with the drug and were subsequently sequenced to identify HEV71 genomic variations. Lastly, 1-day-old HEV71-infected BALB/c pups were treated with the drug and observed for survival, body mass and mean clinical score over a course of 14 days.

Results: The bicistronic plasmid-based transient transfection assays in RD cells was highly reliable and consistent with a Z-factor of 0.859. Through utilizing this assay, MARVAS09 was discovered as the most potent suppressor of IRES-facilitated cap-independent translation with a dose of 20µg/ml. Downstream secondary assays revealed the EC50 and CC50 of MARVAS09 to be 115.3nM and 2715nM respectively, with a high SI of 23.6 and minimal cytotoxicity effects. 1µM of MARVAS09 reduced HEV71 viral titre by 3.5 log10 PFU/ml and prevented HEV71 protein detection at both 6 and 12hpi. Moreover, MARVAS09 (1µM) was able to reduce HEV71 RNA production by 2 and 1.5 log10 units at 6 and 12hpi respectively. Further studies also deduced MARVAS09 to be effective against Enteroviruses but not CHIKV or HSV at its EC50. Sequence analysis of drug resistant HEV71 mutants revealed 5 mutations at positions 165, 166, 178, 369 and 371 of HEV71 IRES region, hence proving MARVAS09 inhibitory action on IRES. When administered at 3mg/kg in murine model, MARVAS09 offered full protection of mice against HEV71 infection.

Conlusion: The HEV71 bicistronic reporter assay can serve as a high-throughput screening platform for identifying antivirals targeting against HEV71 infection. MARVAS09 had potent antiviral activity against HEV71 in vitro and in vivo and may be a promising clinical candidate for further development as an anti-HEV71 therapeutic agent.

Keywords: None

17th International Congress of Virology 185

Abstract Submission

Antivirals PO 881

N-HETEROCYCLIC BORNEOL DERIVATIVES AS INHIBITORS OF MARBURG GLYCOPROTEIN-MEDIATED VSV PSEUDOTYPE ENTRY S. Cheresiz, A. Kononova 1,*, O. Yarovaya 2, A. Chepurnov 3, R. Nikitina 3, A. Pokrovsky 1, N. Salakhutdinov 2, A. Sokolova 2 1Medicine, Novosibirsk State University, 2Institute of Organic Chemistry, 3Institute of Clinical Immunology, Novosibirsk, Russian Federation

Objectives: Our objective was to study the activity of N-containing bornyl ester derivatives as the entry inhibitors of Marburg virus using a VSV capsid-based pseudovirus system (rVSV-ΔG). Methods: The studied library includes borneol itself as well as two groups of N-containing bornyl ester derivatives differing in the length of the aliphatic chain between the nitrogen atom and the ester group. Synthesis of the target compounds was described elsewhere. Marburg glycoprotein-pseudotyped and VSV(G) glycoprotein-pseudotyped VSV pseudoviruses were obtained by the transfection of corresponding expression plasmids into FT293 producer cells with a subsequent infection of transfected cells with VSV(G)-rVSV(Luc) pseudovirus caarying the luciferase reporter. Pseudovirus infectivity in the presence of inhibitors and in control (noninhibited) samples was measured using luminometry, All measurements were performed in triplicate, with calculation of the mean value and standard deviation. Verapamil was used as a reference specific Marburg virus entry inhibitor. Results: For all compounds, the half maximal inhibitory concentration (IC50) was determined for both rVSV-ΔG-MarV and rVSV- ΔG-G* pseudoviruses. Further, the selectivity index (SI), a ratio of compound toxicity to inhibitory activity against the Marburg virus (CC50/ICMarV50), and the inhibitor specificity coefficient (SC), a ratio of half maximal inhibitory concentrations for two pseudoviruses (ICMarV50/ICVSV50), were calculated for each compound. As a reference compound inhibiting filovirus infection but not affecting VSV infection at therapeutic concentrations, we chose an ion channel inhibitor, verapamil. Borneol itself is not active against both pseudoviruses and is nontoxic, whereas its derivatives have varying toxicity and antiviral activity. Among the low-toxic borneol derivatives, six compounds were identified that were relatively specific inhibitors of Marb-GP-mediated infection (SC>10). The derivative containing a methylpiperidine moiety exhibited the highest virus-specific activity with IC50 three times lower than the reference filovirus entry inhibitor, verapamil (4 µM/mL vs . 13 µM/mL). We revealed some relationships between the structure of the heterocyclic moiety and compound properties. The compounds containing methylpiperazine and ethylpiperazine moieties have a high therapeutic index against rVSV-ΔG-MarV exceeding that of the reference compound. In addition, we found that the selectivity index of produced compounds was dependent on the length of the aliphatic chain. Compounds with a longer chain turned to be more active against rVSV-ΔG-MarV and to have a greater selectivity index. Conclusion: Thus, our study demonstrates the Marburg virus entry inhibition by some N-heterocyclic bornyl esters with the efficiency higher or comparable to that of a previously described specific filovirus entry inhibitors (verapamil). Further studies should reveal the molecular mechanisms responsible for infection inhibition, and the obtained data may be useful for development of more active and specific borneol-based inhibitors of filovirus infection.

Keywords: Entry inhibitor, Marburg virus, VSV pseudotype

17th International Congress of Virology 186

Arboviruses

17th International Congress of Virology 187

Abstract Submission

Arboviruses PO638

A Seroepidemiological Study of Dengue virus Infection among elderly after the largest outbreak in 2015 in Kaohsiung, Taiwan Y.-H. Pan 1,*, P.-Y. Chen 1, M.-Y. Liao 1, Y.-W. Chien 2, S.-F. Chang 3, P.-Y. Shu 3, Y.-F. Wang 4, C.-Y. Chi 4, C.-Y. Pan 5, C.-C. King 1 1Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, 2Department of Public Health , National Cheng Kung University , Tainan, 3Centers for Disease Control, R.O.C., Taipei, 4National Health Research Institute, Tainan, 5Department of Health, Kaohsiung City Government, Kaohsiung, Taiwan, Province of China

Objectives: Dengue viral infection has rapidly spread around the world and has become a global public health concern in recent decades. In 2015, Kaohsiung City (a metropolis located in southern Taiwan), had experienced the largest and most severe dengue outbreak since 1987. The objective of this study was to investigate the high-risk area as well as high-risk age group of dengue virus infection in this large city. Methods: The study population was comprised 39- to 96-year old elderly who were the residents in Fengshen, Lingya, Sanmin and Qianzhen Districts in Kaohsiung. The blood samples were collected from local physical examination center during early 2016. The seroprevalence of dengue IgG antibodies in these serum samples was detected by using Dengue NS1 Serotype-specific IgG ELISA developed from the Centers for Disease Control in Taiwan (Taiwan CDC) and commercially available ELISA kits that are more suitable for community-based seroepidemiological studies than clinical tests of patients. The OD readings at the borderline were all reconfirmed by the second laboratory test. The data was analyzed by R software and Geographic Information System (GIS). Results: The overall seroprevalence rate of DENV-IgG among 1498 community residents at 39-96 years of age (mostly elderly) was 52.80%, including 49.64 %, 55.53% and 54.04 % in the Sanmin, LinYa and Qianzhen Districts, respectively. It implies that about 40- 50% of elderly living at the hot-spots of past dengue epidemics in Kaohsiung might be the target population for next epidemic. Interestingly, 660 elderly who were born before1943 (>73 yrs) had significantly higher seroprevalence rate of DENV-IgG than 838 those borne after 1943 [72.73% vs 37.11%, P< 0.0005], confirming the presence of a large-scale epidemic in Taiwan during 1942-43. The serological surveillance among the 85 elderly right after the occurrence of DENV-2(+) case [tested by RT-PCR] in ChenYi Li of FungShan District on Aug. 10, 2016 showed the seroprevalence of DENV-IgG in this neighboring area was 61.18%, indicating the importance of this district as next prevention site. Seroprevalence rate of dengue increased with age in these four districts in Kaohsiung, from 17.14% in elderly aged below 50 years to 73.76% aged over 80 years. Conclusion: The overall seroprevalence of DENV infection was around 50% among elderly in past dengue hot-spot districts of Kaohsiung and was estimated as at least around 70% by 75 years of age. Our results can explain that Kaohsiung had experiences several large epidemics in 1942-1943, 1987-1988, 2001-2002, 2006, 2009-2010, 2014 and 2015, continuous presence of the two major vectors (Aedes aegypti and Aedes albopictus) in Kaohsiung, and past seroepidemiological results confirmed by plaque reduction neutralization test (PRNT). Since secondary DENV infection might be correlated with severe dengue disease, it is important to monitor herd immunity, as well as viral load and co-morbidity to elucidate the importance of each risk factor contributing the severity of dengue among elderly. These findings highlight the importance of post-epidemic serological surveillance to find out the susceptible that would be the high-risk populations in future epidemics.

Keywords: Dengue virus, elderly, seroepidemiology

17th International Congress of Virology 188

Abstract Submission

Arboviruses PO639

ACCUMULATION OF SUBGENOMIC NONCODING RNA OF JAPANESE ENCEPHALITIS VIRUS DOES NOT RELY ON THE ACTIVITY OF EXORIBONUCLEASE XRN1 BUT DOES DEPEND ON VIRAL REPLICATION

Y.-S. Chen 1,*, Y.-H. Fan 1, C.-F. Tien 1, A. Yueh 2, R.-Y. Chang 1 1Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Shoufeng, 2Institute of Biotechnology and Pharmaceutical Researc, National Health Research Institutes, Miaoli , Taiwan

Objectives: All arthropod-borne flaviviruses accumulate abundant small subgenomic RNA (sfRNA) that is collinear with highly conserved regions of the 3’ untranslated region (UTR) in the viral genome. The sfRNA is generated by incomplete degradation of the cellular exoribonuclease XRN1, as demonstrated in several flaviviruses but not in Japanese encephalitis virus (JEV). Characterization of the mechanism for the sfRNA formation in JEV-infected cells was sought.

Methods: We analyzed the effect of XRN1 depletion by RNA interference on sfRNA generation in virus-infected cells, and further investigated the structure and sequence requirements for the production of sfRNA by an in vitro RNA-dependent RNA polymerase (RdRp) assay and mutagenesis studies in the context of a JEV infectious clone.

Results: Knocking down host XRN1 had no effect on the accumulation of sfRNA in JEV-infected cells. In contrast, an infectious clone containing a mutation at the active site (GDD) of the RdRp of the viral genome halted accumulation of the sfRNA indicating that functional RdRp is required for the formation of JEV sfRNA. According to the results of an in vitro RdRp assay, the (-)10431-10566 RNA fragment, representing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Furthermore, mutagenesis studies provided evidence that the stemloop II (SLII) determines the formation of the sfRNA. In addition, minus strand of the sfRNA was detected in the wild-type virus infected cells but not in the sfRNA-deficient virus infected cells.

Conclusion: Our results indicate that the JEV sfRNA is synthesized from the antigenome rather than being produced as a result of degradation of viral genome by exoribonuclease XRN1.

Keywords: sfRNA, noncoding RNA, XRN1

17th International Congress of Virology 189

Abstract Submission

Arboviruses PO640

CHARACTERIZATION OF JAPANESE ENCEPHALITIS VIRUS ISOLATED FROM PERSISTENTLY INFECTED CHICK EMBRYO CELLS

H. Toriniwa*, T. Komiya 1 1R&D Division, Kitasato Daiichi Sankyo Vaccine CO., LTD., Kitamoto, Japan

Objectives: Japanese encephalitis virus (JEV) is a positive-sense single-stranded RNA genome that belongs to the genus Flavivirus of the family Flaviviridae. Persistent infection of JEV was shown in pig blood cells, which act as a natural reservoir of this virus. Our objective was to determine pathogenicity factors involved in persistent infection of JEV by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model.

Methods: We established JEV persistent infections in cells by inoculating the Beijing-1 strain and Nakayama NIH strain of JEV to ddY mouse fetus primary cell cultures, and then repeated infected cell passages and harvested viruses after each passage while monitoring plaque size over the 100th generation. The virus growth rate was compared in Vero, C6/36 and Neuro-2α cells. The pathogenicity was examined in female ICR mice at several ages. Also, we determined the whole genome sequences.

Results: The 134th Beijing-1 derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain, but did not grow well in C6/36 and Neuro-2α cells. However, the 117th Nakayama NIH derived persistent virus (J117) grew at a similar rate to the parent strain in Vero and C6/36 cells. Moreover, J117 grew well in Neuro-2α cells. Differences in pathogenicity after intracerebral inoculation in mice of different ages was not observed, but the number of days of survival was extended in older age mice. Mutations in the persistent virus genomes were found across all regions, but were mainly focused in NS3, NS4b and 3'NCR region with a 34 base-deletion being found in the variable region.

Conclusion: We analyzed the pathogenicity and genome sequences of JE viruses isolated from persistent infections in mouse embryo cells by inoculating Beijing-1 or Nakayama NIH strain of JEV. The short deletion in 3'NCR region seemed to be responsible for the reduced pathogenicity and growth efficiency.

Keywords: Japanese encephalitis virus, Persistance

17th International Congress of Virology 190

Abstract Submission

Arboviruses PO641

COCIRCULATION OF DENGUE VIRUS DURING AN OUTBREAK OF CHIKUNGUNYA VIRUS IN DEMOCRATIC REPUBLIC OF CONGO

E. Ido 1,*, S. Ahuka 2, S. Karhemere 2, K. Ibuki 3, J. J. Muyembe 2 1Department of Molecular Virology, Tokyo Medical and Dental University, Tokyo, Japan, 2National Institute of Biomedical Research, Kinshasa, Congo, The Democratic Republic of the, 3Kyoto University, Kyoto, Japan

Objectives: Along with the unprecedented expansion of human migration and trade on the earth and perhaps due to the global warming too, mosquito-borne viruses appear to be rapidly spreading their natural habitats to new areas where such viral diseases were not existing or at least not well documented. The recent large-scale outbreaks of chikungunya virus and zika virus in Latin America may be good examples of that kind. Dengue virus (DENV) is known to be threatening the large number of people in Mid/South America as well as South/Southeast Asia, but not much information is available in tropical Africa partially due to the lack of adequate research facilities. Here we aimed to examine the existence of DENV in Democratic Republic of Congo (DRC).

Methods: Hundreds of febrile patients visited hospitals in Kinshasa, the capital city of Democratic Republic of Congo (DRC), between March and June in 2012. They were initially suspected of chikungunya virus (CHIKV) based on their clinical symptoms. The stored serum specimens were thus first tested by a CHIKV IgM kit and RT-PCR, and then the specimens were examined by a rapid test for DENV NS1 antigens and some of them were subjected to RT-PCR.

Results: Out of 96 examined, 10 were positive for CHIKV IgM antibodies and 5 were positive for CHIKV by RT-PCR. Nonetheless, 3 different specimens were unexpectedly found to be positive for DENV NS1 antigens. One out of 3 were positive by RT-PCR both in the PrM-E and NS5 regions. Genetic analyses have revealed that this DRC strain can be classified into the group of DENV serotype 1.

Conclusion: The febrile sickness observed in Kinshasa in 2012 was surely identified to be caused by CHIKV. However, the above results have also indicated that DENV infection simultaneously occurred during the outbreak of CHIKV. This is the first genetic report of DENV from DRC and neighboring central African countries. We have also learned a lesson that we need to test both viruses preferably including other mosquito-borne viruses because their clinical manifestations are very similar each other.

Keywords: dengue virus

17th International Congress of Virology 191

Abstract Submission

Arboviruses PO643

DENGUE VIRUS INFECTION CAN LEAD TO CARTILAGE LOSS AND AN OSTEOARTHITIS-LIKE DISEASE P. Rudd 1,*, L. Herrero 1, A. Zaid 1, H. Bielefeldt-Ohmann 2, D. Zhang 3, R. Li 3, A. Supramaniam 1, V. Costa 4, M. Teixeira 4, S. Alonso 5, S. Mahalingam 1 1Institute for Glycomics, Griffith University, Southport, 2School of Veterinary Science, University of Queensland, Gatton, 3College of Medicine, Biology and Environment, Australian National University, Canberra, Australia, 4Department of Biochemistry and Immunology, Biological Sciences Institute, Universidade Federal de Minas Gerais, Belo Horizonte - MG, Brazil, 5Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore

Objectives: Dengue virus (DENV) is a prominent arbovirus and member of the Flavivirus genus. Other well-known human pathogens from that genus include yellow fever virus and West Nile virus. DENV is the causative agent of dengue fever (DF) and of the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Mackenzie et al., 2004). Endemic in subtropical and tropical countries, around half of the global population is currently at risk of contracting DF or DHF/DSS and more than 50 million cases are reported each year (Schmidt et al., 2010; Tolle et al., 2009). Once known as break-bone fever or bone crushing disease myalgias and arthalgias are often associated with illness in addition to high fever, headache, chills, eye pain, rash, nausea/vomiting and mild bleeding. Very few studies have investigated the reasons why severe joint/muscle pain is part of the symptomatology of DF despite one cohort study showing up to 25% of adult patients suffer from arthralgia. The objective of our study was to further investigate the causes explaining these joint pains.

Methods: Towards this, we used a multifaceted approach using both in vivo and in vitro models of DENV infection. In the first instance, we used the AG129 mouse model to assess histopathological findings and qRT-PCR examining cartilage associated factors and pro-inflammatory cytokines, which may be key players in disease. We also examined the susceptibility of human chondrocytes to dengue infection and followed up with both qRT-PCR and BioPlex cytokine assays.

Results: Our results show that mice infected with a type-2 strain of DENV develop arthritis as well as cartilage damage/reduction as seen by safranin O’ staining. Human chondrocytes are susceptible to DENV and display productive infection with peak titers slightly above 10e4 PFU log10mL-1. qRT-PCR and BioPlex data indicate that matrix degradation is indeed occurring and osteoarthritis-like factors are secreted following DENV infection.

Conclusion: In conclusion, DENV seems to target chondrocytes and causes cartilage loss and induces a osteoarthritis-like disease. People with cartilage damage commonly experience joint pain, stiffness, and inflammation therefore the damage caused by DENV could explain the symptoms associated with infection. However, we do hypothesize that this damage would probably be transient in human patients since most recover without any long-term joint/muscle sequelae.

Keywords: cartilage, dengue virus, osteoarthritis

17th International Congress of Virology 192

Abstract Submission

Arboviruses PO644

Difference in responses of synoviocyte fibroblast to infection with various types of chikungunya viruses: clinical isolates, small-plaque, and large-plaque viruses A. Sukkaew*, S. Ubol

Objectives: To elucidate how different plaque sizes CHIKV affect pathogenicity in infected human synoviocyte fibroblasts. Methods: Two clinical isolates CHIKV from patients' sera were once propagated in C6/36 cells. Small- and large-plaque variants of CHIKVwere purified in Vero cells by plaque purification assay. Once we have got homogeneous large- and small- plaque variants, the viral titers were quantified by plaque assay in Vero cells. Human fibroblast-like synoviocyte (HFLS) cells were infected with clinical-isolated, large-plaque, and small-plaque variants of CHIKVs at 0.01 MOI. Supernatant from infected cells was harvested and quantified for the amount of infectious particles using plaque assay. The cytopathic effect induced by the viruses was observed under a microscope and classified into levels, + for 10-25%, ++ for 25-50%, +++ for 50-75%, and ++++ for 75-100% by area.

Results: We showed that HFLS are susceptible to all three CHIKVs with different rate of extracellular virus productions. The replication efficiency of these viruses was compared and small-plaque variants were found to have the highest one, while the clinical isolates had the lowest one. Interestingly, the cytopathic effect (CPE) induced by these viruses did not correlate with the efficiency of replication. Small-plaque phenotypes, as well as clinical isolates showed to cause CPE earlier than the large-plaque viruses one, which showed to cause moderately less CPE in HFLS.

Conclusion: The results indicate the heterogeneity of the clinical isolates of CHIKVs and the impact of each phenotype on responses of the major target cell of CHIKV.

Keywords: Chikungunya virus, plaque size

17th International Congress of Virology 193

Late breaking abstract

Arboviruses PO645

Differentially derived monoclonal antibodies potentially confer protection against Zika virus

C. Y. P. Lee 1 2,*, Y. W. Kam 1, T. H. Teo 1, H. L. Tan 3, A. Choo 3 4, C. I. Wang 1, K. Fink 1, L. Renia 1, L. Ng 1 5 6 1A*STAR Singapore Immunology Network, 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 3A*STAR Bioprocess Technology Institute , 4Department of Biomedical Engineering, Faculty of Engineering, 5Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 6Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom

Objectives: Zika virus (ZIKV) is an arthropod-borne virus that displays typically mild symptoms such as fever and maculopapular rash. However, the recent ZIKV epidemic across several geographical regions has been presented as a global threat, especially with substantial evidence that associates ZIKV with neurological implications, including Guillain- Barré syndrome (GBS) and congenital ZIKV syndrome. With neither vaccines nor antiviral drugs currently available, it is crucial to develop prophylactic and therapeutic agents against ZIKV.

Methods: Due to the high level of homology between ZIKV and its other closely related flavivirus, Dengue virus (DENV), we explored a panel of DENV human monoclonal antibodies (mAbs) for any cross-reactivity with ZIKV, as well as their ability to inhibit ZIKV infection in vitro. Separately, ZIKV-specific mAbs generated from ZIKV-infected mice were also screened for their ability to recognize and neutralize ZIKV infection in vitro. The most potent mAbs were subsequently tested in type I interferon receptor 1 deficient animals (IFNAR-/-) to assess for therapeutic potentials.

Results: Interestingly, mice receiving DENV human mAb, SIgN-3C, one of the potent human DENV mAbs in vitro, were protected from ZIKV infection, whereas untreated ZIKV-infected animals succumbed to infection at 8 days post-infection (dpi). The mutant variant of SIgN-3C, SIgN-3C-LALA, was also able to prevent ZIKV-induced mortality in IFNAR-/- mice with a comparable degree of protective efficacy.

Conlusion: These findings present the protective potential of differentially derived monoclonal antibodies and highlight their capacity to be used as anti-ZIKV therapeutics.

Keywords: Zika virus, monoclonal antibodies, mouse models

17th International Congress of Virology 194

Abstract Submission

Arboviruses PO646

Effect of glucose on dengue virus replication in the mosquito Aedes aegypti S.-C. Weng 1,*, S.-H. Shiao 1 1Department of Parasitology, National Taiwan University College of Medicine, Taipei, Taiwan

Objectives: Dengue is the most rapidly spreading mosquito-borne viral disease in the world and over 2.5 billion people are living in geographic areas where dengue is endemic. In Taiwan, dengue is not yet considered to be endemic. However, most individuals infected with dengue are adults, with dengue hemorrhagic fever (DHF) and related fatality occurring primarily in the elderly. In previous analysis, diabetes mellitus (DM) is a predictor of mortality among the DHF cases. Furthermore, the glycolysis is a critical metabolic pathway for optimal dengue virus (DENV) replication. However, little is known about the effect of glucose on dengue virus replication in the mosquito. The aims of this study were to investigate the impact of glucose on DENV replication in the mosquito Aedes aegypti.

Methods: The DENV2 16681 strain was used for mosquito oral infection. Female mosquitoes uptake an infectious blood meal with additional glucose. The mosquito infectivity and dengue virus genome were determined by real-time PCR at different time point post blood meal. And the mosquito saliva was collected for Western blot analysis to assess the dengue virus E protein levels.

Results: Our data showed that a significant increase of dengue virus genome while mosquitoes uptake an infectious blood meal with additional glucose, suggest blood glucose is an important factor for virus replication. We next examine the effect of blood glucose on virus transmission by determine dengue virus E protein in the mosquito saliva. Interestingly, a significant increase of dengue virus E protein was detected in the saliva at 5 days post an infectious blood meal with a low concentration of blood glucose.

Conclusion: This study raises the possibility that blood glucose will benefit the virus replication and facilitate the virus transmission in the mosquito.

Keywords: Aedes aegypti, Dengue virus, glucose

17th International Congress of Virology 195

Abstract Submission

Arboviruses PO647

ELUCIDATION OF DENGUE VIRUS INTERACTION WITH HUMAN MICROVASCULAR ENDOTHELIAL CELLS – A TRANSCRIPT LEVEL ANALYSIS A. M G 1,*, S. Singh 1, S. Easwaran 1 1MOLECULAR VIROLOGY LABORATORY, RAJIV GANDHI CENTRE FOR BIOTECHNOLOGY, THIRUVANANTHAPURAM, India

Objectives: Dengue virus (DENV) of Flaviviridae family cause one of the most dreadful, arthropod-borne viral diseases in the tropical and subtropical regions of the world. It can cause mild dengue fever to severe dengue. The fatal forms of DENV infection include dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with the hallmarks of vascular leakage, bleeding and shock. The mechanism of DHF and DSS is poorly understood. Researchers focus on the molecular changes upon dengue infection in the vascular barrier. This barrier includes endothelial cells, smooth muscle cells, an extracellular matrix, a basement membrane, cyto-skeleton and cell-cell junctions. Endothelial cells are key players maintaining the vascular barrier. In vitro studies have shown that only 3-15% of the cells are susceptible to dengue infection. In our earlier studies, we have seen that even with such a low percentage of susceptibility, DENV infection of endothelial cells leads to increased vascular permeability. Hence, identification of endothelial cell gene/proteins responding to DENV infection would help in deciphering molecular mechanisms responsible for vascular permeability.

Methods: In the present study, we did a transcript profiling of DENV2-infected human microvascular endothelial cells using Affymetrix human PrimeView GeneChip.

Results: Gene expression analysis revealed modulation of genes in the signalling pathways of type I interferon response, antiviral, chemokine, complement, and coagulation responses and angiogenesis pathways.

Conclusion: The highly up-regulated genes of the IFN-signalling were the Interferon Stimulated Genes (ISGs) indicating their predominant role in restricting infection of endothelium by these viruses and thus limiting pathogenic changes that could lead to vascular leakage.

Keywords: dengue, microarray

17th International Congress of Virology 196

Abstract Submission

Arboviruses PO648

ESTABLISHMENT OF A NOVEL GENE DELIVERY SYSTEM FOR THE EXPRESSION OF VIRAL STRUCTURAL PROTEIN OF CHIKUNGUNYA VIRUS ON MOSQUITO S.-C. Kuo 1,*, T.-Y. Wu 2, Y.-M. Wang 1, P.-Y. Tsui 1, L.-X. Chen 1, C.-C. Lin 1 1 Institute of Preventive Medicine, National Defense Medical Center, Taipei, 2Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan

Objectives: Mosquito-borne infectious diseases such as filarial, malaria, dengue, chikungunya, and Zika pose a public health threat and result in significant economic losses in many countries. Anthrophilic Aedes spps are the most effective transmitters of arboviruses. Comprehensive understanding mosquito–pathogens interactions may provide novel strategies to block virus transmission in nature. Gene delivery is emerging as an indispensable method for many applications on biological researches of mosquito. The lack of efficient approaches to delivery genes into mosquitoes, however, blocks efforts to investigate pathogen-mosquito interactions. The aim of this study is to developing a feasible gene delivery method on mosquito.

Methods: This study proposes using the recombinant baculovirus bearing mosquito promoter driving expression of heterologous gene as a novel gene delivery system on mosquito.

Results: The highly percentage of EGFP-expressing C6/36 cells were observed after transduction of recombinant baculoviruses bearing mosquito promoters driving EGFP expression. The dose-dependent EGFP expressions in transducted-mosquito cells were detected on the transduction assay of the serial diluted baculoviruses. In addition, the abundant expression of CHIKV’s glycoprotein in C6/36 cell was detected after transduction of another recombinant baculovirus harboring a mosquito promoter driving expression of CHIKV’s 26S cDNA.

Conclusion: This novel recombinant baculovirus is an efficient tool to deliver and express exogenous genes in mosquito cell in vitro. It could be potentially useful on studies of pathogen-mosquito interactions and mosquito-derived arbovirus vaccines.

Keywords: Chikungunya, mosquito, gene delivery

17th International Congress of Virology 197

Abstract Submission

Arboviruses PO649

EXPRESSION OF FLAVIVIRUS CAPSID PROTEINS ENHANCE THE CELLULAR ENVIRONMENT FOR VIRAL REPLICATION BY ACTIVATING AKT-SIGNALLING PATHWAYS A. Airo 1,*, M. Urbanowski 2, J. Lopez-Orozco 2, J. Hwan You 2, T. Skene-Arnold 3, C. Holmes 3, N. Malik-Soni 4, L. Frappier 4, T. Hobman 2 1Medical Microbiology & Immunology, 2Cell Biology, 3Biochemistry, University of Alberta, Edmonton, 4Molecular Genetics, University of Toronto, Toronto, Canada

Objectives: Flaviviruses infect hundreds of millions of people each year causing significant morbidity and mortality worldwide. As with all viruses, they depend on multiple host pathways during their life cycle and accordingly, have evolved strategies to avoid the innate immune response. Previously, we showed that the West Nile virus capsid protein plays a role in this process by blocking apoptosis. In the present study, we examined how expression of capsid proteins from Dengue (DENV), St. Louis encephalitis (SLEV), Yellow fever (YFV), tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), Murray Valley encephalitis (MVEV) and Zika viruses (ZIKV) affect apoptosis and other Akt-dependent signalling processes that impact virus replication.

Methods: Myc-tagged capsid proteins from seven flaviviruses were expressed from lentiviral vectors in this study. To determine the effect of capsid expression on apoptosis, lentivirus-transduced cells were challenged with anti-Fas and the proportion of capsid-expressing cells with active-caspase 3 was determined by flow cytometry analysis. To assess if active Akt was required for capsid-mediated protection from apoptosis, cells were treated with a phosphatidylinositol (PI) 3-kinase inhibitor. The levels of active Akt in cells were determined by immunoblotting for Akt serine-473. To assess the effect of capsid-expression on cellular metabolism, total ATP levels were determined and an Alamar Blue assay was used to measure metabolic activity of mitochondria. In addition, levels of glucose transporter 1 (GLUT1) mRNA were quantified by real time PCR. Affinity purification coupled to mass spectrometry (AP-MS) and affinity pulldowns revealed that some of the flavivirus capsids form complexes with protein phosphatase 1 (PP1).

Results: All seven of the flavivirus capsid proteins tested protected cells from Fas-dependent apoptosis via a mechanism that requires activation of Akt. In addition to blocking apoptosis, other Akt-dependent processes were upregulated by capsid protein expression. Specifically, levels of GLUT1 mRNA, total ATP, and mitochondrial metabolism were significantly increased in capsid-expressing cells. A proteomic screen for host factors that interact with DENV capsid revealed the phosphatase PP1 as a capsid-binding protein. In this case, we observed that capsid proteins from DENV, JEV, and MVEV, form stable complexes with the Akt-inactivating phosphatase PP1.

Conclusion: Together, our data indicate that the most plausible mechanism by which flavivirus capsid proteins activate numerous signalling pathways is by sequestering phosphatases away from the pro-survival kinase Akt.

Keywords: Akt-signalling, Capsid, Flavivirus

17th International Congress of Virology 198

Late breaking abstract

Arboviruses PO650

Fluorodeoxyglucose (FDG) imaging of in vivo inflammation associated with Dengue Virus Infection and Treatment Response in AG129 mice S. Watanabe 1,*, A.-M. Chacko 2, K. J. Herr 2, S. Kalimuddin 3, J. Y. Tham 2, J. Ong 2, M. Reolo 2, R. Serrano 2, Y. B. Cheung 4, J. Low 3, S. Vasudevan 1 1Programme in Emerging Infectious Disease, 2Laboratory for Translational and Molecular Imaging, DUKE-NUS, 3Department of Infectious Diseases, Singapore General Hospital, 4Centre for Quantitative Medicine, DUKE-NUS, Singapore, Singapore

Objectives: Development of antiviral therapy against acute viral diseases, such as dengue virus (DENV), suffers from the narrow window of viral load detection in serum during onset and clearance of infection and fever. Therefore the use of viral load reduction or NS1 antigen reduction as biomarkers in dengue clinical trials can be challenging because of the questionable robustness and lack of hard evidence on any tissue specific pathogenesis. We recently showed that infection of AG129 mice with immune complexes of DENV can lead to increased vascular permeability in the small intestine that correlates with increased viral load and cytokine (TNF-a, IL-6) levels (Watanabe et al., 2015). Since 18F-fluorodeoxyglucose (FDG) PET is known to be taken up at a higher rate in cells during inflammation, we explored this as a biomarker in our AG129 mouse models for primary and antibody-dependent enhancement infection with DENV.

Methods: AG129 mice (deficient in IFNα/β and γ receptors) were inoculated with DENV serotype 2 (DENV2). FDG organ biodistribution and FDG PET imaging were performed on Day 0-5 post infection. To assess FDG as a predictive biomarker of antiviral therapy response, celgosivir, an α-glucosidase inhibitor, was injected orally at 50mg/kg, 4 times daily from day 2 post infection (Watanabe et al., 2016). Tissue viral load and cytokine production were also analysed through the course of disease and drug treatment.

Results: FDG uptake measured by ex-vivo terminal biodistribution studies was most prominent in the intestines and correlated with increased virus load and pro-inflammatory cytokines. In agreement with this, the FDG uptake could also be visualized by in vivo PET imaging. Furthermore, a significant temporal trend in FDG uptake was seen in intestines and selected tissues over the time course of infection. Notably, FDG uptake and visualization by PET robustly differentiated treatment-naive groups from drug-treated groups as well as non-lethal from lethal infections with a clinical strain of DENV2.

Conlusion: Taken together, FDG may serve as a novel DENV infection–associated inflammation biomarker for assessing treatment response during therapeutic intervention trials.

References: 1. Watanabe S, Chan KW, Wang J, Rivino L, Lok SM, Vasudevan SG. (2015) Dengue Virus Infection with Highly Neutralizing Levels of Cross-Reactive Antibodies Causes Acute Lethal Small Intestinal Pathology without a High Level of Viremia in Mice. Journal of Virology. 89(11):5847-61. 2. Watanabe S, Chan KW, Dow G, Ooi EE, Low JG, Vasudevan SG. Optimizing celgosivir therapy in mouse models of dengue virus infection of serotypes 1 and 2: (2016) The search for a window for potential therapeutic efficacy. Antiviral Research. 127:10-9. 3. Chacko AM, Watanabe S, Herr KJ, Kalimuddin S, Tham JY, Ong J, Reolo M, Serrano RMF, Cheung YB, Low JGH, Vasudevan SG. (2017) 18F-FDG as an inflammation biomarker for imaging dengue virus infection and treatment response. JCI Insight. 2(9).

Keywords: Dengue virus, inflammation, PET imaging

17th International Congress of Virology 199

Abstract Submission

Arboviruses PO651

Functional analysis of dengue virus host factor in the mosquito Aedes aegypti Y.-C. Hsu*, S.-H. Shiao 1 1Parasitology, National Taiwan University, Taipei, Taiwan

Objectives: Dengue fever is one of the most devastating arthropod-borne diseases. The WHO reported that 2.5 billion people are at risk from dengue and estimates that more than 50 million cases of dengue infection worldwide annually. However, until now, no effective or breakthrough of anti-dengue vaccine and drugs has been published. Therefore, intensive study for potential anti-dengue genes in the mosquito vector and construction of transgenic mosquito together with gene drive technique to replace the vector populations became an alternative strategy to combat dengue virus.

Methods: In this study, we made use of the well-established reverse genetic approach by silencing potential host factors in the mosquitoes. Silence of different candidate genes were performed by microinjection of double-stranded RNA targeted to specific genes into the mosquitoes.

Results: Our results showed that two immune responsive genes, Thioester-containing protein 1 (TEP1) and Leucine-Rich Immune Gene 1 (LRIM1), were highly expressed in response to dengue virus infection in the mosquito midgut. Silencing of TEP1 or LRIM1 resulted in the over-expression of transcript of dengue virus 2 in the mosquito midgets. Immunofluorescent assay revealed that silencing of TEP1 or LRIM1 resulted in the over-expression of DENV E-protein in the mosquito midgets.

Conclusion: This study provide new insights into the understanding of dengue-vector interaction and new strategy for dengue eradication program. Data revealed by this proposal will be crucial for future studies on vector competence and vector control in the field.

Keywords: Dengue virus, Leucine-Rich Immune Gene 1, Thioester-containing protein 1

17th International Congress of Virology 200

Abstract Submission

Arboviruses PO652

Functional importance of dengue virus maturation in the mosquito Aedes aegypti L.-T. Kuo*, S.-H. Shiao

Objectives: Mosquitoes are one of the fatal animals in the world and act as vectors to spread disease to humans, including malaria, dengue fever, West Nile fever, chikungunya and Zika. There are 50 thousand dengue cases been reported in 2014 and 2015 in Taiwan and become a serious public health problem. However, there are no effective medication and vaccine for dengue fever till now. Thus, preventing mosquito bites and developing novel strategies to against mosquito-borne diseases are urgently required. Previous studies indicated that human Furin, a proprotein convertase, plays an important role during dengue virus (DENV) maturation. We identified a vitellogenin convertase (VgC) in the mosquito Aedes aegypti which is highly homologous to human Furin. VgC has been described to be involved in the production of vitellogenin (Vg) during mosquito vitellogenesis. Therefore, the aim of this study is to investigate the effect of VgC on DENV maturation in the mosquito.

Methods: First, we compared the similarity of Vg convertase and human furin and investigated the expression of VgC post a normal and an infectious blood meal. Next, we made use of furin inhibitor to inhibit the activity of Vg convertase, in order to elucidate the efficiency of furin inhibitor and the effect on dengue virus cleavage during maturation.

Results: Our results showed that Vg convertase displayed high similarity to human furin and the transcript of VgC was highly expressed post a normal and an infectious blood meal. In addition, Western blot analysis revealed that the expression of precursor membrane (prM) protein of DENV was increased at day 4 and 5 after furin inhibitor treatment, indicating the cleavage of prM was impaired when Vg convertase was inhibited.

Conclusion: Therefore, our results suggested that Vg convertase is a key enzyme for the maturation of dengue virus in the mosquito Aedes aegypti. This study may provide new insights into the understanding of dengue-vector interaction and the development of new strategy for dengue eradication program.

Keywords: Aedes aegypti, Dengue virus, Furin, Vg convertase

17th International Congress of Virology 201

Abstract Submission

Arboviruses PO653

GENOMIC RNA OF TICK-BORNE ENCEPHALITIS VIRUS IS TRANSPORTED IN NEURONAL DENDRITES VIA NEURONAL GRANULE MACHINERY M. Hirano 1,*, M. Muto 1, M. Sakai 1, H. Kondo 1, S. Kobayashi 1, H. Kariwa 1, K. Yoshii 1 1Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan

Objectives: Encephalitic flaviviruses cause severe neurological diseases with high levels of mortality rate. However, detailed mechanisms of the viral replication and pathogenesis in the central nervous system remain poorly understood. Previously, we reported that the genomic RNA of tick-borne encephalitis virus (TBEV), a member of genus Flavivirus, was transported to and replicated in dendrites of infected neurons, which may be involved in the neuro-pathogenicity of TBEV. In the current study, we analyzed the molecular mechanisms of the transport of the viral genome to dendrites.

Methods: Plasmids were designed to transcribe RNAs of TBEV sequence, and were transfected to PC12 cells, which develop neurites in the presence of neuronal growth factors. Distributions of the RNAs were analyzed by using specific fluorescein-labeled RNA probe. A recombinant virus was constructed by introducing a mutation in the 5′ untranslated region (UTR). Viral replication and neurologic symptoms were analyzed by using primary cultured mouse neurons and intracerebral inoculation to C57BL/6 mice.

Results: RNAs with the 5′ UTR of TBEV were transported to neurites of the transfected PC12 cells. Deletion or mutations in stem-loop (SL)-2 region of the TBEV 5′ UTR drastically reduced the transport of the RNA. TBEV with the mutation in the SL-2 region was incompetent in the genome transport in the infected primary cultured neuron. Mice infected with the mutant virus exhibited attenuated neurological symptom. The genomic RNA of TBEV was co-localized with components of neuronal granules which transport host dendritic mRNA to neuronal dendrites. TBEV infection disturbed the host dendritic mRNA transport by the neuronal granule.

Conclusion: This is the first report to show that the genomic RNA of TBEV was transported in dendrites by the neuronal granule, resulting in the neurological symptoms. These data will contribute to understand molecular mechanisms of neurologic diseases caused by infections of encephalitic flaviviruses.

Keywords: Flavivirus, Neuron, RNA protein interactions

17th International Congress of Virology 202

Abstract Submission

Arboviruses PO654

HIGH THROUGHPUT PROTEOMIC ANALYSIS OF MICROVASCULAR ENDOTHELIAL CELLS UPON DENGUE INFECTION SIGNIFIES THE ROLE OF NUCLEOLAR PROTEINS IN HOST-VIRUS INTERACTION

S. Singh*, A. MG 1, E. Sreekumar 1 1Molecular Virology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, India

Objectives: Dengue virus (DENV) infection remains a major public health problem worldwide. 390 million dengue infections are estimated to occur annually. They cause approximately 500,000 cases of a potentially lethal form of dengue which causes severe capillary plasma leakage. Virus infection of endothelial cells lining the capillaries could be a key element that controls the vascular leakage. We used a proteomics approach to gain a better understanding of the host-virus interaction in these cells.

Methods: Using a label-free LC MS/MS analysis, and by stringent, high cut-off filtering of data from triplicate experiments, we short-listed 244 differentially regulated proteins in cultured human microvascular endothelial cells (HMEC) infected with DENV.

Results: We found that 95 of these proteins were up-regulated and 149 were down-regulated. There was a decrease in the expression of the proteins involved in cell adhesion and motility, transcription, translation, immune response and nucleotide metabolism. An increase was observed in proteins involved in host-virus interaction and platelet aggregation. The nucleolar proteins were found to be up-regulated in the proteome of infected cells. Transcript level analysis corroborated with the increased expression of these nucleolar proteins during infection.

Conclusion: We presume that there is interaction of nucleolar proteins with dengue viral proteins or replication complexes and subsequent trapping of them in cytoplasmic compartments. Our study provides large scale quantitative information of the proteome during DENV infection in endothelial cells. This would prove to be useful for further understanding the mechanism of disease and vascular leakage observed in severe cases.

Keywords: dengue, nucleolar proteins, proteomics

17th International Congress of Virology 203

Abstract Submission

Arboviruses PO655

HOST-RESTRICTION AND TRANSMISSION DYNAMICS OF A NEWLY DISCOVERED FLAVIVIRUS IN THE YELLOW FEVER VIRUS GROUP A. Colmant*, L. Vet 1, S. Hall-Mendelin 2, A. van den Hurk 2, H. Bielefeldt-Ohmann 1, C. O'Brien 1, J. Hobson-Peters 1, T. Piyasena 1, A. Khromykh 1, R. Hall 1 1School of Chemistry and Molecular Biosciences, University of Queensland, 2Public Health Virology, Forensic and Scientific Services, Department of Health, Queensland Government, Brisbane, Australia

Objectives: To investigate the biodiversity of viruses in Australian mosquitoes we screened archival mosquito homogenates for novel viruses. Bamaga virus (BgV), a new species of flavivirus, was discovered and subsequently characterized to determine its phylogenetic and antigenic relationship to other members of the genus and examined its host range and mode of transmission.

Methods: Illumina next generation sequencing, phylogenetic analysis, growth kinetics, assessment of replication in a range of arthropod and vertebrate cells at different incubation temperatures, construction of chimeric viruses using CPEC, pathogenicity assays in mice and transmission assays in mosquitoes were used to characterize BgV.

Results: Bamaga virus (BgV) was isolated from Culex annulirostris mosquitoes collected from northern Australia. Phylogenetic analysis of the complete nucleotide sequence of the BgV genome revealed it clustered with the vertebrate infecting flaviviruses (VIFs), in particular the yellow fever virus (YFV) group, and was most closely related to Edge Hill virus, another Australian flavivirus, with 61.9% nucleotide and 63.7% amino acid sequence identity. However, in contrast to VIFs, BgV displayed restricted growth in a range of vertebrate cell lines with no or relatively slow replication in inoculated cultures, suggesting a singularly narrow host range. Additionally, it was shown that BgV replication is greatly influenced by temperature in vitro. BgV also displayed restricted replication in vivo, notably in virus-challenged mice (intraperitoneally or intracranially) but could be horizontally transmitted horizontally in mosquitoes. Using infectious DNA technology a series of chimeric viruses between BgV and WNV were also produced to investigate the mechanisms of BgV host-restriction in vitro and in vivo.

Conclusion: Our results indicate that BgV is an evolutionary divergent member of the YFV group of mosquito-borne flaviviruses that exhibits an unusual pattern of host restriction. This BgV represents a unique model virus to study mechanisms of flavivirus host-restriction and transmission.

Keywords: Flavivirus, Host-restriction, Yellow fever group

17th International Congress of Virology 204

Abstract Submission

Arboviruses PO656

How to Define the “Epidemic Potential” of Dengue Viruses? - From Taiwan’s Experiences to Global Health

C.-C. King 1,*, H.-Y. Ko 1, Y.-T. Lee 1, C.-H. Chin 1, S.-Y. Lin 1, P.-W. Shih 2, P.-Y. Chen 1, Y.-C. Chang 1, C.-L. Kao 1, T.-C. Chan 3, S.-C. Lee 4, C.-C. Ku 5, G.-J. J. Chang 6, E.-E. Ooi 7, D. J. Gubler 7 1Institute of Epidemiology and Preventive Medicine, 2Dept. of Public Health, National Taiwan University, 3Research Center for Humanities & Social Science, Academia Sinica, 4Institute of Molecular Medicine, College of Medicine, 5Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan, 6Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States, 7Program in Emerging Infectious Diseases, Duke-NUS, Singapore, Singapore

Objectives: Epidemics of dengue in Taiwan and many parts of the world involving severe dengue cases occur only in certain years or areas. Global epidemiology also shows that specific strains or genotypes of the same serotype of dengue viruses (DENVs) did increase in epidemic severity. Therefore, this study aimed at better defining the “epidemic potential” of DENV through epidemiological virological, and immunological characterization that will be worthwhile pursuing for future prediction of outbreaks.

Methods: We isolated the DENV-2 strains from patients presented as clinically mild dengue fever (DF) versus severe dengue hemorrhagic fever (DHF) cases with different epidemiological conditions (e.g. temporal and spatial attributes) during the same epidemic in the same city for better monitoring dynamics of viral genetic and phenotypic changes through an epidemic process. On the other hand, different clades of DENV-2 with viral sequence variations were used for spatial analyses on viral spreading by using geographical information system (GIS) and reproductive numbers of R0.

Results: The results showed that DHF cases appeared mostly during climbing up and peaking time of dengue cases through temporal and spatial evolution of an epidemic. Interestingly, DENV-2 variations occur at different days after onset of dengue illness of the same patient at micro-level as well as in different time periods of the same epidemic at macro-level of population dynamics. On the other hand, different DENV-2 isolates with viral sequence variations did have differences in viral growth kinetics, viral transmissibility and spatial spreading. The epidemiological conditions are more important than immune status of primary or secondary DENV infection as driving forces associated with viral changes and severe dengue cases, particularly in areas with low incidence of dengue in previous years.

Conclusion: Taiwan has not been an area of dengue-endemicity, however, most epidemics of dengue have been caused by a single serotype of DENV. Therefore, this provides us with a very valuable opportunity to integrate clinical, epidemiological, viral and immunological findings from those time-dependent DENV isolates to generate causal inference to fully understanding the mechanism of increasing epidemic severity. It is particularly of importance to monitor DENV viral changes through international collaboration for those genotypes with greater inter-country spread.

Keywords: Dengue virus, Taiwan

17th International Congress of Virology 205

Abstract Submission

Arboviruses PO657

IDENTIFICATION OF CELLULAR GENES SUPPRESSING DENGUE VIRUS REPLICATION BY A GAIN-OF-FUNCTION CDNA LIBRARY SCREEN APPROACH

Y. Suzuki 1,*, H. Wu 1, T. Nakano 1, K. Sano 1 1Department of Microbiology and Infection Control, Osaka Medical College, Takatsuki, Japan

Objectives: Dengue virus (DENV) belongs to the genus Flavivirus of the Flaviviridae family, which is a large family of enveloped, positive-stranded RNA viruses. Immunologically distinct serotypes of DENV have recently emerged as significant pathogens that can cause dengue fever and dengue hemorrhagic fever in humans. Yet, an effective medication for dengue has not already been established. As seen in other RNA viruses, DENV replication is reported to be limited by the IFN response. In this study, we intended to identify IFN-induced cellular factors that potentially restrict DENV infection.

Methods: We employed a gain-of-function screen using a cDNA library derived from type I IFN-treated cells to identify the anti-DENV genes. A pool of cDNA was generated from the mRNA of type I IFN-treated HeLa cells and lentivirally transduced into Huh7.5 cells that exhibited massive cell death by DENV infection. IFN cDNA library-transduced Huh7.5 cells were then challenged with DENV, and cDNAs expressing in survived cells were identified by sequencing analysis.

Results: The result of DENV challenge in transduced Huh7.5 cells revealed that about 60% of surviving cell clones contained cDNA encoding a previously uncharacterized gene, C19orf66, which we named Repressor of yield of DENV (RyDEN). Then, it was confirmed that RyDEN/C19orf66 was an IFN-stimulated gene (ISG) whose expression confers resistance to DENV infection in human cells. Sequencing analysis further found that cDNA of several other genes including C19orf53, LINC00052, IFN-α-inducible protein 27, and a deletion mutant of DNAJC14 were transduced in the DENV resistant clones. Subsequent analysis showed that overexpression of some of the genes likely to inhibit DENV replication, suggesting that these were potential antiviral ISGs against DENV infection.

Conclusion: Our expression cloning approach of cDNA obtained from IFN-treated cells identified novel cellular genes having inhibitory effects on DENV infection. Although the detailed mechanisms by which these potential antiviral factors inhibit DENV infection are currently under investigation, our data suggest that various types of cellular factors target DENV replication process during the IFN response. Exploiting the function and viral target of the individual antiviral factor will provide a clue to the development of new therapies against dengue.

Keywords: Cellular factors, Dengue virus, Interferon response

17th International Congress of Virology 206

Abstract Submission

Arboviruses PO658

IDENTIFICATION OF HOST PROTEINS INTERACTING WITH THE 3' UTR OF TICK-BORNE ENCEPHALITIS VIRUS M. Muto 1,*, W. Kamitani 2, M. Sakai 1, M. Hirano 1, S. Kobayashi 1, H. Kariwa 1, K. Yoshii 1 1Laboratory of Public Health, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 2 International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Objectives: Tick-borne encephalitis virus (TBEV) is a member of genus Flavivirus. The virus causes severe neurological disease in human, but the pathogenic mechanism is largely unclear. We previously reported that the conformational structure of the 3’ untranslated region (UTR) of TBEV was associated with the virulence. In this study, we tried to identify host proteins which could interact with the 3’ UTR of TBEV.

Methods: Biotinylated TBEV RNAs were prepared from the construct having the 3’ UTR of low or high virulent TBEV strains. Cellular lysate of HEK293T cells was mixed with each RNA and pulled down by streptavidin beads. Co-precipitated proteins were subjected to SDS-PAGE analysis and identified by Mass spectrometry (MS) analysis. The interaction of the identified proteins and the 3’ UTR of TBEV were confirmed by specific antibodies followed by the pull-down assay and RNA immunoprecipitation.

Results: Several specific bands were detected by the pull-down assay with the RNA for the 3’UTR of the low virulent strain, but not of the high virulent strain. The proteins that bound specifically to the 3’ UTR of the low virulent strain were identified by MS analysis, and cold shock domain containing protein E1 (CSDE1) showed the highest score. The pull-down assay and RNA immunoprecipitation using specific antibodies confirmed the interaction between the 3’ UTRs of the low virulent strain and CSDE1.

Conclusion: It has been reported that CSDE1 is involved in RNA metabolism and neurological functions. Further analysis of roles of the host proteins in the TBEV pathogenicity would lead to clarify the detailed mechanism of neurologic disease of TBEV.

Keywords: 3' UTR, host factor, TBEV

17th International Congress of Virology 207

Abstract Submission

Arboviruses PO659

INFECTION AND INJURY OF HUMAN NEURONS AND ASTROCYTES BY TICK-BORNE ENCEPHALITIS VIRUS D. Ruzek 1,*, M. Palus 2, M. Vancova 3, T. Bily 3 1Department of Virology, Veterinary Research Institute, Brno, 2Laboratory of Arbovirology, 3Laboratory of Electron Microscopy, Institute of Parasitology, The Czech Academy of Sciences, Ceske Budejovice, Czech Republic

Objectives: Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, genus Flavivirus, causes tick-borne encephalitis (TBE) in humans, a neuroinfection prevalent in large areas of Europe and North-eastern Asia. Humans develop a febrile illness, and a subset of cases progress to neurological manifestations ranging from mild meningitis to severe encephalomyelitis. Despite the medical importance of TBE, some crucial steps in the development of encephalitis remain poorly understood. Neurons are primary targets after TBEV enters the CNS, but other brain cells, like astrocytes, may also be infected. However, knowledge about this viral infection and virus-induced injury of CNS cells is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons and astrocytes.

Methods: Primary human neurons and astrocytes were infected with TBEV at m.o.i. of 5. Virus-mediated CPE was investigated using light microscopy. Viral growth was investigated using plaque assay. Activation of astrocytes was measured by GFAP expression using flow-cytometry. Expression of cytokines and chemokines was measured by qRT-PCR and ELISA. Morphological changes in TBEV-infected cells were investigated using three-dimensional electron tomography.

Results: Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus. In case of TBEV-infected astrocytes, the infection induced a marked increase in the expression of GFAP, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key pro-inflammatory cytokines/chemokines (e.g. tumour necrosis factor α, interferon α, interleukin (IL)-1β, IL-6, IL-8, interferon γ-induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) was upregulated.

Conclusion: Both neurons and astrocytes are sensitive to TBEV infection and the infection causes a number of dramatic ultrastructural changes in the infected cells. The infected astrocytes might be a potential source of pro-inflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood–brain barrier breakdown that occurs during TBE.

17th International Congress of Virology 208

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Keywords: electron tomography, pathology, tick-borne encephalitis virus

17th International Congress of Virology 209

Abstract Submission

Arboviruses PO660

INFECTIOUS VIRAL DNAS DRIVEN BY INSECT OR MAMMALIAN PROMOTERS FACILITATE NOVEL STUDIES ON THE HOST RESTRICTION OF AN INSECT-SPECIFIC FLAVIVIRUS T. Piyasena 1,*, Y. Setoh 1, J. Hobson-Peters 1, B. McLean 1, H. Bielefeldt-Ohmann 1, N. Newton 1, L. Vet 1, A. Khromykh 1, R. Hall 1 1Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Objectives: Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are responsible for significant mosquito-borne disease. However recently, insect-specific flaviviruses (ISF) which only replicate in mosquito hosts have been identified. Of particular interest, mosquitoes persistently infected with some of these ISFs are less capable of transmitting pathogenic flaviviruses. This study aimed to generate infectious cDNA constructs to identify the genes responsible for host restriction in ISFs.

Methods: A novel technique known as circular polymerase extension cloning (CPEC) was used to generate an infectious DNA constructs from fragments of viral cDNA. A modified insect (OpIE2) promoter was used to drive transcription in C6/36 insect cells. Infectious DNA constructs were transfected into cells and characterised using a combination of IFA, RT-PCR and ELISA. Viral authenticity was confirmed using sequencing and antibodies specific to each of the viruses.

Results: Infectious virus was successfully recovered from C6/36 mosquito cells transfected with a cDNA construct of the Australian ISF, Parramatta river virus (PaRV) using a modified OpIE2 insect promoter. A similar infectious cDNA under transcriptional control of a mammalian promoter was used to directly transfect mouse embryo fibroblasts and revealed that, in both the absence or presence of a type I IFN response, PaRV did not initiate replication in vertebrate cells even when cell attachment and virion entry steps were by-passed.

Conclusion: These infectious ISF DNA constructs provide novel tools to elucidate the mechanisms restricting ISFs to mosquito hosts and are currently being manipulated to identify the viral host factors associated with this restriction. These studies will also deliver novel insights into flavivirus evolution.

Keywords: Flavivirus, Infectious DNA, Insect-specific flavivirus

17th International Congress of Virology 210

Late breaking abstract

Arboviruses PO661

Macropinocytosis-Dependent Entry of Chikungunya Virus into Human Muscle Cells R. Lee*, K. M. Hussain, J. J. H. Chu

Objectives: To develop susceptibility profiles of various cell lines upon CHIKV infection. To determine the specific entry pathways of CHIKV into human muscle cells.

Methods: Establishment of growth kinetic studies on three different CHIKV strains was carried out on 18 different cell lines. Immunofluorescence assay was carried at 24hpi to look at the infectious entry of CHIKV into cells after 24h. After which, highly susceptible cell lines, SJCRH30 was selected for drug entry studies. Finally, gene knockdown was carried using western blot analysis and siRNA transfection to further confirm the important gene associated with entry studies.

Results: Pre-treatment of cells with receptor-mediated endocytic inhibitor, monodansylcadaverine and clathrin-associated drug inhibitor, dynasore, caveolin-related drug inhibitor, fillipin showed no inhibition or minimal inhibition of CHIKV infection was observed with SJCRH30 cells. On the other hand, dose-dependent inhibition was observed with the use of macropinocytosis-related drug inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Additionally, an siRNA-based gene knockdown on SNX9 targeting macropinosomes revealed CHIKV infectious entry primarily via macropinocytosis. Finally, a FITC-dextran assay was carried to map the entry process of CHIKV in macropinosomes.

Conlusion: This study revealed, for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by the macropinocytosis pathway. Altogether, this study paves the way for the development of specific inhibitors that targets entry process of CHIKV.

Keywords: Chikungunya, Macropinocytosis Entry, siRNA gene knockdown

17th International Congress of Virology 211

Abstract Submission

Arboviruses PO662

MOLECULAR EPIDEMIOLOGY OF AFRICAN SWINE FEVER IN AREAS PERCEIVED TO BE NON-ENDEMIC IN ZAMBIA (1989-2015) E. Simulundu*, A. Takada, A. Mweene

Objectives: African swine fever (ASF) is a highly infectious and deadly viral hemorrhagic disease that mostly affects domestic pigs. It is caused by ASF virus (ASFV), a member of the family Asfarvidae, genus Asfivirus. In Zambia, ASF was first reported in 1912 in Eastern Province and is currently believed to be endemic in that Province only. In 1989, an outbreak occurred for the first time outside the endemic Province. Since then, sporadic outbreaks have been experienced almost throughout the country. These events have brought into acute focus our limited understanding of the epidemiology of ASF. In this study, we investigated the epidemiology of the disease in areas considered to be ASF-free (non-endemic) from 1989 to 2015.

Methods: Comprehensive sequence analysis was conducted on genetic data (i.e. p72, p54, p30 and central variable region of B602L genes) of ASFVs that have been detected in domestic pigs and those that were obtained from soft ticks in Zambia.

Results: We found that p72 genotypes I, II, VIII and XIV have been involved in causing ASF outbreaks in domestic pigs in Zambia between 1989 and 2015. With the exception of the 1989 outbreak, we found no evidence of transmission of ASFVs from Eastern Province to other parts of the country. Analysis of molecular and epidemiological data revealed a complex epidemiology of the disease, with trade and/or movement of pigs and their products both within and across international borders being the major factor in ASFV dissemination.

Conclusion: Overall, our analyses suggests that ASFVs with potential to cause countrywide, and possibly regional outbreaks could emerge outside Eastern Province and thus the current ASF control policy in Zambia requires a dramatic shift to ensure a more sustainable pig industry.

Keywords: African swine fever virus, Asfarviridae, Molecular epidemiology

17th International Congress of Virology 212

Abstract Submission

Arboviruses PO663

MUTATIONS REDUCING CYTOTOXICTY OF CHIKUNUNYA VIRUS AFFECT VIRAL RNA REPLICATION AND ANTIVIRAL INTERFERON RESPONSE A. Utt*, A. Merits

Objectives: To analyze the mechanisms by which different alphaviruses activate and counteract host antiviral responses.

Methods: To select the noncytotoxic mutants, we adapted Chikungunya virus (CHIKV) replicons for noncytotoxic growth in mammalian cells. Adaptive mutations were identified and introduced into CHIKV genomes and replicase expression constructs. HS 633T cells were infected with wt and mutant viruses; the IFN-α/β levels were measured using HEK-Blue IFN-α/β cell-based assay. To analyze IFN production by CHIKV and SFV trans-replicase systems COP-5 cells were transfected with plasmids encoding corresponding replicases and template RNAs; the amount of secreted IFN-β was measured using VeriKine-HSTM Mouse IFN Beta Serum ELISA Kit. The ability of wt and mutated replicases to amplify CHIKV mini-genome was analyzed by measuring the activity of Gluc reporter expressed from the subgenomic RNA synthesized by viral replicase activity

Results: Mutation P718G (PG) in CHIKV nsP2 was found to be necessary but to sufficient to generate CHIKV replicon with a noncytotoxic phenotype; for this combination of PG and additional mutations were required. These mutations affected known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, leading to a reduced capacity for RNA replication. In infected cells CHIKV, harboring such mutations, caused early and prominent production of IFN-β indicating that mutant viruses were unable to suppress this antiviral response. Selected mutations were also analyzed using uncoupled replicase expression/RNA replicaton assay. This method allows analysis of effects of the mutations on the ability of viral replicase to perform RNA synthesis. In this system the mutations caused only moderate reduction of viral replicase activity and failed to induce excessive IFN response. Most likely it indicates that in the context of virus infection IFN response leads to suppression of RNA replication. On another hand, mutations in nsP1 and nsP2 that had no effect on RNA replication but boosted IFN response were identified. Thus, activity of nsP2, or more likely stability of viral non-structural polyprotein, is also crucial for IFN activation. Coherent with this assumption, the increased induction of IFN was also observed when infected cells were treated using chemical compounds inhibiting protease activity of nsP2.

Conclusion: Our results provide novel insights about interactions between alphaviruses and innate immune response pathways. Comparison of this data with that available for related Semliki Forest virus (SFV) it was found that two closely related alphaviruses (SFV and CHIKV) are differently recognized by the host cell.

Keywords: Chikungunya virus, Interferone response, RNA replication

17th International Congress of Virology 213

Abstract Submission

Arboviruses PO664

REGULATION OF THE STABILITY OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 4B BY UBIQUITIN- PROTEASOME PATHWAY H. Takahashi*, Y. Suzuki 1, Y. Sameshima 2, S. G. Vasudevan 3, N. Yamamoto 4, T. Sawasaki 2 1Department of Microbiology and Infection Control, Osaka Medical College, Osaka, 2Proteo-Science Center, Ehime University, Matsuyama, Japan, 3Program in Emerging Infectious Diseases, DUKE-NUS Medical School, 4Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Objectives: Dengue virus (DENV), a member of the Flaviviridae family, contains a single-stranded RNA genome, which encodes three structural and seven nonstructural (NS) proteins. Recently, it has been reported that the function and stability of several viral proteins of other flaviviruses family such as hepatitis C virus were regulated by ubiquitination, which is one of the important post-translational modifications in cells. In the case of DENV, we have found that NS4B, which possesses multiple transmembrane domains and functions as scaffold of viral replication complex on ER membrane, was ubiquitinated and degraded by ubiquitin-proteasome pathway, whereas the physiological significance of the NS4B degradation is still unclear. To know the molecular mechanism of ubiquitination and subsequent degradation of NS4B in cells, we aimed to determine the host E3 ligases targeting NS4B in this study.

Methods: Here a biochemical screening for identification of novel E3 targeting NS4B was performed. At first, a protein array containing recombinant proteins of 250 E3 ligases from human and mouse (termed E3 protein array) was prepared with a wheat cell-free protein synthesis system. Then, the E3 ligases bound to NS4B were screened from the E3 protein array by luminescent-based high-throughput binding assay, AlphaScreen. The interaction of NS4B and the E3s identified by AlphaScreen assay were further validated with functional analysis using human cultured cells.

Results: As a result of the in vitro screening, fifteen E3 ligases were found to bind to NS4B. Then, to validate that these E3 ligases indeed targeted NS4B in cell, HEK293T cells were co-transfected with NS4B and each of these 15 E3 ligases. The result showed that at least one E3 ligase, which was reported to localize on ER membrane, significantly decreased the expression level of NS4B in its E3 ligase activity-dependent manner. Furthermore, the replication of DENV was suppressed when the wild-type but not activity deficient mutant of the E3 ligase was overexpressed in HeLa cells.

Conclusion: The results obtained here suggested that the E3 ligase we found in this study is likely to function as a novel host antiviral factor for DENV through ubiquitination and subsequent degradation of NS4B. Further functional analysis to investigate the physiological significance of the interaction between NS4B and this E3 for DENV replication is ongoing.

Keywords: Dengue virus, host factor, ubiquitin/proteasome

17th International Congress of Virology 214

Abstract Submission

Arboviruses PO665

RNA-seq analysis highlights unique host-pathogen interaction following Zika virus infection of primary human Sertoli cells D. Strange 1,*, R. Green 2, D. Siemann 1, M. Gale Jr. 2, S. Verma 1 1Department of Tropical Medicine, Medical Microbiology, and Pharmacology, University of Hawaii at Manoa, Honolulu, 2Department of Immunology and Department of Microbiology, Washington School of Medicine, Seattle, United States

Objectives: Reports of Zika virus (ZIKV) in seminal fluid without viremia and confirmed cases of sexual transmission provides indirect evidence that ZIKV crosses the blood-testes barrier (BTB) and establishes infection in seminiferous tubules of the testes. The BTB is comprised of Sertoli cells connected by tight junctions and adherens junctions that protect germ cells from cytotoxic agents, pathogens and autoimmune response. ZIKV is shown to infect mouse Sertoli cells in vivo, but the role of human Sertoli cells (HSECs) in ZIKV infection is still unknown. Here, we utilized RNA-sequencing technology to identify changes in key pathways associated with immune response and cell-to-cell signaling in HSECs following ZIKV infection.

Methods: RNA-seq was performed on RNA extracted from ZIKV-infected (MOI 1) primary HSECs at different time points post-infection. Raw alignments were mapped to ZIKV and host genomes. Differentially expressed genes (DEGs) were identified using voom/limma package in R/Bioconductor. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) and JEPETTO in Cytoscape.

Results: A total of 1,053 genes were differentially modulated by ZIKV in HSECs as compared to controls by log2 fold- change (p<0.05). At 12 hours post infection (hpi), virus replication was not detected; however, we observed downregulation of several cell-growth associated pathways, including DNA replication/repair, cell cycle control, and granzyme A signaling. Peak viral reads at 48 and 72 hpi correlated with significant upregulation of anti-viral response pathways, including activation of IRF signaling (IRF7, ISG15, STAT1/2), IFN signaling (MX1, IFIT1, IFIT3), RIG1-like receptor signaling (LGP2, MDA-5) and IL-6 signaling. Majority of the downregulated genes at 72 hpi clustered into cell-to-cell junction pathways; however, ZIKV did not affect the expression of key tight junction genes (OCLN, CLDN, ZO1).

Conclusion: The present study identifies differences in the timing and magnitude of several pathways induced by ZIKV including cell growth, cell-to-cell adhesion and antiviral defense response, and provides unique mechanistic insights into the role of Sertoli cells in testicular infection of ZIKV that warrants further experimental investigation.

Keywords: RNA-seq, Sertoli cells, Zika virus

17th International Congress of Virology 215

Abstract Submission

Arboviruses PO666

SFRNA INCREASES DENV MOSQUITO TRANSMISSION BY ALTERING SALIVARY GLANDS IMMUNITY J. Pompon*, M. Garcia-Blanco 1 2 1Department of Biochemistry and Molecular Biology, UTMB, Galveston, United States, 2Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore

Objectives: Dengue virus (DENV) is transmitted by mosquitoes, mainly Aedes aegypti species. Upon infection, DENV generates a subgenomic flaviviral RNA fragment (sfRNA) from its 3'UTR that can antagonize the mosquito antiviral response and favor virus replication. Here, we set to characterize how sfRNA influences mosquito transmission to determine virus epidemiological fitness.

Methods: We made use of an existing bank of serotype 2 DENV isolates collected in Puerto Rico that have different epidemical fitness and variable 3’UTR sequences. Upon oral infection of A. aegypti with 9 isolates, we quantified virus infectivity and ratios of sfRNA:gRNA (genomic RNA). We then selected two isolates with low and high epidemiological fitness to quantify sfRNA:gRNA ratios in salivary glands, midgut and carcass at different time points following oral infection, estimate immune response in salivary glands and quantify saliva infectivity. Using reverse genetics, we identified the role of 3'UTR sequence variation in sfRNA:gRNA ratio, immune response and saliva infection rate.

Results: Higher epidemiological fitness was associated with higher sfRNA:gRNA ratio but not with higher infectivity to mosquitoes. The tissue-specific kinetic experiment further revealed that sfRNA:gRNA ratio was higher in salivary glands than in midgut or carcass regardless of the isolates, and that the isolate with higher epidemiological fitness was associated with higher sfRNA:gRNA ratio in salivary glands. After infection with the epidemic isolate, the immune response was disrupted in salivary glands and saliva infection rate was higher. Using chimeric viruses with similar genomic backbone but epidemic or non-epidemic 3'UTR sequences, we demonstrated that the higher sfRNA:gRNA ratio in salivary glands, the immune disruption and the higher saliva infectivity were caused by variation in the epidemic 3'UTR.

Conclusion: Our study revealed that 3’UTR variation increases sfRNA in salivary glands, which disrupt the immune response to enhance transmission and epidemiological fitness of viruses.

Keywords: Dengue virus, Mosqsuito, Subgenomic flaviviral RNA

17th International Congress of Virology 216

Late breaking abstract

Arboviruses PO667

Specific biomarkers associated with neurological complications and congenital CNS abnormalities from Zika virus- infected patients in Brazil Y.-W. Kam 1,*, J. A. Leite 2, F.-M. Lum 1, J. J. Tan 1, B. Lee 1, C. C. Judice 2, L. Rénia 1, J. L. Proença-Modena 2, L. F. P. Ng 1, F. T. M. Costa 2 1SINGAPORE IMMUNOLOGY NETWORK (SIgN), Singapore, Singapore, 2Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas (Unicamp), Campinas, São Paulo, Brazil

Objectives: Zika virus (ZIKV) infections have been linked to different levels of clinical outcomes ranging from mild rash and fever to severe neurological complications and congenital malformations. Brazil, hit by the ZIKV epidemics since 2014, accounted more than 200,000 probable ZIKV cases with almost 2,000 cases of microcephaly in 2015-16. Until now, the association between clinical profile and immune response upon ZIKV infection in patients remain unknown. Therefore, we aim to analyze the clinical and immunological response by analyzing the cytokine/chemokine profiles of ZIKV-infected patients from Brazil.

Methods: This study was approved by the Research Ethics Committee of the University of Campinas (CAAE: 56793516.0.0000.5404) with written informed consent from all participants. We investigated the clinical and immunological response, focusing on the immune mediators profile in 95 acute ZIKV-infected adult patients from Campinas, Brazil. Among the 95 patients, there were 6 pregnant women who later delivered during the course of this study. Clinical observations were recorded during hospitalization. Levels of 45 immune mediators were quantified using multiplex microbead-based immunoassays. One-way ANOVAs were conducted on the logarithmically transformed concentration with post hoc t tests corrected using the method of Bonferroni.

Results: While 11.6% of patients had neurological complications, 88.4% displayed mild disease of rash and fever. Several immune mediators were specifically higher in ZIKV-infected patients, while levels of IL-10, IP-10, and HGF could differentiate between patients with and without neurological complications. Interestingly, higher levels of IL-22, MCP-1, TNF-α, and IP- 10 were observed in ZIKV-infected pregnant women carrying babies with fetal growth associated malformations. Notably, babies with congenital CNS deformities had significantly higher levels of IL-18 and IP-10 but lower HGF compared to normal babies from ZIKV-infected mothers.

Conlusion: This study identified several key markers for the control of ZIKV pathogenesis. This will allow a better understanding of the molecular mechanisms of ZIKV infection in patients.

Keywords: Zika virus, cytokines, biomarkers

17th International Congress of Virology 217

Abstract Submission

Arboviruses PO668

SPECTRUM OF ZIKA VIRUS INFECTIONS IN TRAVELLERS RETURNING TO THE UK – AND THE UTILITY OF SEROLOGICAL TESTING N. Wong 1, O. Ahmed 1, F. Elsanousi 1, J. Veater 1, A. Ahmed 2, J. Osborne 2, E. Aarons 2, J. Tang 1,* 1Clinical Microbiology/Virology, University Hospitals of Leicester, Leicester, 2Rare and Imported Pathogens Laboratory, Public Health England, Porton Down, United Kingdom

Objectives: We present a case series of Zika virus infections in travellers returning to the UK, all of whom had serologically diagnosed Zika virus infection. Polymerase chain reaction (PCR) testing was only positive in one of these patients in an acute sample (urine in Case 5), though another patient was PCR positive in semen on follow-up testing (Case 3).

Methods: Five patients were included in this case series. Case 1 (52-yr old female): Travelled to Antigua between 1/7/16-5/8/16, developed fever, rash, headache, polyarthralgia and swollen feet on return to the UK. Case 2 (40-yr old male): Presented with fever, rash and arthralgia following travel to Jamaica between 26/7/16-9/8/16. Case 3 (30-yr old male): Travel led to Barbados 13/7/16-1/8/16, developed fever and headaches on return to the UK. Case 4 (49-yr old female): Travelled to St Vincent between 29/6/16 and 27/7/16. Symptoms included a pruritic rash, fever, headache, joint stiffness and conjunctivitis. Case 5 (38-yr old male): Travelled extensively on a cruise around the Caribbean, visiting Barbados, Bonaire, Aruba, Grenada, St Vincent, St Lucia, Dominica, Antigua, and St Martin between 5/11/6-20/11/16. His symptoms included fever, rash, headache, sore throat and neck lymphadenopathy.

Results: The test results for these five patients were as follows: Case 1 (52-yr old female): Serum taken three days after onset was Zika IgM positive. Renal function, liver function and C- reactive protein were normal, and the full blood count showed a mild leucopaenia. Follow up testing on day 12 post-symptom onset showed Zika IgG seroconversion with persisting IgM. Case 2 (40-yr old male): Zika serology at day 10 post symptom onset was IgM and IgG positive. Full blood count, renal and liver function tests were all normal. Semen analysis six weeks after the infection was negative for Zika RNA. Case 3 (30-yr old male): Zika serology 15 days after onset was both IgM and IgG positive. Semen samples were PCR positive on days 29 and 45, and negative on days 93 and 136. This man’s wife conceived while abroad, but developed some mild symptoms after returning to the UK. She eventually tested negative for Zika virus by serology. Case 4 (49-yr old female): Zika serology on day 4 after symptoms showed a low level IgG response, but the IgM was negative. A repeat sample on day 17 was Zika IgM and IgG positive. Zika PCR in both serum and urine was negative. Case 5 (38-yr old male): His serology on day 7 after symptom onset was Zika IgM positive, and his urine was Zika RNA PCR positive as well. Follow up serology on day 31 remained IgM positive, and his IgG converted to positive.

Conclusion: In the UK, Zika virus PCR testing was the only test for diagnosing Zika virus infection for several months. However, the utility of this test was limited to the few sample types (urine and semen, and possibly blood and saliva very early in infection) taken within the few 1-2 weeks of illness onset. The introduction of a Zika virus serology panel (IgG, IgM) broadened the diagnostic window considerably. However, consistent with WHO guidance, this testing is not currently provided for asymptomatic pregnant women in the UK, despite it being available in the USA. Given the accumulating evidence that asymptomatic infections in pregnancy can also have adverse fetal sequelae, extending serological testing to asymptomatic pregnant women returning to the UK from Zika virus-affected areas would permit appropriate intensive monitoring of infected women whilst alleviating the anxiety that the uninfected women experience, and removing the need for extra ultrasound follow-up.

Keywords: Asymptomatic testing, Serology, Zika virus

17th International Congress of Virology 218

Abstract Submission

Arboviruses

PO670

STUDIES ON CHIKUNGUNYA VIRUS NON-STRUCTURAL PROTEIN 2

K. Rausalu 1,*, A. Utt 1, P. K. Das 2, A. Merits 1

1Institute of Technology, University of Tartu, Tartu, Estonia, 2University of Helsinki, Helsinki, Finland

Objectives: Chikungunya virus (CHIKV) is a mosquito-transmitted arbovirus that belongs to the genus Alphavirus, family Togaviridae. It has an approximately 12 kb long positive-strand RNA genome. In infected cells, the genome is first translated into non-structural polyprotein (P1234), which is cleaved by nsP2 protease domain into individual non-structural proteins 1- 4 (nsP1-nsP4), which form the viral replicase. It was recently suggested that CHIKV nsP2 protease might not be a cysteine- protease. Therefore, we analyzed the critical amino acid residues in nsP2 protease’s active site. As little is known about CHIKV replicase activity in mosquito cells we also characterized the role of CHIKV nsP2 for CHIKV RNA replication in Aag2 cells.

Methods: CHIKV nsP2 variants, harbouring mutations in protease active site residues, were first characterized by cell-free protease assays. These were carried out using recombinant protein, FRET-based peptide and in vitro translated polyprotein substrates. In cell based assays CHIKV trans-replicase and construction and rescue of recombinant viruses were used. For analysis of role of nsP2 in mosquitoes we constructed and used mosquito-specific trans-replication systems.

Results: We found that substrate requirements of CHIKV nsP2 protease are similar to these of nsP2 from related Semliki Forest virus (SFV). CHIKV nsP2 cleaved fluorogenic peptide corresponding to P10-P5’ residues of nsP3/nsP4 cleavage site, recombinant protein substrate containing the same sequence or that from nsP1/nsP2 cleavage site. Similarly to SFV nsP2 CHIKV nsP2 preferred the nsP3/nsP4 substrate for cleavage; the observed cleavage rate was ≈0.08 s-1 or 4.8 min-1 and Km was found to be 2.5±0.1 μM. Short substrates representing nsP2/nsP3 cleavage site could not be cleaved by nsP2, for this cleavage to occur the nsP3 macro-domain must be present in the substrate.

In contrast to recent report we found no evidence that Ser482 residue of CHIKV nsP2 contributes to its protease activity. Substitution of Ser482 residue with Ala had very little negative effect on the CHIKV nsP2 protease activity; in contrast substitution of Cys478 or Trp481 with Ala abolished nsP2’s ability to process recombinant protein or peptide substrates or P1234 polyprotein. Consequently, the latter mutations also abolished CHIKV RNA replication and infectious virus rescue; in contrast Ser482 to Ala mutation had no such effects.

Different mutations were inserted either in the sequence nsP2 or into nsP2 cleavage sites and the effects of these mutations on CHIKV RNA replication in Aag2 cells were analysed. Again, Cys478 to Ala mutation abolished RNA replication; in contrast most of other studied mutations had a moderate effect. Interestingly, three types of mutations that boost the replication in Aag2 cells were identified: i) the insertion of EGFP into central part of nsP2, ii) mutation blocking the cleavage between nsP2 and nsP3, and iii) the combination of two mutations affecting the speed of nsP1/nsP2 site processing. None of the mutations, that boost replication in Aag2 cells, have similar effect in mammalian cells.

Conclusion: The data from CHIKV nsP2 protease studies unambiguously confirmed that CHIKV nsP2 is a cysteine protease. Furthermore, our data indicates that the role of nsP2 protease activity in replication complex assembly/functioning is different in different host cell types.

Keywords: Chikungunya virus, nsP2, protease

17th International Congress of Virology 219

Abstract Submission

Arboviruses

PO671

SYLVATIC CHIKUNGUNYA IN PHILIPPINE MACAQUES (Macaca fascicularis) FROM THE DAVAO REGION

M. C. Otero 1,*, L. D. Sarol 1, R. Puentespina, Jr. 2, A. K. Sy 3, A. Tandoc, III 3

1Dept. of Medical Microbiology, College of Public Health, University of the Philippines Manila, Manila, 2Animal Solutions Veterinary Hospital, Davao City, 3Virology Laboratory, Research Institute for Tropical Medicine, Department of Health, Muntinlupa City, Philippines

Objectives: Chikungunya is one of the important re-emerging arboviral diseases in the Philippines, causing almost yearly urban outbreaks. The Chikungunya virus (CHIKV) can be transmitted via an urban epidemic cycle and an enzootic or sylvatic cycle. Recent studies have reported spilling over of CHIKV from non-human primates to human populations (and vice versa) in Africa and in Asia. These findings suggest that non-human primates are reservoirs of CHIKV in between outbreaks. Thus, the chance of contracting CHIKV infections increases in areas where humans and animal reservoirs, like monkeys, are in close association since the vectors have access to both hosts. In this study, Chikungunya was detected from captive and semi-wild Philippine macaques in Davao City, Tagum City, and Kapalong, Davao del Norte.

Methods: A total of forty-five caged and semi-wild Philippine macaques were captured and bled in the 3 sites. Whole blood was transported on ice to the laboratory in Davao City, and serum was separated via centrifugation. All serum samples were then brought for laboratory analyses at the Research Institute for Tropical Medicine in Muntinlupa City.

Results: Two monkeys tested positive for anti-CHIKV IgM, and one of these monkeys was febrile during blood sample collection. However, no RT-PCR amplification of the E1 and Nsp1 genes was observed for all the serum samples, possibly due to RNA degradation during transport.

Conclusion: These results support the sylvatic or enzootic transmission of CHIKV in monkeys in the Philippines.

Keywords: Chikungunya in Monkeys, sylvatic transmission

17th International Congress of Virology 220

Abstract Submission

Arboviruses

PO672

THE WEST NILE VIRUS HELICASE DOMAIN REGULATES INTERFERON RESPONSE IN INFECTED CELLS

Y. X. Setoh 1,*, A. Khromykh 1

1School of Chemistry and Molecular Biosciences, THE UNIVERSITY OF QUEENSLAND, Brisbane, Australia

Objectives: West Nile virus (WNV), a mosquito-borne virus, is the causative agent of an outbreak in New York City in 1999 (WNVNY99) responsible for the most extensive spread of arboviral encephalitis in the Americas. In Australia, an endemic and mildly virulent strain of WNV, Kunjin virus (WNVKUN), circulates in the northern parts of the country. WNVKUN infection is usually benign and asymptomatic, but in 2011, a newly emerged virulent strain of WNVKUN appeared in southeastern Australia (WNVNSW2011) and resulted in more than a thousand equine cases with a 10-15% case mortality. In vivo virulence studies showed that WNVNSW2011 possess an intermediate virulence compared to WNVKUN and WNVNY99. We hypothesised that further accumulations of genetic mutations could render WNVNSW2011 to become more virulent.

Methods: Using a method called Circular Polymerase Extension Cloning (CPEC), we generated chimeric viruses between WNVNSW2011 and WNVNY99 and compared the growth and virulence of these chimeras in mouse and human cells, and in mice.

Results: We found that the chimeric virus encoding all non-structural (NS) genes (NS1-NS5) of WNVNY99 on the WNVNSW2011 background produced the most enhanced growth and virulence. The most significant contributor being the WNVNY99 NS3 protein, specifically the helicase domain, which encoded the ability to inhibit IFN signaling in infected cells.

Conclusion: We identified the NS3-helicase domain of WNV as another viral protein that inhibit host IFN signaling, and further mutations in this region of WNVNSW2011 could have the potential to render this Australian strain of WNV more virulent.

Keywords: helicase, NS3, West Nile virus

17th International Congress of Virology 221

Abstract Submission

Arboviruses

PO673

UNIQUE DISTRIBUTION OF PHOSPHORYLATION SITES IN THE C-TERMINAL DOMAIN OF NSP3 OF CHIKUNGUNYA VIRUS

M. Teppor 1,*, E. Žusinaite 1, A. Merits 1

1Institute of Technology, TARTU UNIVERSITY, Tartu, Estonia

Objectives: Chikungunya virus (CHIKV) is a medically important mosquito-transmitted pathogen belonging to the genus Alphavirus (Togaviridae). The alphaviral life cycle crucially depends on four non-structural proteins (nsP1–4), which form the virus-specific part of the viral replicase and mediate a wide range of host-pathogen interactions. For example, nsP-s are important determinants of virus host specificity, cytotoxicity and pathogenicity. From all ns-proteins specific functions of nsP3 are least understood. This protein is essential for RNA replication and interacts with multiple cellular proteins. nsP3 of Semliki Forest virus (SFV) and Sindbis virus (SINV) is also known to be phosphorylated by host cell kinases; the same has been assumed to be the case for nsP3 of CHIKV. However, several recent studies have indicated that at least in respect of virus-host cell interactions the nsP3 proteins of these viruses are clearly different. Therefore the aim of this study was to investigate the phosphorylation status and to map probable phosphorylation sites of CHIKV nsP3.

Methods: Radiolabelling of CHIKV infected cells followed by immunoprecipitation and immunoblotting was used to determine the incorporation of labelled phosphate groups into the nsP3 of CHIKV. Regions containing phosphorylation sites were mapped using deletion- and scanning mutagenesis. Complementary data supporting the initial results was collected by the use of mass-spectrometry.

Results: It was confirmed that the nsP3 of CHIKV belonging to both the East/Central/South African (isolate LR 2006 OPY1) and Asian (isolate CNR20235) genotypes is indeed a phosphoprotein. Additionally, O'nyong'nyong virus (phylogenetically the closest relative of CHIKV) also appeared to have a phosphorylated nsP3. Deletion of the region corresponding to the beginning of the C-terminal "tail" of nsP3, which in SFV has been shown to include all phosphorylated amino acid residues, did not abolish phosphorylation of CHIKV nsP3. Thus, the location of the phosphorylation sites of CHIKV nsP3 differs from those of SFV, and similar biological properties (in vitro and in vivo attenuation) of SFV and CHIKV lacking corresponding regions are not caused by the removal of nsP3 phosphorylation. Our data is in agreement with sequence data for these two viruses: the heavily phosphorylated region at the beginning of the C-terminal part of SFV nsP3 has an abundance of serine and threonine residues, while the respective region of CHIKV nsP3 contains less of such residues. In contrast (and unlike the nsP3 of SFV), the nsP3 of CHIKV contains a number of eligible sites for host kinases outside of this region. Their importance for CHIKV nsP3 phosphorylation was confirmed by the use of an additional set of viruses harbouring deletion and substitution mutations.

Conclusion: Our data confirmed that similar to its SFV and SINV counterparts, CHIKV nsP3 is clearly a phosphoprotein. However, unlike the nsP3 of SFV, CHIKV nsP3 does not contain a heavily phosphorylated cluster of amino acids at the beginning of its C-terminal tail; instead phosphorylation sites are more dispersed along the protein. As a consequence, the nsP3 of an attenuated CHIKV vaccine candidate, designed on the basis of an SFV counterpart, is still phosphorylated and the attenuation of this virus originates from different mechanism(s). Links between nsP3 phosphorylation, location of viral replicase complexes in infected cells and in vivo properties of mutated CHIKV constructs represent topics for additional studies.

Keywords: Alphavirus, Chikungunya virus, nsP3 phosphorylation

17th International Congress of Virology 222

Abstract Submission

Arboviruses

PO674

VECTOR COMPETENCY OF AEDES AEGYPTI AND AEDES ALBOPICTUS FROM THE AMERICAS FOR VARIOUS STRAINS OF ZIKA VIRUS

S. Azar 1,*, C. Roundy 1, S. Rossi 1, K. Hanley 2, N. Vasilakis 1, S. Weaver 1

1Pathology, University of Texas Medical Branch, Galveston, 2Biology, New Mexico State University, Las Cruces, United States

Objectives: To evaluate the potential role the domestic and peridomestics vectors Aedes aegypti and Aedes albopictus play in the transmission of Zika virus (ZIKV) in the Americas and to determine whether different ZIKV strains differred in their ability to infect mosquitoes, we undertook a vector competency analysis.

Methods: Minimally colonized mosquito populations from the Americas were orally exposed to artificial or in vivo blood meals containing one of 5 strains of ZIKV. Mosquitoes were maintained for up two weeks. Bodies, legs and saliva were collected at various time points post-feeding and monitored for infection, dissemination, and transmission potential respectively via focus forming assay. Positive salivary samples were further evaluated to estimate the salivary titer.

Results: We found evidence in Ae. aegypti that in vivo blood meals prove more infectious than artificial bloodmeal. Additionally, in the populations tested the African strain DAKAR 41525 was significantly more efficient at infection, dissemination and transmission. Additionally we determined a maximum salivary titers of Ae. aegypti to be 3.0log10 focus forming units per collections, while the maximum salivary titer of Ae. albopictus was determined to be be 3.72 log10 FFU/collection.

Conclusion: Overall, our data recapitulates the finding that different mosquito populations of the same genus vary in their ability to be infected by and transmit ZIKV. Additionally, our data indicates that Ae. aegypti populations tested are more competent vectors of ZIKV than Ae. albopictus populations.

Keywords: arbovirus vectors, zika virus

17th International Congress of Virology 223

Abstract Submission

Arboviruses

PO675

VIRAL POPULATION REPLACEMENT OF DENGUE SEROTYPE 2 VIRUSES IN TWO CONSECUTIVE OUTBREAKS IN TAIWAN

H.-Y. Ko 1,*, S.-Y. Lin 1, Y.-T. Lee 1, C.-H. Chin 1, Y.-H. Pan 1, C.-C. King 1 1Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan

Objectives: Dengue virus (DENV), a mosquito-borne human pathogen, poses an important threat to global health. In Taiwan, the epidemic of dengue/dengue hemorrhagic fever (DHF) in 2001 to 2003 caused by dengue virus serotype 2 (DENV-2) was the most severe epidemic since 1942 in Kaohsiung. This dengue epidemic has unique characteristics: it crosses three years from 2001 winter to 2003 spring, including two epidemic peaks, overwintering transmission. In addition, the second year epidemic has higher DHF percentage and larger number of infected population. We thus propose a hypothesis that epidemic potentials of different DENV-2 isolates varied within the same epidemic in Kaohsiung and would correlated with their virological characteristics. In this study, we try to understand the origin of the circulating viruses, transmission dynamics and the factors boosting the epidemiological and clinical severity.

Methods: Totally 2234 blood samples from 1565 dengue-confirmed patients were collected from 2001 to 2003 during dengue epidemic period. To study the phylogenetic relationship of dengue viruses in this epidemic, totally 104 sequences (1485 nt.) of E protein gene and 43 polyprotein open reading frame (ORF) (10176 nt.) of DENV viruses had been sequenced. Time-scaled phylogenies were inferred by the Bayesian Markov Chain Monte Carlo (MCMC) method.

Results: Three virus groups according to the phylogenetic analyses and conserved amino acid residues: Ia and II groups were representative of prototype DENV-2 in 2001 and 2002, respectively, whereas Ib as an intermediate strain. Phylogenetic analyses show the most common ancestor of these three groups of viruses clustering with virus isolated in Philippian with an emerging time point prior to the epidemic in 2001. Ib viruses had lower transmissibility throughout 2001- 2003 compared to the other two groups of virus, which started their transmissions in 2001 and 2002, separately; however, the transmission of Ib sustained across the winter with its R0 resurged in early 2002.

Conclusion: In this study we provided an investigation of dengue virus maintain its transmission during inter-epidemics, and found the properties of maintenance strains. Our study not only delineates the evolution process of DENV in different scales but also provide new insights into increasing severity as viral transmission.

Keywords: Dengue virus, Epidemic potential, Viral Population replacement

17th International Congress of Virology 224

Abstract Submission

Arboviruses

PO810

GLYCOBIOLOGY OF ARBOVIRUSES: DO GLYCANS AFFECT THE TRANSMISSION OF ROSS RIVER VIRUS?

W. S. Lee 1,*, A. Van Den Hurk 2, L. Herrero 1

1Institute for Glycomics, Griffith University, Gold Coast, 2Virology, Public and Environmental Health, Forensic and Scientific Services, Department of Health, Queensland Government, Brisbane, Australia

Objectives: Mosquito-transmitted viral diseases (arboviruses) are a leading cause of morbidity and mortality worldwide. In Australia, Ross River virus (RRV) causes thousands of cases of polyarticular disease annually, the highest incidence of any circulating arboviruses in the country. Arboviruses are maintained in nature by a continuous cycle of transmission between mosquitoes and susceptible vertebrate hosts. To be transmitted by a mosquito, the virus in an infected blood meal must invade the mosquito’s midgut epithelium by crossing the midgut infection barrier (MIB). Once in the midgut epithelium, virus replicates and disseminates to other secondary tissues, such as the salivary glands, allowing subsequent infection of a susceptible vertebrate host during feeding. The midgut epithelium is decorated with a dense array of glycoconjugates and many arboviruses target host cells by attachment of the envelope glycoprotein to glycans on the luminal face of the midgut epithelium to gain entry and initiate infection. On the virus itself, N-linked glycosylation is the most common type of protein modification on the envelope glycoprotein. This glycosylation has been found to be involved in biological functions, such as proper folding of protein, binding to host surface receptors, entry into host cells, trafficking in the ER, as well as assembly and budding of virus. The envelope glycoproteins of RRV contain more than one N-glycosylation sites. Despite the importance of these early interactions, the binding targets and key interactions involved are still unknown. In this study, we identified the glycans and lectins present on the surface of RRV and evaluated the role of novel viral N-linked glycosylation sites on the envelope glycoproteins of RRV.

Methods: We used site-directed mutagenesis to make a number of unique mutant viruses lacking the glycosylation sites of the E1 and E2 glycoprotien to evaluate the role of novel viral N-linked glycosylation in viral infectivity both in vitro and in vivo. We then determined the changes in glycan composition and glycan binding specificity caused by removal of the viral envelope glycosylation sites using lectin and glycan array technology, respectively. The interactions between RRV and glycans were also determined using surface plasmon resonance (SPR).

Results: Our study revealed different glycan and lectin-binding profiles of viral mutants lacking the glycosylation sites in either E1 or E2 glycoproteins. The viral mutants lost the ability to bind 23 glycan structures of different classes. We have also demonstrated the role of glycosylation on the E2 glycoprotein for dissemination of RRV in a common mosquito vector, Aedes vigilax.

Conclusion: This study would provide insights on the specificity of carbohydrate binding proteins and the cell surface glycosylation on RRV through the use of glycan and lectin array technology, respectively. This study also defines the role of N-linked glycosylation on the envelope glycoproteins of RRV for viral attachment and entry into host. Finally, the results of this study would provide the groundwork for further study into the glycan-lectin interactions between viruses and host cells to establish successful infection.

Keywords: Envelope glycoprotein, N-linked glycosylation, Ross River virus

17th International Congress of Virology 225

Abstract Submission

Arboviruses

PO874

THE NOTCH SIGNALING PATHWAY IS ESSENTIAL FOR THE REGULATION OF MOSQUITO REPRODUCTION S.-H. Shiao*

Objectives: Mosquitoes are important vectors for several infectious diseases such as malaria and dengue fever. All together kill more than one million people a year, due to the unavailability of effective vaccines and the development of insecticide and drug resistance to vectors and pathogens. Therefore, there is an urgent need to explore every possible avenue for developing novel control strategies against these mosquito-borne diseases.

Methods: Notch signaling pathway is an evolutionary highly conserved cell-cell signaling pathway. Notch acts as both transmembrane receptor and transcription activator. It has been reported that Notch signaling may activate through a ligand or CSL independent (non-canonical) mechanism. However, its detail mechanism remains largely unknown.

Results: Our previous results demonstrated that silencing of Notch in the mosquito Aedes aegypti resulted in significant inhibition of egg tanning and hatching rate. To further confirm the role of Notch signaling in egg tanning and embryogenesis, we made use of DAPT, a Notch signaling inhibitor, to examine the effect on mosquito reproduction. To elucidate the role of non-canonical Notch signal in the mosquito reproduction, a non-canonical pathway factor (JNK) was inhibited by SP600125 to examine the effect on egg tanning. Our results showed that inhibition of JNK activity resulted in the reduction of egg tanning and hatching rate. Interestingly, the activity of chorion peroxidase, which catalyzing chorion protein cross-linking during chorion hardening, was also reduced in non-tanning eggs.

Conclusion: In the future, we will investigate the non-canonical Notch-mediated mosquito reproduction. Data revealed by this study will be crucial for future study on vector competence and vector control in the field.

Keywords: mosquito, reproduction

17th International Congress of Virology 226

Abstract Submission

Arboviruses

PO892

Elucidation of host innate immune response during Japanese encephalitis virus infection S. K. Saxena*

Please specify what speaker category are you in?: Workshop chair Objectives: Recently there was a major epidemic of Japanese encephalitis in the northern states of India causing at least 6097 cases and 1398 deaths. Our laboratory has found a novel strain of JEV (JEV-GP05, accession no: FJ979830) which has significant variation as compared to JEV-GP78 (endemic strain of that area). Earlier studies have shown that JEV replicates in macrophages and microglial cells. Macrophages have been implicated to play an important role during early innate response via induction of nitric oxide (NO) in various viral infections. NO has an inhibitory effect on Ectromelia virus, Vaccinia virus, Herpes simplex virus type-1, Influenza virus and Vesicular stomatitis virus replication. Methods: Therefore, we have planned to elucidate the role of macrophages towards host-defense during novel JEV-GP05 infection as per the standard protocols. Results: Our data suggest that macrophages produced NO after JEV-GP05 stimulation. The response was sensitive to nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The enhanced level of NF-kB observed in the early phase of JEV infection correlated well with the enhanced activity of iNOS. Pretreatment of cells with L-NMMA increased the mortality in JEV infected cells and inhibited NO production in vitro. However, NO donor Sodium nitroprusside (SNP) stimulated macrophages inhibited virus replication with high levels of NO production. Ascorbic acid (an essential antioxidant) treatment increased the survival rate of JEV infected macrophages significantly (P<0.05). Conclusion: These observations demonstrated the protective role of macrophages induced NO during JEV infection. This induction was mediated via iNOS gene expression involving NF-kB activation pathway, and may be a key mediator in host innate immune response to infection. This may be one of the important mechanisms of host-defense and natural immunity in controlling the initial stages of novel JEV-GP05 infection by macrophages.

Keywords: Flavivirus, Japanese encephalitis virus, Viral immunology

17th International Congress of Virology 227

DNA Viruses

17th International Congress of Virology 228

Late breaking abstract

DNA viruses

PO678

Differential innate immune signaling in macrophages by wild type vaccinia mature virus and a mutant virus deleting A26 protein

S. K. Kasani 1, H.-Y. Cheng 1, K.-H. E. Yeh 1, S.-J. Chang 1, P. W.-C. Hsu 1, S.-Y. Tung 1, C.-T. liang 2, C. H. Yang 1,*, W. Chang 1

1Institute of Molecular Biology, Academia Sinica, 2National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan

Objectives: The Western Reserve (WR) strain of mature vaccinia virus contains an A26 envelope protein that mediates virus binding to cell surface laminin and subsequent endocytic entry into HeLa cells. Removal of A26 protein from mature virus generates a mutant WRΔA26 that enters HeLa cells through plasma membrane fusion.Here, we infected murine bone marrow-derived macrophages (BMDM) with wild-type WR (WT-WR) and WRΔA26 and analyzed viral gene expression and cellular innate immune signaling. In contrast to previous studies in which HeLa cells infected by WT-WR or WRΔA26 both expressed abundant viral late proteins, we found that WT-WR expressed much less viral late protein than WRΔA26 in BMDM. It remains unclear why BMDM executes differential restriction on viral late protein expression between thse two viruses.

Methods: We therefore isolated total RNA from BMDM infected with either WT-WR or WRΔA26 at 1,2,4 and 8hours post infection for microarray analyses in order to understand host cell responses.

Results: Microarray analysis of cellular transcripts in BMDM induced by virus infection revealed that WT-WR preferentially activated interferon receptor (IFNAR)-dependent signaling, but WRΔA26 did not. Consistently, we detected a higher level of soluble IFNβ secretion and phosphorylation of Stat1 protein in BMDM infected with WT-WR compared to WRΔA26. When IFNAR knockout BMDM were infected with WT-WR, late viral protein expression increased, confirming that IFNAR-dependent signaling was differentially induced by WT-WR and in turn restricted viral late gene expression. Finally, wild-type C57BL/6 mice were more susceptible to mortality from WRΔA26 than WT-WR infection, whereas IFNAR knockout mice were equally susceptible to WT-WR and WRΔA26 infection, demonstrating that the ability of WRΔA26 to evade IFNAR signaling has an important influence on viral pathogenesis in vivo.

Conlusion: In conclusion, we showed that WT-WR infection induced a robust innate immune responses that activates interferon receptor (IFNAR)-dependent cellular genes in BMDM, whereas WRΔA26 did not. We further demonstrated that differential activation of IFNAR-dependent cellular signaling between WT-WR and WRΔA26 is not only important for differential host restriction in BMDM but is also important for viral virulence in vivo. Our study reveals a new property of the WRΔA26 in regulating host antiviral innate immunity in vitro and in vivo.

Keywords: bone marrow -derived macrophage, vaccinia virus

17th International Congress of Virology 229

Abstract Submission

DNA viruses

PO679

GENETIC POLYMORPHISM OF VARICELLA-ZOSTER VIRUS IN IN VITRO CELL PASSAGING AND ATTENUATION

J. S. Park*, I. K. Kim, H. R. Hwang, S. H. Yeon, C. H. Lee

Objectives: Varicella-Zoster Virus(VZV) is a member of herpesviruses, with a genome size about 125 kbp. Primary infection of VZV results in varicella and reactivation from latency often leads to zoster. Both varicella and zoster can be prevented by live attenuated vaccines, but the molecular mechanisms of attenuation is not clearly understood. In this study, genetic polymorphism of VZV were investigated in order to get an insight into the molecular mechanism of attenuation.

Methods: We attempted to understand the nature of VZV genetic polymorphism by determining the relative frequencies of 4 nucleotides at each position of VZV whole genome using next-generation sequencing technique.Clinical strains YC01, YC02 and YC03 isolated from Korean patients were passaged in vitro cell culture up to 110 times. Different passages of YC01 (p14, p110), YC02 (p14, p110) and YC03 (p6, p110) were subjected to next-generation seqeuncing (NGS) and their full genome sequences were compared with each other.

Results: Genetically polymorphic site (GPS) was defined as the site where the proportion of minor base exceeds 5%. The number of GPS increased in high passaged strains. The GPS was not distributed evenly among the genome, and appeared to be clustered in ORF11, ORF14, ORF22, and ORF62/71. The most prevalent pairs of major and minor base were A/g, C/t, G/a, and T/c where major bases shown as capital and minor bases were shown as small letters. Conclusion: VZV genomes are highly polymorphic and further studies of genetic polymorphism in vaccine and high- passage clinical strains will help to elucidate the molecular mechanisms of VZV attenuation.

Keywords: attenuation, polymorphism, VZV

17th International Congress of Virology 230

Abstract Submission

DNA viruses

PO680

INTERACTION BETWEEN OV20.0 AND ADENOSINE DEAMINASES ACTING ON RNA 1

G.-R. Liao*, F.-Y. Lin 1, Y.-Y. Tseng 2 3, W.-L. Hsu 2 1Department of Beauty Science, MeiHo University, Pingtung , 2Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taichung, Taiwan, 3Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, Australian National University, Canberra , Australia

Objectives: Orf virus (ORFV) OV20.0 is an important virulent factor. By interacting with protein kinase R (PKR) and its activator, namely PACT, OV20.0 inhibits PKR activation to promote virus replication. OV20.0, PKR and PACT all harbor dsRNA binding domains (DRBDs) and are classified as dsRNA binding proteins (DRBPs). It has been proposed that DRBPs regulate cell physiology by formation of protein dimer. A DRBP, designated adenosine deaminases acting on RNA 1 (ADAR1), catalyzes deamination of adenosine (A) to produce inosine (I) in dsRNA substrate in nucleus. Due to its nature of binding dsRNA, we postulated that OV20.0 possibly interacts with ADAR1. Therefore, we studied the impact of OV20.0 on cellular function of ADAR1 and their effects on ORFV pathogenesis.

Methods: Immunoprecipitation was performed to identify cellular proteins interacting with OV20.0. A series of constructs containing deletions of individual functional domains were used to determine the minimal criterial for intermolecular interaction are determined. To further confirm their cellular distribution, transient expression of GFP-tagged OV20.0 and mCherry-tagged ADAR1 followed by immunofluorescence assay was performed.

Results: In current study, association of ADAR1 with OV20.0 was evidenced for the first time. Consistently, C-terminal DRBD of OV20.0, and the first DRBD of ADAR1 are critical for this interaction. Furthermore, we found mCherry-tagged ADAR1 colocalized with two OV20.0 isoforms, the full length and N terminal truncated OV20.0 (sh20), in nucleus. Interestingly, ARAD1 was also shuttled to cytoplasm in the proximity of sh20 in a subset of cells that indicates the interaction between ADAR1 and sh20 possibly alters their cellular distribution.

Conclusion: OV20.0 associates with cellular ADAR1 protein via their DRBD. Furthermore, among the three DRBDs, the first DRBD of ADAR1 is critical for their interaction. The consequence of interaction leads to re-distribution of ADAR1 to cytoplasm, indicating that OV20.0 possibly influences the function of ADAR1.

Keywords: ADAR1, orf virus, OV20.0

17th International Congress of Virology 231

Late breaking abstract

DNA viruses

PO681

PURIFICATION OF VACCINIA VIRAL A26 PROTEIN FOR BIOCHEMICAL ANALYSIS AND CRYSTALLIZATION

Y.-C. Luo 1,*, H.-C. Wang 2, W. Chang 1 1INSTITUTE OF MOLECULAR BIOLOGY, ACADEMIA SINICA, 2Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

Objectives: Vaccinia virus is the prototypic member of the Orthopoxvirus genus of the family Poxviridae. It contains a double-stranded DNA genome of approximately 190 kb and encodes more than 200 viral proteins. Our previous studies showed that vaccinia mature virus (MV) of Western Reserve (WR) strain is endocytosed into HeLa cells and subsequently viral membrane and vesicular membrane is fused to release viral core into cytoplasm. Furthermore, when a viral A26 protein was deleted from vaccinia virus genome, the resulting WR ΔA26 virus altered the entry route from endocytosis to plasma membrane fusion suggesting that removal of A26 protein eliminated virus dependence on acidification for membrane fusion. Further experiments showed that viral A26 binds to viral entry fusion complex in an acid-dependent manner and supported a model that A26 is a low-pH sensitive fusion suppressor during MV endocytosis into HeLa cells. Our current studies showed that A26 protien contains an acid sensitive N-terminal region that responds to low pH rendering virus-mediated fusion under control of enviromental cue. When the N-terminal region of a26 was deleted or mutated vaccinia virus became unresponsive to low pH and mature virus infectivity was reduced more than 10 fold. To understand how A26 protein functions as an acid-sensitive fusion suppressor of vaccinia virus, we plan to express vaccinia viral A26 protein in E. coli, purify and crystallize the protein to obtain 3D structure in order to understand its structure and funcational relationship.

Methods: Vaccinia virus full length A26 open reading frame was cloned into pET16b and dequenced correctly. The recombinant A26 protein will be induced using IPTG and purified using Ni-NTA affinity chromatography. Further purification will be employed if needed.

Results: Recombinant A26 protein fused with His10 tag at the N-terminus was purified using Ni-NTA affinity chromatography using imidazole gradient solution. The eluted protein was separated on 12% SDS-PAGE to verify the yield and purity.

Conlusion: The yield of recombinant A26 was relatively low. We will scale up the protein expression level to obtain large amount of protein for further structure analyses. We will also run the protein on size exclusion chromatography to assess its oligomeric state.

Keywords: None

17th International Congress of Virology 232

Abstract Submission

DNA viruses

PO682

VARIABLE NUMBER TAMDEM REPEATS (VNTRs) IN THE CYPRINID HERPESVIRUS-3 (CyHV-3) GENOME: A MOLECULAR EPDEMIOLOGICAL TOOL

Stone, D M, Schofield M, Martin P, Wood G and Way K Cefas, Weymouth, Dorset, UK.

D. Stone*

Objectives: Cyprinid herpes virus-3 (CyHV-3) is a highly contagious viral disease which causes significant morbidity and mortality in common carp and its ornamental domesticated form, koi carp. Until 2003, KHV had only been detected and isolated from sites in the UK holding imported ornamental carp. However, during investigations into mortalities in angling waters in England since 2003 the Cefas Weymouth laboratory detected CyHV-3 in common carp suggesting that the introductions of KHV to these waters may be linked to either the rearing or holding of common carp, destined for restocking fisheries, alongside ornamental varieties of carp, or the stocking of waters with a susceptible ornamental species. Molecular epidemiological investigations have sought to discriminate between the different geographical strains of CyHV-3 to better understand the transmission routes. Here, we have used variable number tandem repeats (VNTR) to investigate the genetic variability in KHV genome and to establish links between outbreaks of KHV disease in the UK.

Methods: Fifteen uninterrupted VNTRs displaying at least 4 repetitions were identified in the CyHV-3 genome by alignment of the three complete genome sequences for CyHV-3 (accession no. DQ657948.1 DQ177346.1 and AP008984.1). Four of the loci, were studied in detail.

Results: A total of 75 distinct VNTR profiles were observed in the UK between 2003 and 2016, suggesting large numbers of independent introductions in to the UK, and highlights the potential for even greater genetic diversity of the CyHV-3 strains circulating globally. During our investigations, novel CyHV-3 like DNA sequences were also detected in carp and koi tissues when using a generic PCR primers capable of detecting a wide range of cyprinid herpesviruses. The same samples were negative for CyHV-3 when using the recognised diagnostic assay based on the thymidine kinase gene sequence.

Conclusion: The same VNTR profile was observed in all viruses identified during a single disease episode, indicating that they represent suitable markers for epidemiological studies and outbreak tracing. Importantly, the same VNTR was observed in viruses isolated from a fishery site after long periods without clinical disease indicating that the virus can remain dormant for several years, and raises concerns that the virus may be more widespread than originally thought but not manifest as a clinical disease outbreak. This will have serious implications for surveillance for CyHV-3 and how transmission of CyHV-3 infection is controlled. The implications of the novel CyHV strains for common carp are not known, however, most of the CyHV-3 variants were detected in apparently healthy animals. Only in a few cases the animals exhibited signs usually attributed to a CyHV-3 infection which suggests either that the animals are asymptotic carriers or survivors of infection with low virus loads, or that the variants represent low-pathogenic strains of CyHV-3 that do not warrant control in the same way as conventional CyHV- 3 isolates. The potential origins of these novel CyHV-3 viruses and the impact on surveillance for CyHV-3 distribution and prevalence will be discussed.

Keywords: Alloherpesviridae

17th International Congress of Virology 233

Abstract Submission

DNA viruses PO692

SUPPRESSION OF THE GUT MICROBIOME ACCELERATES LETHAL MOUSE HERPESVIRAL INFECTION; MODIFIED MYXOMAVIRAL SERPIN PEPTIDE IMPROVES SURVIVAL

A. Lucas*, S. Ambadapadi 1, C. Jobin 2, S. Karst 3, S. Tibbetts 3, J. Yaron 1, G. McFadden 4 1Center for Personalized Diagnostics, Biodesign Institutre, Arizona State University, Tempe, 2Medicine, 3Molecular Genetics and Microbiology, University of Florida, Gainesville, 4Center for immunology, Vaccines and Virotherapy, Biodesign Institutre, Arizona State University, Tempe, United States

Objectives: Inflammatory vascular syndromes (IVS), giant cell arteritis and Takayasu’s disease, are rare but devastating arterial disorders. Mouse gamma herpesviral (MHV68) infection in interferon gamma receptor knock out mice (IFNgR-/-) is a model for IVS. The role of the bacterial microbiome in IVS and treatment is not defined. In prior work myxomavirus- derived serine protease inhibitor (serpin), Serp-1, significantly improved survival and reduces aortic inflammation in MHV68 -/- infected IFNgR mice. An anti-inflammatory peptide, S-7 (G305TTASSDTAITLIPR319), derived from the Serp-1 reactive center also improves survival

Methods: We examined survival and disease progression in 56 MHV68 infected IFNgR-/-mice, both with and without Serp- 1, serpin peptide (S-2, S-7 or S-8) or modified S-7 peptide treatments with and without suppression of gut bacteria with oral antibiotics.We also examined immune cell and microRNA response.

Results: Depletion of gut bacteria accelerated MHV68 induced IVS increasing early mortality, reducing survival from 60 to 20 days (P<0.036). Suppression of gut bacteria reduced efficacy of Serp-1 and S-7 treatment, decreasing survival from 70% at 150 days to 20% at 30 days for Serp-1 (P < 0.0028) and to 0% at 30 days for S-7 (P<0.0001). S-2 peptide was inactive and S-8 produced a non-significant trend toward improved survival with antibiotic treatment (N=14). However a modified S-7 peptide (N = 10) designed to improve serpin blockade significantly improved survival in MHV68 infected mice (P < 0.01). Antibiotics suppressed aerobic stool bacterial growth, but did not alter Serp-1 inhibition of uPA. HASmiRNA 126 and 136 were significantly decreased and 335 increased with Serp-1. Antibiotic treatments modified TH1, Treg and M1/ M2 levels.

Conclusion: Suppression of gut bacteria accelerates MHV68 infection, suggesting interaction between herpes infection and the microbiome. Depletion of gut bacteria also reduces serpin and serpin peptide treatment efficacy, but a modified serpin peptide is capable of improving survival. The microbiome may have a central role in viral sepsis and IVS.

Keywords: MHV68, Microbiome, serpin

17th International Congress of Virology 234

Abstract Submission

DNA viruses

PO783

THE ROLE OF RECEPTOR-SMAD PROTEINS DURING VACCINIA VIRUS INFECTION

C. Abbott 1,*, T. Newsome 1

1Department of Microbiology, The University of Sydney, Sydney, Australia

Objectives: Vaccinia virus (VACV) possesses many different techniques to facilitate viral movement in the host. These include subversion of host cell systems such as the microtubule network and actin cytoskeleton. Induction of cell migration following infection with VACV is often observed in concert with a suite of morphological changes. These changes are strikingly similar to the host phenomenon known as epithelial-mesenchymal transition (EMT), a process that occurs during embryonic morphogenesis, cancer metastasis, and wound healing. Previous observations in our lab have indicated elements of the TGF-β/Smad pathway, a key EMT regulatory pathway, as active following VACV infection, potentially playing an important role in facilitating virus spread throughout the host. To this end, the role of the receptor-Smad proteins, Smad2 and Smad3, was investigated.

Methods: Firstly, phosphorylation status of Smad2 and Smad3 proteins was deteremined. To assess the role of Smad2 and Smad3 during VACV infection, multiple approaches such as CRISPR/Cas9 and transient delivery of siRNA were employed to deplete levels of either Smad2 or Smad3. Cell populations were then assessed for effectiveness of plaque formation and changes in infected cell morphology.

Results: Not only are receptor-Smad proteins phosphorylated following infection, but their absence impacts strongly upon successful viral infection. We observed that deficiencies in the receptor-Smad proteins not only severely attenuated VACV infection but also prevented the characteristic morphological changes normally observed following infection.

Conclusion: Ultimately, these results support a role for VACV-mediated Smad signalling in eliciting an EMT-like phenotype to promote virus spread. While current trends in EMT-related research are largely within the contexts of either of developmental biology and cancer metastasis, these findings indicate a potentially important, and as-yet under-researched, role for EMT in pathogenesis.

Keywords: host-pathogen interactions, Smad signalling, vaccinia virus

17th International Congress of Virology 235

Late breaking abstract

DNA viruses

PO879

Investigation of vaccinia virus entry pathways and pathogenesis in vivo S. Khadijah Kasani*

Objectives: Vaccinia virus (VV) is a large DNA virus that infects many cell cultures in vitro and animal species in vivo. We have recently showed that vaccinia A26 protein of Western Reserve (WT-WR) strain is the determinant for virus entry into HeLa cells through fluid-phase endocytosis whereas a mutant virus, WRΔA26, entered HeLa cells through plasma membrane fusion. Here, we extended our studies to mouse model infection of WT-WR vaccinia virus in comparison to the mutant vaccinia virus, WRΔA26, to investigate whether different entry pathways of vaccinia virus affect pathogenesis in vivo.

Methods: We inoculated BALB/c mice intranasally with WT-WR or WRΔA26 with 105 PFU per mouse and monitored for body weight change and mortality. We extracted mice nasal turbinates, lungs, brains, spleens, ovaries and thymus to check for virus titers. We also analyzed for immune infiltrates and cytokine expression in lungs of the infected mice.

Results: Our results showed that WRΔA26 virus caused higher body weight loss and mortality than WT-WR virus did. Consistently, immunohistochemistry also showed more extensive necrosis in the nasal turbinates and lungs of mice infected with WRΔA26 virus compared to WT-WR virus. Comparable virus replication was observed in the nasal turbinates and lungs although WR∆A26 seemed to spread faster into secondary organs, ovaries and thymus. We further analyzed the lungs, of infected mice. We observed significant changes in the immune cells homeostasis (myeloid and lymphoid populations) and inflammatory cytokines expression of WT-WR compared to WRΔA26 infected mice.

Conlusion: In vivo infection of WT-WR and WR∆A26 resulted in different immune responses due to differential entry pathways; mice infected with WR∆A26 showed enhanced inflammatory responses that include, increased neutrophils, inflammatory monocytes, increased inflammatory cytokines and thymus atrophy.

Keywords: None

17th International Congress of Virology 236

Double Strands RNA Viruses

17th International Congress of Virology 237

Abstract Submission dsRNA Viruses

PO683

FIRST ISOLATION AND CHARACTERIZATION OF PTEROPINE ORTHOREOVIRUSES IN FRUIT BATS IN THE PHILIPPINES

S. Taniguchi 1,*, K. Maeda 2, T. Horimoto 3, J. Mansangkay 4, R. Puentespina Jr. 5, K. Egawa 1, S. Fukushi 1, Y. Yoshikawa 6, M. Shimojima 1, M. Saijo 1, S. Kyuwa 7

1Virology I, National Institute of Infectious Diseases, Japan, Tokyo, 2Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, 3Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 4College of Veterinary Medicine, University of the Philippine Los Baños, Los Baños, 5University of the Philippines Mindanao, Davao, Philippines, 6Department of Animal Risk Management, Chiba Institute of Science, Chiba, 7Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

Objectives: Pteropine orthoreovirus (PRV) belongs to the genus Orthoreovirus, family Reoviridae and causes respiratory tract illness (RTI) in humans in Malaysia and Indonesia. Up to date, several PRV strains were isolated from fruit bats in Australia, China, Malaysia, and Indonesia. In this study, we investigated the prevalence of PRV infection in wild bats in the Philippines.

Methods: Ninety-one wild bats were captured on the Mindanao, Samal, and Talikud Islands, the Philippines, in August 2013 under the approval of the Department of Environment and Natural Resources, the Philippines. Viral isolation from throat swabs and serological survey against PRV were performed. All isolated viruses were identified as PRV by orthoreovirus specific RT-PCR. The nucleic acid sequence determination of the isolates’ whole genome was performed by Sanger Sequencing method. Furthermore, the characteristics of the PRVs were analyzed in comparison with those of Miyazaki- Bali/2007 strain, which was isolated from an RTI patient who returned to Japan from Indonesia in 2007.

Results: PRVs were isolated from throat swabs collected from 9 of 91 wild bats. Phylogenetic analyses of the isolates’ amino acid sequences indicated that the isolated PRVs were novel strains in which viral genome re-assortment events occurred. Serum specimens collected from 76 of 84 bats were positive for PRV neutralization antibody. The characteristic comparison between isolates and Miyazaki-Bali/2007 strain revealed that the Philippines bat-borne PRVs had similar characteristics in terms of antigenicity to those of Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro).

Conclusion: We isolated novel PRV strains from wild bats in the Philippines. The results of viral isolation and serological assay suggested the high prevalence of PRV in wild bats in the Philippines. This is the first report that describes the epidemiological features of PRV in wild bats in the Philippines. Recently, PRV was detected in the oropharyngeal samples of outpatients with upper RTI in a suburban setting in Malaysia. The impact of the Philippines bat-borne PRVs should be studied in human RTI cases in the Philippines.

Keywords: bat, Philippines, Pteropine orthoreovirus

17th International Congress of Virology 238

Abstract Submission

Orthoreo/Orbi/Rotaviruses

WHOLE GENOMIC CHARACTERIZATION OF G9P[8] ROTAVIRUS IN WUHAN, CHINA, FROM 2004 THROUGH 2016

Yuan Hong Wang* 1, Xuan Zhou1, Bei Bei Pang1, Xue Ying Wang2, Cheng Chen3, Yu Ting Zuo1, Nobumichi Kobayashi4

1Division of Microbiology, Wuhan Centers for Disease Prevention & Control, 2Department of Laboratory , Hubei University of Chinese Medicine, 3Department of Laboratory, Hubei University of Chinese Medicine, Wuhan, China, 4Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan

Objectives: Group A rotavirus (RVA) is a major etiologic agent of severe acute gastroenteritis in children. This study was conducted to analyze the fluctuation of the genotypes of RVA in Wuhan, 2011-2016, and whole genomic evolution of G9P[8] rotaviruses collected in Wuhan from 2004 through 2016.

Methods: Fecal specimens were collected continually from outpatients with acute gastroenteritis in Wuhan, China from Jun. 2011 through Nov. 2016. Rotavirus was screened by detection of 11 RNA segments in polyacrylamide gel electrophoresis. G and P types of RVA were determined by RT-PCR or sequencing. Sequences of genes were determined directly with RT-PCR products. Multiple alignments of sequences and phylogenetic analysis were performed using MAFFT program and MEGA software, respectively.

Results: Detection rates of RVA were 25.9% (816/3148) and 11.7% (177/1514) in children and adults, respectively, during 2011-2016. Among 993 positive specimens, the most frequent genotype was G9P[8] (69.1%). The proportion of G9 in children (71.6%) was higher than that in adults (58.2%) (P<0.01) among all genotypes. The whole genomic sequences of total 65 G9P[8] strains, that were chosen in each year from 2004 through 2016, were determined. Genotypes of 11 genes of these 65 strains were assigned to G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. The phylogenetic analysis of individual genes revealed at least two lineages in gene segments encoding VP1, VP4,VP6, VP7 and NSP1-NSP5. In contrast, only a single lineage was detected for VP2 and VP3 genes. Remarkable lineage shift was observed for VP7 and NSP1 genes. G9 rotaviruses emerged in Jul. 2004 in Wuhan, whose VP7 genes were genetically close to those of globally spreading G9 strains, and were mostly combined with OP-354 like P[8], P[6] or P[4] with very low frequency. In contrast, the strains with variant G9-VP7 genes emerged in Jan. 2010, and became predominant from 2012 until now. The variant G9-VP7 genes were genetically close to recent human G9 strains detected in Asia, America and Africa showing 99% identity, while their identity to historical globally spreading human strains was 93%. The VP7 gene of variant human G9 rotaviruses clustered with the contemporary Chinese porcine rotaviruses in the same lineage with 94-96% nucleotide identities. Difference in deduced amino acids in partial antigenic regions of VP7 was found between old G9 strains and new variants. Except for VP7 gene, the remaining 10 genes were very close to human G1P[8] and G3P[8] strains detected in Wuhan during the same period.

Conclusion: Two VP7 gene lineages of G9 Rotavirus co-circulated in Wuhan from 2010. It was suggested that the VP7 genes of variant G9 were introduced to human Wa genogroup rotaviruses with G3 and/or G1 by reassortment, followed by adaptation and spread widely among humans, especially in children.

Disclosure of Interest: None Declared

Keywords: G9P[8], Genome evolution, Rotavirus

17th International Congress of Virology 239

Hepatitis Viruses

17th International Congress of Virology 240

Abstract Submission

Hepatitis viruses

PO686

CATHEPSIN L IS REQUIRED FOR ENTRY STEP OF HEPATITIS E VIRUS

S. Nagashima*, M. Takahashi, T. Kobayashi, T. gis, T. Nishizawa, T. Nishiyama, P. P. Primadharsini, H. Okamoto

Objectives: Hepatitis E virus (HEV), a member of the genus Orthohepevirus in the family Hepeviridae, is the causative agent of acute or chronic hepatitis E. Although HEV particles in feces and bile are non-enveloped, those in circulating blood and culture supernatant have been found to be covered with a cellular membrane, similar to enveloped viruses. We previously demonstrated that HEV entry into PLC/PRF/5 cells requires Rab5 and Rab7 regardless of particulate morphology, suggesting that these particles need to reach the late endosome. Furthermore, the membrane-associated HEV enters the cells with an acidic pH-dependent step and activity of Rab9. In this study, we investigated the role of cathepsins on HEV internalization.

Methods: HEV particles in culture supernatant containing a cell culture-adapted JE03-1760F strain (genotype 3) were utilized as membrane-associated particles. On the other hand, membrane-unassociated particles were generated from HEV particles in the culture supernatant following treatment with sodium deoxycholate and trypsin. Requirements of cathepsin B or L in HEV entry were examined using several chemical inhibitors. The growth inhibition of HEV by teicoplanin was evaluated using a cell culture system. After virus inoculation, the extracellular or intracellular HEV RNA levels were quantified by real-time RT-PCR.

Results: The infectivity of both enveloped and non-enveloped HEVs was not affected by cathepsin B. The addition of cathepsin L inhibitors exhibited the inhibition of the membrane-associated HEV infection, but no detectable effect on the infectivity of the membrane-unassociated HEV. HEV RNA replication and virion release were not affected by the drug treatments. Teicoplanin, which is known to inhibit the activity of cathepsin L, potently prevented viral replication of both enveloped and non-enveloped HEV particles in a dose-dependent manner in cultured cells. Furthermore, when inoculated at low viral loads, HEV replication was completely inhibited by teicoplanin.

Conclusion: These results indicate that cathepsin L is necessary for entry step of enveloped HEV particle. Cathepsins are delivered from the trans-Golgi network (TGN) to the late endosome by mannose-6-phosphate receptors (M6PRs), and recycling of M6PRs back to TGN depends on Rab9. Currently, we are analyzing the importance of M6PRs on HEV entry. Furthermore, teicoplanin blocks HEV entry into the cells by specifically inhibiting the activity of cathepsin L, suggesting that teicoplanin is applicable to not only HEV but also a wide range of cathepsin L-dependent viruses.

Keywords: Cathepsin, Hepatitis E virus, Virus entry

17th International Congress of Virology 241

Abstract Submission

Hepatitis viruses

PO687

Comparison of changes in host transcript profile caused by Hepatitis B virus infection for genotype C and D viruses

C. W. Sze*, Z. Zheng 1, C. W. Yew 1, Y.-J. Tan 1 2

1Monoclonal Antibody Unit, Institute of Molecular and Cell Biology, 2Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: Hepatitis B virus was discovered half a decade ago but the knowledge on its molecular virology is lacking due to the absence of an efficient viral infection model. HBV is classified into genotypes A to J with distinct geographical distributions for certain genotypes. Genotype C is mainly found in the Southeast Asia whereas genotype D is most prevalent in the Europe. In addition, ample studies have shown a strong association between HBV genotypes and the pathogenesis of HBV infection. For instance, genotype C infection is associated with increased liver inflammation, fibrosis and HCC while genotype D infection leads to increase liver complications such as cirrhosis and HCC. In this study, we sought to determine the differences in the pathogenesis of HBV genotype C and D, as well as between HBV and HCV, by using various PCR arrays to determine the host transcriptional profile following infection to identify factors that are differentially regulated.

Methods: Various PCR arrays corresponding to different host signaling pathways were used in this study. Host transcriptional profiles were also compared between cells infected with Hepatitis B and C viruses.

Results: Differences in host transcriptional profile were observed upon infection with HBV of different genetoypes. Using the same PCR array, we showed that HBV and HCV infection induce different host gene changes in infected cells.

Conclusion: HBV exerts different level of pathogenesis on host cells in a genotype-dependent manner, leading to differential expression of host transcriptional profile. Even though both hepatitis B and C virus tagert the same cell type, their pathogenesis mechanism differ from one another.

Keywords: Apoptosis, gene array, hepatitis B

17th International Congress of Virology 242

Abstract Submission

Hepatitis viruses

PO688

DHCR24 AUTO-ANTIBODY AS A NEW BIOMARKER FOR PROGRESSION OF HEPATITIS C

S. Ezzikouri 1, K. Kimura 2, S.-I. Kaneko 3, M. Kohara 4, K. Tsukiyama-Kohara 5,*

1Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco, 2Liver Unit, Komagome Hospital, Tokyo, 3Faculty of Medicine, Kanazawa University, Kanazawa, 4Tokyo Metropolitan Institute of Medical Science, Tokyo, 5Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan

Objectives: New biomarkers are needed to identify the stage of hepatitis C virus (HCV)-infected diseases in order to reduce the mortality rates. Herein, we investigated whether serum 3β-hydroxysterol Δ24-reductase antibody (DHCR24 Ab) may serve as a prognostic marker for hepatitis C infection progression to hepatocellular carcinoma (HCC).

Methods: Serum DHCR24 Abs from 395 HCV-positive patients, including 133 chronic hepatitis (CHC), 85 liver cirrhosis (LCC), and 177 HCC (HCC-C) patients; 232 hepatitis B virus (HBV)-positive patients, including 103 chronic hepatitis (CHB), 56 liver cirrhosis (LCB), and 73 HCC (HCC-B) patients; and 24 healthy controls, were measured using enzyme-linked immunosorbent assay.

Results: The serum DHCR24 Ab levels were significantly higher in patients with CHC than in healthy controls, in LCC than in CHC, and in LCC than in HCC-C (P<0·0001 for all). The concentration of serum DHCR24 Ab in HCC-B patients showed no significant difference compared to CHB and LCB patients (P=0·1247). The DHCR24 Ab levels were significantly higher in early HCC-C than CHC or LCC patients and in late HCC-C compared to early HCC-C patients. The sensitivity of the DHCR24 Ab for HCC-C detection (70·6%) was higher than that of alpha-fetoprotein (AFP; 54·8%) and protein induced by vitamin K absence or antagonist-II (PIVKA-II; 42·5%). Moreover, DHCR24 was up-regulated in HCV-positive, but not HBV- positive tissues or HBV-negative, HCV-negative HCC specimens.

Conclusion: DHCR24 auto-antibody represents a potential noninvasive biomarker for HCV-related liver disease and may facilitate the diagnosis of PIVKA-II and AFP-negative HCC.

Keywords: autoantibody, DHCR24, HCV

17th International Congress of Virology 243

Abstract Submission

Hepatitis viruses

PO690

Pan-genotype structural analysis of HCV NS5B polymerase to elucidate similarities/differences across genotypes through in silico docking of nucleoside/non-nucleoside inhibitors

S. Mansoor 1,*, H. Dar 1, A. Javed 1, A. Ali 1

1ASAB, NUST, ISLAMABAD, PAKISTAN, ASAB, NUST, ISLAMABAD, PAKISTAN, ISLAMABAD, Pakistan

Objectives: Chronic HCV infections exert a severe toll on the patient both physically and economically. The latest in therapeutic effort against chronic HCV infections, are the direct acting antivirals (DAAs). Direct inhibition of viral polymerase (NS5B), protease (NS3/NS4A) and non-structural protein 5A (NS5A) with nucleoside/nucleotide and non-nucleoside analogs have yielded promising response, however there pan-genomic activity and drug resistance is still an issue. Currently inhibitors of HCV polymerase like Sofosbuvir have shown promise and it is desired that more similar molecules are engineered which have good pan-genomic activity and robust drug resistance barrier.

The main aim of this investigation is to determine the pan-genotype similarities and differences in HCV NS5B polymerase, through the in-silico docking of nucleoside/non-nucleoside inhibitors. This will facilitate in a better understanding of the molecular aspects behind inhibition and subsequent drug resistance. Studies such as this will certainly aid in the development of improved DAAs based therapeutics.

Methods: Homology models of HCV NS5B polymerase for HCV genotypes 1, 2, 3 and 4 were constructed using Modeller (version 9.18) with 4WTG (pdbID) and 5CZB (pdbID) as templates. Models with the best ERRAT score and acceptable Ramachandran plot were selected for further analysis. Energy minimized SOFOSBUVIR (a nucleoside analog) and LIGAND IDX17119-5 (a non-nucleoside analog) inhibitor molecules were docked in the polymerase using Molecular operating environment (moe, release 2010.10) and AutoDock vina (version 1.1.2). The ligand protein interaction clusters were analyzed for best inhibitor binding by comparing it with protein-ligand interaction in 4WTG and 5CZB for nucleoside and non-nucleoside inhibitors, respectively. Finally the active site and the inhibitor binding residues were analyzed for each model using moe.

Results: Sofosbuvir is a very promising uridine (nucleoside) analog pro-drug. The docking analysis revealed that the inhibitor binding site and the polymerase residues involved across genotypes are relatively conserved. The inhibitor Sofosbuvir binds within the main active site competing with nucleoside binding. In contrast LIGAND IDX17119-5, a non- nucleoside HCV polymerase inhibitor, which binds within the thumb region of the polymerase. The docking analysis showed that the residues within the thumb region are less conserved across genotypes meaning LIGAND IDX17119-5 binds and inhibits the polymerase from different genotypes with different efficacies.

Conclusion: Nucleoside analog inhibitors show better pan-genomic activity with robust drug resistance barrier however since they are nucleoside analogs they may also compete for host polymerases posing therapy risks. Non-nucleoside inhibitors on the other hand are tailored specifically against viral polymerase but since there is less conservation, there is less pan-genotype activity and more drug resistance. Therefore, non-nucleoside inhibitors developed should be genotype specific and against more conserved locations within and around the polymerase active site.

Keywords: HCV, NS5B polymerase, sofosbuvir

17th International Congress of Virology 244

Abstract Submission

Hepatitis viruses

PO691

Rad51 recombinase interacts with nonstructural 3 protein of hepatitis C virus and regulates viral propagation

S. Hwang*, K. Son 1, Y.-S. Lim 1

1Ilsong Institute of Life Science, HALLYM UNIVERSITY, Anyang, Korea, Republic Of

Objectives: Hepatitis C virus (HCV) is the leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Since HCV exploits host cellular machinery for viral propagation, we selected and characterized host protein in an attempt to develop host-targeting antivirals.

Methods: We screened a siRNA library targeting 131 genes that control the cell cycle in cell culture-grown HCV-infected cells.

Results: In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of DNA double-strand breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was co-precipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 at the lipid raft.

Conclusion: These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV.

Keywords: cellular factors, hepatitis C, siRNA screening

17th International Congress of Virology 245

HIV and Retroviruses

17th International Congress of Virology 246

Abstract Submission

HIV and Retroviruses

PO693

CHARACTERIZATION OF L100V AND I202V MUTATIONS ON IN VITRO AND IN VIVO VIRAL SUSCEPTIBILITY TO NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NNRTI)

S.-Y. Chang*, C.-C. Hung 1, P.-H. Lin 2, T.-W. Chang 2

1Internal medicine, National Taiwan University Hospital, 2Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan

Objectives: Increasing trends of resistance-associated mutations (RAMs) to non-nucleoside reverse-transcriptase inhibitors (nNRTIs) have caused concerns about the effectiveness of the regimens in resource-limited countries. The L100V mutation is a rarely detected mutation, as compared to the other well-known L100I mutation, which was known to cause reduced viral susceptibility to the first generation of nNRTIs by in vitro assay. However, a drastic increase of L100V mutation was detected in treatment-naïve HIV patients in Taiwan in 2012. In this study, we aimed to determine the impacts of L100V and L100I mutations on viral replication and the viral susceptibility to the second generation of nNRTIs.

Methods: The L100V and L100I mutations were introduced individually or in combination into the HXB2 backbone by site- directed mutagenesis. Viral replication was determined using peripheral blood mononuclear cells and U87-CD4+-CXCR4+ cell line. The half maximal effective concentrations (EC50) of nevirapine, efavirenz, ripilvirine, and etravirine.against the wild-type HXB2 and mutant viruses were determined using peripheral blood mononuclear cells.

Results: Mutant viruses harboring the L100V mutation had a significantly reduced replication profile as compared to the wild-type and L100I viruses, although no changes in the virus compositions were observed in viral lysates by western blot. Like L100I, the presence of L100V mutation did reduce viral susceptibility to nevirapine (EC50=1.48uM, fold change=16.81), although only a modest effect was observed against efavirenz(EC50=15.51nM, fold change=4.14). Both L100V and L100I had no significant effects on viral susceptibility to the second generation of nNRTI, ripilvirine (EC50=3.93 nM, fold change=2.75; EC50=3.14nM, fold change=2.19) and etravirine (EC50=3.67 nM, fold change=1.81; EC50=4.43nM, fold change=2.19).

Conclusion: The presence of L100I mutation did significantly reduce viral replication and susceptibility to the first and the second generation of nNRTIs. Emergence of these mutations among antiretroviral-naïve patients warrants continued surveillance of transmitted drug resistance in Taiwan.

Keywords: None

17th International Congress of Virology 247

Abstract Submission

HIV and Retroviruses

PO694

HIV-1 Pol-phylogenetic diversity and drug resistance mutation profile in two different cohorts (adult and infants) from Yaoundé, Cameroon.

J. Gichana*, G. Jacobs 1, G. Ikomey, D. Njenda 1

1Pathology, University of Stellenbosch-Tygerberg campus, Cape Town, South Africa

Objectives:

Study and characterize the molecular epidemiology of HIV in Cameroon.

Determine the HIV resistance profile of all study participants.

Methods: We collected 125 samples from HIV-1 positive patients (55 infants < 6 years of age and 70 adults) from the CSCCD research institute and other peripheral clinics within Yaoundé. The CD4 count ranged between 500-2000 cells/m3 and the HIV-1 viral load between 3000-6000 copies/ml. To study the HIV-1 diversity and resistance in our two cohorts, we targeted the pol Protease (PR), Reverse Transcriptase (RT) and Integrase (IN) regions of HIV-1 for sequencing, through well-established methods.

Results: For infants, a total of 37/55 (67.3%) patient samples could be amplified for at least one of the HIV-1 pol fragments. This includes 29/55 (52.0%) for the PR, 27/55 (49.0%) for the RT and 28/55 (51.0%) for IN. The most predominant strain was CRF02_AG (n = 17; 63.0%). Other subtypes detected include subtype A (n =3, 11.1%), C (n =2, 7.4%), and F2 (n =2, 7.4%). Three sequences (11.1%) could not be assigned to any subtype with confidence. Only one patient sequence (3.7%) had RAMs against NRTIs- K65R mutation conferring intermediate to high level resistance to TDF, DDI, ABC and d4T with low to intermediate-level resistance to 3TC and FTC. For adults, we analysed 30 PR and 32 RT genome fragments with the following observed results; HIV-1 subtype G accounts for 56% (n = 18), Subtype A 19% (n = 6), Subtype C and J 9% each (n = 3) and subtype D 6% (n = 2). Thirteen samples could not be assigned to any subtype with confidence. Three patients had NNRTI drug resistance mutations; V90I and G190R which conferred intermediate to high- level resistance to NVP and EFV.

Conclusion: Central Africa is seen as the birth place of HIV, with a wide genetic diversity of the virus found in this region. The genetic variability makes it challenging to study the viral strains found in Cameroon and to implement optimized diagnostic assays for the country. Our study gives us an opportunity to characterize the HIV diversity in Cameroon, while studying HIV resistance patterns resulting from ART.

Keywords: adults, HIV, infants

17th International Congress of Virology 248

Abstract Submission

HIV and Retroviruses

PO695

Involvement of of Sp1/CBP in butyric acid-producing bacteria-induced HIV-1 reactivation

K. Imai*

Objectives: The ability of HIV-1 to establish latent infection and its re-activation is considered critical for the progression of HIV-1-associated diseases. Accumulating evidence has indicated that histone acetylation and deacetylation regulates HIV- 1 gene expression. The bacterial metabolite butyric acid has been shown to be a potent inhibitor of histone deacetylases (HDACs), leading to transcription of HIV-1 proviruses. We previously demonstrated that butyric acid-producing bacteria in the vaginal and oral cavities and gut can strongly induce histone acetylation and HIV-1 replication from latently infected cells by inhibiting HDAC; thus, co-infection with anaerobic bacteria is an important risk factor for progression of AIDS. However, the molecular mechanism by which butyric acid activates HIV-1 gene expression is not well understood.

Methods: Luciferase and chromatin immunoprecipitation assays were employed to analyze the transcription factors involved in HIV transcription. Human ACH-2 cells were incubated with butyric acid and HIV proteins were detected by immunoblot and ELISA.

Results: Sp1 binding sites are considerably involved in butyric acid-mediated activation of HIV-1 gene expression as indicated by the results of a luciferase assay using HIV-1 LTRs of various mutants. Sp1 knockdown by siRNA and the Sp1 inhibitor mithramycin A inhibited the effects of butyric acid. Furthermore, we observed that CBP was involved in butyric acid- induced Sp1-dependent HIV-1 gene expression. A chromatin immunoprecipitation assay analysis revealed that Sp1 and HDAC1 are present in the HIV-1 LTR promoter in TZM-bl cells and dissociate from the promoter concomitantly with the association of acetylated histone H3 and CBP upon butyric acid stimulation. Furthermore, siRNA knockdown of Sp1 resulted in decreased recruitment of CBP to the promoter.

Conclusion: These results suggest that butyric acid stimulates HIV-1 promoter activity through the inhibition of Sp1- associated HDAC activity and recruitment of CBP to the Sp1 sites on HIV-1 LTR. Sp1 should be considered as a therapeutic target in new anti-viral therapies against HIV-1 infection aggravated by butyric acid-producing bacteria.

Keywords: butyric acid-producing bacteria, HIV, Sp1

17th International Congress of Virology 249

Abstract Submission

HIV and Retroviruses

PO697

SUPPRESSING VIRAL RESERVOIR BY VACCINE INDUCED CD8+ T CELLS IS CRITICAL FOR LONG-TERM PROTECTION OF SIV-INFECTED RHESUS MACAQUES

C. Sun 1,*, Z. Chen 2, L. Zhang 3, L. Chen 4

1Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health), Chinese Academy of Sciences, Guangzhou, China, 2AIDS Institute , University of Hong Kong, Hong Kong, Hong Kong, 3Comprehensive AIDS Research Center , Tsinghua University, Beijing, 4Center for Infection and Immunity, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences, Guangzhou, China

Objectives: The latent viral reservoir has been recognized as one of the major obstacles for eradicating HIV-1 infection. However, it is not well defined if vaccine induced immune responses can affect the integrated reservoir.

Methods: Previously, we demonstrated that priming with a modified vaccinia Tiantan (MVTT) vectored SIV vaccine and boosting with an adenovirus vectored SIV vaccine elicited robust immune response and afforded a significant control of virus infection upon challenge with SIVmac239 (Sun et al, J of Virology, 2013,87:5669). In this study, we performed a long- term follow-up experiment to study the mechanism for this protection.

Results: Our follow-up study showed that 60% (7/12) of vaccinated macaques survived more than 1800 days without obvious AIDS symptoms after rectal challenge with high dose (50,000TCID50) SIVmac239. In contrast, most of non- vaccinated macaques (7/8) died of AIDS progression. Strikingly, both SIV RNA and integrated SIV DNA fell to below detection limits in four vaccinated “elite” macaques. Further study revealed that this suppression correlates with vaccination- elicited SIV-specific immune responses, especially effector memory CD8+ T cells. Moreover, there was a transient viral rebound in these vaccinated “elite” monkeys after depletion of CD8+ lymphocytes using an anti-CD8 monoclonal antibody, which confirmed that viral reservoir is effectively inhibited by CD8+ T lymphocytes-mediated immune responses.

Conclusion: Our study provided strong evidence that the SIV reservoir can be effectively inhibited and persistently suppressed by vaccine induced immunity, in which the effector memory CD8+ T cells play a critical role. We proposed that a practical HIV vaccine may be designed to achieve long-term control of viral reservoir before having a vaccine with sterile protection.

Keywords: HIV, Reservoir, Vaccine

17th International Congress of Virology 250

Abstract Submission

HIV and Retroviruses

PO698

Tracing the ancient cat’s migration by analyzing retroviral integration sites

S. Shimode 1,*, S. Nakagawa 2, T. Miyazawa 3

1Graduate School of Science, Technology and Innovation, Kobe University, Kobe, 2Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, 3Lab. of Virus-Host Coevolution, Institute fir Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

Objectives: There are about 100 breeds of domestic cats in the world. Their common ancestor is Felis silvestris lybica living in the Middle East, where agriculture started about 10,000 years ago. Then, cats are believed to spread over the world as humans migrated, but details about how they migrated and how they diverged into various breeds have remained unknown.In this study, we used a family of endogenous retroviruses (ERVs) as a genetic marker to trace ancient cats’ migration. ERVs are remnants of ancient retroviral integration into the host genome of germline cells and transmitted from parents to offspring in a Mendelian fashion.In our previous study, we identified one of the ERV families, termed RD-114 virus-related sequences (RDRSs), in domestic cats’ genomes. We found that all domestic cats have an RDRS on chromosome C2 (named RDRS C2a), but the other RDRSs have not been fixed in domestic cats' genomes (i.e., they are insertionally polymorphic). RDRSs can be used as genetic markers because the presence of each ERV in the host genome is the result of a single integration event in one individual.

Methods: We analyzed genomic DNA samples collected from 244 cats belonging to 23 breeds, crossbred (hybrid) and mongrel (random-bred). Mongrel cats samples were collected from cats living in different geographical regions within Japan (n=66) or Vietnam (n=17). We developed a touchdown PCR method to differentiate newer RDRSs’ env (infectious-type env) on any loci from the defective C2a-type env. We designed a reverse primer to step over the region of the RDRS C2a- specific 15-bp insertion. The unidentified RDRS locus in Persian and Persian-derived cat genomes were investigated by inverse nested PCR targeting RDRS env. An insertional polymorphic distribution of each RDRS was determined by using PCR primers based on the flanking sequence of each RDRS and the RDRS env sequence.

Results: We revealed that 47.1 and 56.4% of domestic cats in Europe and North America have infectious-type env, respectively; 18.7 and 18.8% in Asia and Middle East, respectively. Asian domestic cats containing infectious-type env were mongrel cats and Oriental shorthair which is a member of the Siamese breed group but also established by crossing with a number of foreign feline breeds. These results suggested that newer RDRSs have integrated into the genomes of cats moving to Europe after split from the Middle East.By inverse PCR of Persian cat genomic DNAs, we identified an additional RDRS (designed RDRS D2). RDRS D2 has the entire open reading frames (ORFs) for gag and env, and a 1-bp insertion at the the pol coding region, resulting in frameshift.

Within newer RDRSs harboring infectious-type env, RDRS D2 had by far the highest frequency in infectious-type env positive samples (57.0%); RDRS E3 was presented in 15.1%; RDRS C2b, D4, and C1 was detected in only 1.1 to 3.2 % of the same samples. We found that each cat had 1 to 3 copies of RDRSs. Many cats kept either RDRS D2 or E3, but only one cat was holding both of them, suggesting that RDRS D2 and E3 independently had invaded different populations of the ancestry of domestic cats.

Conclusion: We identified seven RDRSs in cat genomes. All cats have RDRS C2a in common, but the other RDRSs have not yet been fixed in domestic cat’s genome. Our results provide useful information to reveal the history of domestication and migration of cats across the world.

Keywords: endogenous retrovirus, genome 17th International Congress of Virology 251

Influenza Virus

17th International Congress of Virology 252

Abstract Submission

Influenza virus PO699

AN IN VITRO MODEL OF FULLY DIFFERENTIATED HUMAN NASAL EPITHELIAL CELLS REVEALS THE TRANSCRIPTOMIC SIGNATURES OF THE NASAL EPITHELIUM FOLLOWING HUMAN INFLUENZA H3N2 INFECTION

Y. Yan*, K. S. Tan 1, H. Koh 2, H. Choi 2, C. Li 1, T. Tran 3, R. Sugrue 4, V. Chow, D.-Y. Wang 1 1Otolaryngology, 2Saw Swee Hock School of Public Health , 3Physiology, National University of Singapore, 4School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Objectives: The nasal epithelium is the first line of defense against invading respiratory pathogens. The physical and immunological barriers of the nasal epithelium are thus important in preventing escalation into infection of the lower airway. However, despite its relative importance, not much research focused on the nasal epithelium due to its relatively smaller size. Therefore, there is an obvious demand for a model that can provide a platform to elucidate nasal responses at the transcriptomic level in order to supplement clinical data.

Methods: Our lab previously generated an in-vitro differentiated, air-liquid interface culture of human nasal epithelial cells (hNECs) derived from nasal epithelial stem cells of healthy donors, which are able to form a fully functioning multi-layered structure of the nasal epithelium. These differentiated nasal epithelial cells are permissive to infections with different respiratory viruses, and exhibit potent innate immune responses. Building on this, we performed microarray analysis on the hNECs model to ascertain the transcriptomic changes in the nasal epithelial cells infected with human IAV under controlled experimental conditions.

Results: Analyses revealed 253 up-regulated and 146 down-regulated genes in the nasal epithelium following infection, which may constitute susceptibility factors of individuals during IAV infection. The gene ontology (GO) analysis identified conserved and essential host defense and inflammatory mechanisms, including type I, II, III interferon (IFN) signaling, JAK- STAT signaling, Toll-like receptor signaling, MDA5 signaling, NF-κB signaling, EGF signaling, and cell death pathways. In addition, the down-regulated genes reflected impaired nasal epithelial functions following IAV infection, including metabolic processes, mucociliary functions, tight junctions, and pre-microRNA processing. Furthermore, the analyses revealed differential expression of certain gene signatures in different individuals, mirroring clinical data.

Conclusion: In conclusion, this study supports the utility of the hNECs model to elucidate the critical innate immune responses of the nasal epithelium, the first line of defense against respiratory virus infections at the population level.

Keywords: human nasal epithelial cells, influenza H3N2 virus, transcriptome

17th International Congress of Virology 253

Abstract Submission

Influenza virus PO700

ANALYSIS OF ANTIBODY REPERTOIRE IN RHESUS MACAQUES AFTER IMMUNIZATION WITH INFLUENZA VIRUS VACCINE USING NGS

X. Niu 1,*, J. Luo 2, Q. Wang 2, Z. Yao 2, L. Chen 2 1State Key Laboratory of Respiratory Disease, , First Affiliated Hospital of Guangzhou Medical University, 2Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences,, Guangzhou, China

Objectives: The emergence of new avian influenza A viruses such as H7N9 infection in humans posed a need to understand the antibody response to HA protein, the key target antigen for neutralizing influenza virus. In this study we explored the antibody immune responses and dynamic antibody repertoire during vaccine immunizaiton.

Methods: We immunized Chinese rhesus macaques with an inactivated whole virus H7N9 experimental vaccine. PBMC samples were collected before and at different time points after immunization. The antibody repertoires at various time points were analysed following next-generation sequencing (NGS) using barcoded primer sets designed for human immunoglobulins as the two species share high degree of homology.

Results: More than 4 million antibody heavy chain sequences were retrieved, which allowed us to analyse the antibody repertoire following immunization. We also identified six funcitonal antibodies by single B cell cloning and picking top CDR3 number cloning, which showed binding and/or neutralizing activities to H7N9 virus. four of antibodies could retrieve in antibody reperotire of different time points. Tracing the sibling of the antibodies in the repertoire showed similar or better binding activity compared with original antibodies.

Conclusion: Therefore, neutralizing monoclonal antibodies influenza viruses could be identified and traced to their clonal expansion and maturation through mining the NGS data. Our study demonstrated that rhesus macaque can be used as a more human relevant model for evaluating antibody repertoire in responding to a vaccine or after infection with a pathogen.

Keywords: Influenza virus rhesus macaque antibody repertoire

17th International Congress of Virology 254

Abstract Submission

Influenza virus PO701

ANTIGENIC AND GENETIC ANALYSES OF NOVEL CLADE 2.3.4.4 H5 HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES ISOLATED IN JAPAN AND VIETNAM

K. Soda 1,*, U. Thi Hong Trang 2, N. Le Khanh Hang 2, T. Usui 1, H. Ito 1, H. Ozaki 1, T. Yamaguchi 1, L. Thi Quynh Mai 2, T. Ito 1 1Avian Zoonosis Research Center, Faculty of Agriculture, TOTTORI UNIVERSITY, Tottori, Japan, 2Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam

Objectives: Highly pathogenic avian influenza (HPAI) caused by novel clade 2.3.4.4 H5 influenza viruses have been reported since late 2013 in worldwide. Japan experienced several HPAI outbreaks in wild and domestic birds in 2014-15. Causal viruses were also isolated in our laboratory thorough pathological appraisals of environmental samples in chicken farms and one dead wild duck. Moreover, a similar H5 virus (Tottori/C6) was isolated from the fecal sample of swan under annual bird-flu surveillance in 2014. Meanwhile, we have continued virological survey in wet markets in Vietnam, and a number of clade 2.3.4.4 viruses have been isolated from domestic ducks since 2013 spring. In the present study, antigenic and genetic characters of these virus isolates were assessed to provide helpful information for diagnosis and prevention of epidemics in future.

Methods: Full genome sequences and phylogenic analyses of clade 2.3.4.4 H5 virus isolates in Japan (JP) and Vietnam (VN) were performed. The antigenicity of H5 viruses was examined by cross haemagglutinin-inhibition and neutralization tests with polyclonal chicken antisera.

Results: HA genes of clade 2.3.4.4 virus isolates were phylogenically divided into several groups (JP-A, JP-B, VN-A, VN- B and more). Notably, Tottori/C6 strain, classified into JP-B, had similar HA gene with North American isolates, suggesting that novel clade 2.3.4.4 viruses have been disseminated by migratory birds via their wintering spots such as Siberia and Alaska. The antigenicity of clade 2.3.4.4 viruses was distinct from those of previous dominant clade 2.3.2.1 viruses. Accordingly, the antiserum against a novel clade 2.3.4.4 virus should be kept especially in reference laboratories/institutes for definitive diagnosis hereafter. Antisera against clade 2.3.4.4 VN-A viruses showed relatively low reactivity to VN-B virus, suggesting that antigenically different clade 2.3.4.4 viruses were kept in poultry in Vietnam. Similarly, JP-A viruses were antigenically different from JP-B and Vietnamese isolates. These results indicate that novel clade 2.3.4.4 viruses have antigenic variety, even in the isolates in same country.

Conclusion: Genetic and antigenic diversities were observed in novel clade 2.3.4.4 HPAI viruses. In Japan, antigenically different clade 2.3.4.4 HPAI viruses were invaded in parallel in 2014-15. Although the viruses were already eradicated in Japan at that time, HPAI outbreaks caused by clade 2.3.4.4 H5N6 viruses reoccur in 2016-17 winter season. As of Jan.16 2017, over 170 cases in wild birds and 8 cases in poultry farms had been reported since Nov. 2016 in Japan. Antigenic and genetic characters of the causal viruses will be also included in this presentation. In Vietnam, genetically and antigenically different clade 2.3.2.1 and 2.3.4.4 HPAI viruses have been circulating in domestic poultry, indicating that antigenic shift of circulating viruses has progressively occurred. Continuous surveillance is important to monitor the changing epidemic strains.

Keywords: H5, highly pathogenic avian influenza

17th International Congress of Virology 255

Abstract Submission

Influenza virus PO702

Antigenic changes found in influenza A(H1N1)pdm09 hemagglutinins during 2009-2016 in Japan R. Yoshida*, M. Ishijima 1, H. Miyamoto 1, A. Shigeno 1, M. Igarashi 1, R. Manzoor 1, A. Takada 1 1Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan

Objectives: Influenza A(H1N1)pdm09 has caused a pandemic in 2009 and continued to circulate in humans as a seasonal influenza virus. Monitoring the antigenic evolution of A(H1N1)pdm09 is particularly important for infection control by vaccine strain selection. Hemagglutinin (HA), a membrane glycoprotein of influenza virus is a critical target to induce neutralizing antibodies by vaccination or infection. To examine antigenic changes of influenza A(H1N1)pdm09 isolates in Japan during 2009-2016,we analyzed antigenicities of their HAs using monoclonal antibodies (MAbs) developed against a A(H1N1)pdm09 strain, A/Narita/1/2009 (Narita).

Methods: We generated 20 neutralizing and hemagglutination-inhibiting MAbs recognizing epitopes in the antigenic site Sa, Sb, and Ca (13, 4, and 3 MAbs, respectively). Neutralizing activities of the MAbs to Influenza A(H1N1)pdm09 isolates in Japan during 2009-2016 seasons (10, 10, 4, and 2 isolates in 2009/10, 2010/11, 2013/14, and 2015/16) were evaluated by microneutralization assay. The HA amino acid sequences of A(H1N1)pdm09 isolates were compared to Narita strain.

Results: Neutralizing activities of these MAbs against all tested 10 clinical isolates of 2009 were almost similar to those against the Narita strain. Interestingly, 6 of 13 Sa-recognizing MAbs lost their neutralizing activities against 3 of 10 clinical isolates of 2010/11. Following amino acid substitutions were found in HA; N142D, G172E, N173D, K180E, G219E, T201N, and G219E. Four Sb-recognizing MAbs showed similar reactivities to all of the 2011 isolates. All 3 Ca-recognizing MAbs did not react 1 isolate that had an amino acid substitution (S160G). The MAbs recognizing the Sa antigenic site did not neutralize six clinical isolates of 2013/14 or 2015/16, which had K180Q or K180Q/S179N amino acid substitutions, respectively.

Conclusion: Our data show that viruses with the amino acid substitutions observed in the Sa antigenic sites on HAs of the 2010/11 isolates did not become dominant in the 2013/14 and 2015/16 seasons. It is important to carefully monitor antigenic changes of A(H1N1)pdm09 by using MAb panels.

Keywords: antigenic change, Influenza A(H1N1)pdm09

17th International Congress of Virology 256

Abstract Submission

Influenza virus PO703

BOOSTING THE EFFICACY OF INFLUENZA SUB-UNIT VACCINE BY TARGETING M2E TO CLEC9A ON DENDRITIC CELLS

K. Kalamba Arachchi 1 2,*, E. Ang 1 2, A. Ker 1 2, H. Bin Mohamad Nasir 1 2, H.-Y. Park 3 4 5, I. Caminschi 3 4 5 6 7, K. Shortman 2 5 8 9 10, M. Lahoud 3 4 5, S. Alonso 1 2 1Microbiology & Immunology, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, Singapore, Singapore, 3Infection and Immunity Program, Monash Biomedicine Discovery Institute, Monash University, 4Biochemistry and Molecular Biology, Monash University, 5Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, 6Microbiology & Immunology, The University of Melbourne, 7Peter Doherty Institute for Infection and Immunity, Melbourne, Australia, 8Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 9The Walter and Eliza Hall Institute, 10Medical Biology, The University of Melbourne, Melbourne, Australia

Objectives: Over the past century there have been at least four devastating pandemics caused by Influenza A that took the lives of millions, and the threat of the next great pandemic remains a top global health concern today. Currently available vaccines need to be annually reviewed and updated to match circulating strains, and are accompanied by long and complicated production cycles and limited production capacity. These are impediments to its practical use in the face of pandemics that demand mass vaccination in a short time-frame. Our research hopes to overcome these limitations by working with a subunit vaccine that targets the conserved M2e epitope to Clec9A on Dendritic Cells (DC).

Methods: M2e is a 24 non-glycosylated ectodomain of the M2e matrix protein 2 from Influenza A virus, but is also displayed on the surface of infected cells. Our novel construct shuttles this epitope to CD8+ DCs, which was shown to elicit a prolonged CD4 T cell proliferation and T follicular helper cell generation. In this work we studied the immunogenicity of the Clec9A- M2e construct co-administered with different adjuvants. Challenge experiments with a lethal dose of H1N1/PR8 virus were conducted to evaluate the protective efficacy of the different combinations.

Results: Preliminary results show a 60-85% survival rate in mice primed with the Clec9A-M2e construct and infected with a lethal dose of H1N1/PR8 virus, as compared to unprimed control mice.

Conclusion: A "universal vaccine" that can be used to fight infection with different strains of Influenza A is highly desirable, and this Clec9A-targeted approach suggests a potential new therapeutic strategy in combating future pandemics.

Keywords: Dendritic Cells, Influenza A, Vaccine

17th International Congress of Virology 257

Abstract Submission

Influenza virus PO704

Characterization of Influenza A(H1N1)pdm09 Viruses Isolated from Hospitalized Cases in the 2015/16 Season

C. Kawakami 1,*, S. Fujisaki 2, E. Takashita 2, K. Shimizu 1, H. Ozawa 1, T. Momoki 1, M. Saikusa 1, S. Usuku 1, S. Watanabe 2, T. Odagiri 2 1Yokohama City Institute of Public Health, Yokohama, 2Influenza Virus Research Center National Institute of Infectious Diseases, Tokyo, Japan

Objectives: In Japan, influenza A and B viruses co-circulated in the 2015/16 season. Surveillance in Yokohama, Japan revealed that A(H1N1)pdm09 viruses were isolated from 84.6% of the hospitalized cases, and 63.6% of them were severe cases. We analyzed the clinical characteristics and virus properties of these severe hospitalized cases.

Methods: Clinical specimens were obtained from severe hospitalized cases in Yokohama. Nine A(H1N1)pdm09 viruses were isolated from the specimens. We sequenced the whole genome of the viruses, and performed phylogenetic analysis. Antigenic characterization of the viruses was carried out by hemagglutination inhibition test. Susceptibilities of the viruses to neuraminidase (NA) inhibitors were determined by using fluorescent NA inhibition assay.

Results: The ages of severe hospitalized case patients ranged from 7 to 82 and 67 % of them were below the age of 15. The severe cases consisted of 5 cases of pneumonia, 1 case of encephalopathy, and 1 case of myocarditis. The antigenicity of the viruses was similar to that of the vaccine strain, A/California/07/2009, and the hemagglutinin genes were classified into subclades 6B.1, 6B.2, and 6B, which were the main subclades of epidemic strains in Japan. All viruses were susceptible to NA inhibitors, but resistant to adamantane. We could not find characteristic amino acid differences in the polymerase basic protein (PB) 1, non-structural protein and PB2 between viruses isolated from severe cases and circulating viruses.

Conclusion: Although no major antigenic changes of the A(H1N1)pdm09 virus have occurred since it emerged in 2009, there have been repeated epidemics. In the 2015/16 season, the majority of hospitalized cases were among children. We have not found any characteristic amino acid substitutions in the severe hospitalized cases. Consequently, continued surveillance is necessary.

Keywords: A(H1N1)pdm09 viruses , Characterization, severe hospitalized cases

17th International Congress of Virology 258

Abstract Submission

Influenza virus PO705

CLINICO-EPIDEMIOLOGICAL STUDY OF INFLUENZA A (H1N1) PANDEMIC OUTBREAK AT A TERTIARY CARE INSTITUTE OF NORTHERN INDIA, 2015

S. Saxena 1 2,*, D. SINGH 1, A. zia 1, J. umrao 1, S. Dwivedi 1, M. Shukla 1, T. N. dhole 1, R. A. S. kushwaha 2 1MICROBIOLOGY, SGPGIMS, 2pulmonary, KGMU, LUCKNOW, India

Objectives: The study aims to investigate the epidemiology and clinical manifestation of the influenza A pdm H1N1outbreak among patients presenting influenza like illness (ILI) at a tertiary care institute, SGPGIMS Lucknow, Uttar Pradesh.

Methods: Throat swab/nasal swab were collected from clinically suspected cases of all age groups during January –June 2015. RNA was extracted and analyzed for Influenza A pdm H1N1 as per CDC protocol. Clinical data were collected by standardized questionnaires. The cases were further categorized with different parameters such as age/gender/symptoms/geographic distribution.

Results: Total 2879 samples were collected, 2496 (83.33%) from OPD and 365 (12.65%) from IPD. Out of 25.8% (744/2879) were positive for Influenza A pdm H1N1 (mean 19.93; range10.61-38.33 %). Maximum positivity was in March (38.33%) followed by February (23.60%) All the patients were categorized in six different age groups (0-10, 11-20, 21-30, 31-40, 41-50, ≥51). The infection rate was found to be higher in the age group 31-40 years (Mean age 32.19; range 1-89), males were predominated. The median onset of illness before hospitalization was 7 days (range 2-30 days). The most common symptoms were fever in 680 (91.39%), cough in 713 (95.83%), nasal congestion563 (75.67%), sore throat 486 (65.32%) breathlessness in 532 (71.50%), headache 365 (49.05%), body ache 355 (47.71%) and vomiting 93 (12.5%) and diarrhea 53(7.1%). Headaches, sore throat, shortness of breath were the significant symptoms of Influenza A pdm H1N1. All samples were collected from Barabanki, Unnao, Raibareilly, Kanpur, Sultanpur, Gorakhpur, Allahabad, Bareilly, but Lucknow was found to be the major affected city with Influenza A pbmH1N1.

Conclusion: Age was found to be an important risk factor that affects the positivity of Influenza A pdm H1N1. Increased positivity in higher age group needs to be evaluated.

Keywords: H1N1, Influenza virus, RESPIRATORY ILLNESS

17th International Congress of Virology 259

Late breaking abstract

Influenza virus PO706

Cytokine status of the mice immunized with live cold-adapted influenza vaccine in combination with chitosan derivatives

N. Akhmatova 1, O. Lebedinskaya*, E. Akhmatova 1, Y. Troynich 2 1I.I. Mechnikov Research Institute for Vaccines and Sera, Moscow, 2Hystology, ACAD. E.A. WAGNER PERM STATE MEDICAL UNIVERSITY, Perm, Russian Federation

Objectives: Aim. To estimate influence of adjuvants in combination with vaccines on mice cytokine status.

Methods: Experiments were performed on male CBA mice weighing 10-12 g. Study design included 5 groups: cold-adapted (CA) strain vaccination with 1% solution of chitosan glutamate adjuvant; CA strain vaccination with 1% suspension of chitosan sulfate adjuvant; CA strain vaccination in glutamate buffer solution (control). There were also two adjuvant control groups that received only the derivatives of chitosan with no CA strain vaccine, and no-vaccine no-adjuvant glutamate buffer control. The mice were placed under light ether anesthesia and immunized with different doses of intranasal strain with or without chitosan by introducing 25 mkl of the vaccine. Immunization was repeated 20 days after the first CA vaccine administration

Results: Splenocytes of immunized mice were pre-incubated for 24 h in growth medium RPMI 1640 or RPMI 1640 and PHA (5 mg/ml), then spontaneous and induced cytokine production was analyzed in supernatants. The moderate activation was observed for cytokines IFN-g, IL-1b, IL-2 and IL-10 in case of intranasal immunization with cold-adapted strain (H2N2). Especially activity augmentation by 4,0-, 2,0-, 2,5-, and 3,7-fold on the 7th day after first immunization, respectively. We could detect only a very slight increase in the activity of GM-CSF, IL-4, IL-17, IL-5 (increase be 1,3-, 1,6-, 1,5-, and 1,3-fold, respectively). The TNF-α level was dramatically reduced in the first week after first immunization. However, it should be noted that after the second intranasal immunization by CA strain there was observed marked elevation of most cytokines: INF-γ, TNF-α, GM-CSF, IL-4, IL-2, IL-5, and IL-6 (the increase by 40-, 63-, 17,8-, 10,3- , 5,0-, 11,7-, 3,3-, and 3,2-fold, respectively). Intranasal administration of live cold-adapted influenza vaccine in combination with chitosan glutamate resulted in the elevation of most cytokines; however this level was low than the level being observed under intranasal immunization with live influenza vaccine alone. The case of micro/nanoparticles of chitosan sulfate suspension in live CA influenza vaccine was accompanied by synthesis inhibition of some cytokines after first immunization: IFN-γ, TNF-α, IL-17, IL-5 (the synthesis decrease by 2,7-, 10,0-, 5,0-, and 2,1-fold, respectively) and moderate activation of other cytokines under study: GM-CSF, IL-1b, IL-2, IL-10 (the increase by 1,6-, 1,7-, 1,9-, and 3,6-fold). After the second immunization pronounced elevation of TNF-α, IL-6 and IL-10 was observed (the synthesis increase by 15,3-, 37,0-, and 18,0-fold, imrespectively), as well as moderate increase in other analyzed cytokines.

Conlusion: Chitosan possess immunomodulatory effect and in most cases, increases the production of cytokines in in combination with influenza vaccine.

Keywords: adjuvant, cytokine status, influence

17th International Congress of Virology 260

Abstract Submission

Influenza virus PO707

DEVELOPMENT OF HIGH-FIDELITY INFLUENZA A VIRUS VACCINE STRAIN

T. Naito 1,*, H. Ushirogawa 1, M. Saito 1 1Department of Microbiology, Kawasaki Medical School, Kurashiki, Japan

Objectives: Vaccination represents the most effective prophylactic option against influenza. The main antigenic epitopes of the influenza A virus are located on the hemagglutinin (HA) and neuraminidase (NA). The reconstituted vaccine strain containing the HA- and NA-encoding genes is designated a 6:2 reassortant virus, as it comprises 6 genome segments from a high-growth master virus and 2 genome segments (HA and NA) from the circulating wild-type virus. A high-growth virus PR8 (A/Puerto Rico/8/34) has been developed for as the backbone master virus. In order to improve antigen yields of the reassortant vaccine virus in eggs, the vaccine seed virus may be required egg adaptation, which involves multiple passages in eggs. The egg adaptation procedure is generally applied to any low-producing vaccine candidate strain. However, viral replication during the egg adaptation process frequently results in gene mutations, which may alter the antigenic properties of the HA and NA proteins derived from wild-type viruses of concern. Optimal vaccine production requires that seed viruses exhibit high growth in eggs, without significant changes in their antigenic properties. We hypothesized that applying a high- fidelity polymerase to vaccine master virus would be a rational strategy for reduction of the frequency of antigenic alterations during vaccine production. It has been reported that an influenza virus encoding high-fidelity RNA polymerase was generated by screening ribavirin-resistant H3N2 viruses, and that the single amino acid substitution of V43I on the PB1 subunit increased selectivity to guanosine RNA substrate. In this study, by introducing the PB1-V43I substitution into the PB1 of the high-growth PR8 master strain, we generated a master influenza A vaccine virus with both high growth property and high genetic stability in eggs.

Methods: In order to compare the RNA polymerase activity and fidelity of the PR8 master viruses with that of the PB1-wild- type and PB1-V43I, we generated a 6:2 reassortant viruses with V43I substitution in the PB1 segment, using pPol1-plasmids encoding HA and NA cDNAs derived from A(H1N1)pdm09, and compared their biological properties with those of the wild- type.

Results: We conducted the serial passages of the reassortant viruses in eggs until a high hemagglutination titer was obtained. The PB1-V43I-6:2 virus gained a high HA titer similar to that of the PB1-wild-type-6:2 virus. Deduced amino acid substitutions occurred in both viruses after passages in eggs; seven in HA and three in NA of the PB1-wild-type virus while three in HA and none in NA of the PB1-V43I mutant virus. An egg-adaptation mutation leading to Q223R substitution in HA was found to have occurred in the passaged viruses encoding both PB1-wild-type and PB1-V43I. The Q223R mutation in the receptor binding site improves infectivity in egg and in human cells without changing HA antigenicity. Interestingly, the K154N and the G155E antigenic mutations were introduced into the HA of the PB1-wild-type vaccine virus following passages; however, these mutations were not found in the PB1-V43I vaccine strain. This finding was attributed to the increased fidelity of RNA synthesis exhibited by the PB1-V43I virus relative to the PR8 wild-type.

Conclusion: These results imply that the PB1-V43I-substitution approach may be applied to the construction of genetically stable high-fidelity vaccine backbone viruses with reduced frequency of antigenic mutations during egg adaptation. Our findings may be additionally applied to mammalian cell-based systems for improving the efficiency of vaccine production.

Keywords: Influenza virus, RNA polymerase, vaccinia virus

17th International Congress of Virology 261

Abstract Submission

Influenza virus PO708

IDENTIFICATION OF HOST HISTONE DEACETYLASE 1 AS A NOVEL ANTI-INFLUENZA A VIRUS FACTOR

P. Nagesh 1, M. Husain 1,* 1Microbiology and Immunology, University of Otago, Dunedin, New Zealand

Objectives: Host cells produce many antiviral proteins that inhibit the influenza A virus infection. We recently identified host histone deacetylase (HDAC), HDAC6 as an anti-influenza virus factor. HDACs catalyse the deacetylation of acetylated proteins and consequently are involved in multiple cellular processes. HDACs are a large family of proteins and are divided into four classes. The objective of this study was to identify a potential antiviral role of host HDAC1, a class I HDAC, in influenza A virus infection.

Methods: Human lung epithelial cells A549 and influenza virus A/PR/8/34/H1N1 strain were mainly used to carry out this study. The expression of HDAC1 was altered by either RNA interference or plasmid overexpression in A549 cells and its effect on influenza A virus growth kinetics was assessed by plaque assay. Further, the expression of HDAC1 and its role in host innate antiviral response was analysed by western blotting.

Results: We found that about 97% knockdown in HDAC1 expression increased the influenza A virus growth kinetics by 6- fold after 48 h of infection. Conversely, the overexpression of HDAC1 decreased influenza A virus growth by more than 2- fold. HDAC1 has been described as a co-activator of type I interferon response and expression of interferon-stimulated genes (ISG). Consistent with this property, we found that the knockdown of HDAC1 expression decreased the expression of viperin, an ISG, in influenza A virus infected cells by 58%, whereas the overexpression of HDAC1 increased viperin expression by 55%.

Conclusion: Our data demonstrate that like HDAC6 (a class II HDAC), HDAC1 is a novel anti-influenza A virus host factor. Further, HDAC1 exerts its antiviral function by being an important component of influenza virus-induced host type I interferon antiviral response.

Keywords: histone deacetylases, host antiviral factors, HDAC1

17th International Congress of Virology 262

Late breaking abstract

Influenza virus PO709

Immune status of the mice immunized with live cold-adapted influenza vaccine in combination with chitosan derivatives

N. Akhmatova 1, O. Lebedinskaya*, Y. Troynich 2, E. Akhmatova 1 1I.I. Mechnikov Research Institute for Vaccines and Sera, Moscow, 2Hystology, ACAD. E.A. WAGNER PERM STATE MEDICAL UNIVERSITY, Perm, Russian Federation

Objectives: Despite progress of modern medicine, millions of people annually die because of infections. The task of scientists is to find a method how the efficiency of vaccines can be increase. Aim. To study the influence of adjuvants in combination with vaccines on immunophenotype of mice immunocompetent cells.

Methods: Experiments were performed on male CBA mice weighing 10-12 g divided in 5 groups. The mice were placed under light ether anesthesia and immunized with different doses of intranasal cold-adapted (CA) strain with or without chitosan by introducing 25 mkl of the vaccine. Immunization was repeated 20 days after the first CA vaccine administration. Study design included three arms: CA strain vaccination with 1% solution of chitosan glutamate adjuvant; CA strain vaccination with 1% suspension of chitosan sulfate adjuvant; CA strain vaccination in glutamate buffer solution (control). There were also two adjuvant control groups that received only the derivatives of chitosan with no CA strain vaccine, and no-vaccine no-adjuvant glutamate buffer control. Flow cytometry analysis was performed using monoclonal antibodies.

Results: Immunophenotyping of the mice immunized with live CA influenza vaccine in combination with chitosan derivatives revealed the moderate increase in the number of lymphocytes displaying CD16 (NK), CD3/CD16 (NKT), CD71 and CD21 biomarkers. The intranasal immunization of mice with live CA influenza vaccine was accompanied by the decrease in the number of T cells with markers CD3 (61±5.2 vs. 35±2.0, p<0.01) on the 7th day after the first immunization, this trend continued at the 1st and 7th day after the second immunization. The number of CD4-positive T cells also decreased two fold (36±2.1 vs. 17±1.5, p<0.01) during the period of observations. Additionally, an inhibition of B1-, CD8-positive T-lymphocytes and MHC II positive cells was noted. The number of NK-cells increased by threefold (11±0.9 vs. 38±3.3, p<0.01), and while the number of immune cells with γδ-TCR markers increased twofold (4±0.4 vs.10±0.7, p<0.01; and 4±0.4 vs. 11.0±0.7, p<0.01), as measured on the 1st and 7th day after immunization, respectively. Intranasal immunization of mice with live CA influenza vaccine in combination with chitosan glutamate was accompanied by the following changes in subpopulations of mononuclear leukocytes: an increase in subpopulations of T cells expressing CD3 (61±5.2 vs. 47.0±3.5, p<0.01; 61±5.2 vs. 58.0±4.6, p>0.05 as measured on the 1st and 7th day after the 2nd immunization, respectively), CD8 (24.0±2.1 vs. 15.0±1.1, p<0.01 on the 7th day after the 1st immunization) and CD16 (11.0±0.9 vs.48.0±4.4, p<0.01 times on the 7th day after the 1st immunization). B1 lymphocyte subpopulations also increased in their numbers as evident by CD5-positive cell increase on the 1st day after the 1st immunization (4.6±0.3 vs. 5.8±0.6, p>0.05) and on the 7th day after the 2nd immunization (4.6±0.3 vs. 6.3±0.9, p>0.05). With the micro/nanoparticles of chitosan sulfate as an adjuvant, live CA influenza vaccine immunizations resulted in a similar trendency: an increase of T-lymphocyte subpopulations with markers CD3, CD8, CD16 and CD4 and B1-lymphocytes subpopulations.

Conlusion: Chitosan in combination with live CA influenza vaccine possess immunomodulatory effect on immunocompetent cells.

Keywords: chitosan derivatives , immunophenotype , influenza

17th International Congress of Virology 263

Abstract Submission

Influenza virus PO710

Infections of Human Respiratory Tract Primary Epithelial Cells Identify Patient-Related Characteristics Response to Avian Influenza A (H7N9) Virus

C.-G. Huang 1 2 3 4,*, L.-A. Lee 5 6, Y.-C. Wu 5 7, M.-J. H. Hsiao 2 3, K.-C. Tsao 2 3, S.-R. Shih 1 2 1Research Center for Emerging Viral Infections, college of medicine, 2Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang-Gung University, 3Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, 4Graduate Institute of Biomedical Sciences, College of Medicine, 5Faculty of Medicine, College of Medicine, Chang-Gung University, 6Department of Otorhinolaryngology - Head and Neck Surgery, 7Department of surgery, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan

Objectives: Avian influenza A (H7N9) virus infections are a serious public health threat. It remains unclear whether cultures of human respiratory tract primary epithelial cells may be helpful to understand Avian Influenza A(H7N9) virus tropism and pathogenesis as well as to evaluate how patient-related characteristics can modulate the host’s response to infection.

Methods: We utilized ex vivo cultures of primary epithelial cells harvested from patients (n = 30) undergoing upper or lower airway surgery. Cells were experimentally infected with the H7N9 virus (multiplicity of infection: 0.01) for 72 h. H7N9 viral titers and cytokine levels were determined in culture supernatants and analyzed in relation to both the anatomical sites from which cells were collected and patient-related characteristics.

Results: H7N9 more productively replicated in primary epithelial cells harvested from the inferior turbinate and paranasal sinus compared with those from the larynx and bronchus (P = .002). Viral titers at 72 hours were significantly higher in younger (< 65 years) than in older (≥ 65 years) individuals (P < .001), but no effects of sex, medical comorbidities, and obesity were evident. H7N9-infected cultured cells released at least 18 cytokines within 72 h, mimicking the onset of a cytokine storm. Increased levels of certain mortality-related cytokines (e.g., IL-1β, IL-6, IL-8, IFN-γ, IP-10, MCP-1, MIP-1α, and MIP-1β) were associated with patient-related characteristics under investigation.

Conclusion: These findings illustrate the utility of this primary culture approach to investigate how patient-related characteristics can modulate the host’s response to H7N9 virus infection. Our model may be helpful to understand virus pathogenesis as well as to evaluate tailored therapeutic options.

Keywords: primary epithelial cells, avian Influenza, H7N9, respiratory tract

17th International Congress of Virology 264

Abstract Submission

Influenza virus PO711

INFLUENZA A VIRUSES CIRCULATING IN THE POPULATIONS OF WILD BIRDS OF KAZAKHSTAN (2014-2015)

A. Kydyrmanov*, K. Karamendin 1, K. Zhumatov 1, M. Sayatov 1, S. Asanova, Y. Kassymbekov 1, K. Daulbaeva 1, S. Suleimenova 1, Y. Khan 1 1Virology, Institute of Microbiology and Virology, Almaty, Kazakhstan

Objectives: Ecological and phylogenetic studies have shown that the birds of water and wetlands are harboring influenza viruses in the biosphere. The several transcontinental flyways of migratory birds overlap and pass through the territory of Kazakhstan, for this reason, virological surveillance of avifauna in this region is important in study of antigenic diversity and natural variability of influenza A viruses.

Methods: Determination of antigenic formula of influenza A viruses was carried out by means of BLAST analysis of nucleotide sequences of HA and NA genes of isolates in GenBank.

Results: A total of 54 influenza A virus isolates were allocated from two geese, six duck, six gull, two shorebird, and by one from flamingo, coot and Phasianidae species as the result of virological screening 937 samples from 48 bird species collected in different parts of Kazakhstan during 2014-2015. Influenza viruses with seven hemagglutinin (H1, H3, H4, H5, H10, H13, H16) and six neuraminidase (N1, N2, N3, N6, N7, N8) subtypes in nine different combinations were identified. Influenza A/H1, A/H3 and A/H4 viruses isolated from ducks, shorebirds and gulls in South, South-East and Central Kazakhstan. Same time influenza A/H1 viruses sporadically isolated from coot and pheasant. Highly pathogenic avian influenza (H5N1) was isolated from duck species in South-Eastern Kazakhstan in 2014; from flamingo and gull species in Caspian Sea coastal area in 2015. The HA genes of H5N1 isolates related to the highly pathogenic strains circulating among domestic and synanthropic birds in South Asia, West Africa and Middle East. Amino acid substitutions on neuraminidase composition of A/flamingo/Caspian Sea/6570/2015 (H5N1) virus were similar to the oseltamivir resistant epidemic virus A/Hanoi/30408/2005 (H5N1). It leads to the conclusion about circulation in Kazakhstan avifauna oseltamivir-resistant variants of highly pathogenic H5N1 viruses. Influenza A/H10 viruses with neuraminidases N2, N7 and N8 isolated from feral ducks and geese for the first time in Kazakhstan simultaneously in North and South–East Kazakhstan in 2014. Contrary of them A/H13 viruses are isolated annually from gulls on islands of Northern Caspian. The viruses of H16N3 subtype detected second time among birds of Western Kazakhstan in 2015 since first isolation in same place in 2007.

Conclusion: These analyses indicate that the populations of wild birds surveyed in Kazakhstan are actively involved in the process of global spreading of influenza A viruses and their evolving.

Keywords: None

17th International Congress of Virology 265

Abstract Submission

Influenza virus PO712

KAMPO HERBAL MEDICINES COMMONLY USED FOR RESPIRATORY DISEASES AND THEIR CRUDE DRUGS ACTUALLY INHIBIT INFLUENZA REPLICATION.

T. Nomura 1,*, T. Irie 1, M. Fukushi 1, K. Oda 1, T. Sakaguchi 1 1Department of Virology, Graduate school of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan

Objectives: Epidemics by influenza virus causes hospitalization of people in the world every year. Furthermore, there is always a threat that a novel pandemic influenza virus may emerge. Several antiviral drugs such as neuraminidase inhibitors have been developed for treatment of influenza. However, there are some problems such as high cost and possible emergence of drug-resistant mutants. To prepare multiple remedies for influenza virus, we should continue to search for anti-influenza drugs. Kampo medicines, originating from China, have been used as traditional medicines in Japan. Kampo medicines, a mixture of crude drugs derived from plants such as herbs, are prescribed to patients with respiratory diseases including influenza. However, the effects of Kampo medicines on influenza virus have not been examined in detail. In the present study, we investigated the effects of Kampo medicines and their crude drugs on influenza virus replication.

Methods: Six Kampo medicines including Kakkonto (Tsumura #1), Saikokeishito (#10), and Maoto (#27) together with 13 crude drugs were purchased from Tsumura Corporation (Tokyo, Japan). The medicines were dissolved in Dulbecco’s modified Eagle’s medium (DMEM) for 60 min at 50℃ and filter-sterilized. MDCK cells and A549 cells, which were obtained from RIKEN Cell Bank, were infected with influenza virus (IFV) A/Udorn/72(H3N2). For a cell toxicity assay, lactate dehydrogenase (LDH) in the culture medium was measured by a colorimetric method. MDCK cells and A549 cells were infected with IFV at an m.o.i. of 0.2 or 5. After 1-hour adsorption, cells were maintained in DMEM containing various concentrations of a Kampo medicine or a crude drug together with 30 µg/ml of trypsin. A part of the medium was recovered at various time points, and virus production was determined by measuring hemagglutinating units (HAU) and infectivity by the TCID50 method. Inactivation of IFV particles by the reagents was also evaluated. Viral protein synthesis was investigated by Western blotting using anti-IFV anti-serum.

Results: We selected six Kampo medicines that are prescribed for influenza or common cold. Firstly, cytotoxicity was evaluated with various concentrations of the reagents, and the concentrations at which cytotoxicity was not observed were used for further study. Influenza virus replication was suppressed by four of the six Kampo medicines. We next investigated the effects of crude drugs that constitute the four Kampo medicines. Influenza virus replication was suppressed by three of the four crude drugs. None of the reagents directly inactivated virus particles.

Conclusion: Four of the six Kampo medicines investigated suppressed influenza virus replication, support the rationale to prescribe Kampo medicines to influenza patients. Two of the four medicines have not been reported to be effective for treatment of influenza virus, and this is the first report for these two medicines. Three of the four crude drugs including Glycyrrhiza suppressed influenza virus replication. Glycyrrhiza was previously reported to inhibit influenza replication, and it was the most effective drug in this study. All of the effective reagents appear to work on infected cells, not on the initial steps such as adsorption and membrane fusion. Many of the reagents suppressed protein synthesis of the influenza virus. The mechanism of inhibition of influenza virus remains to be determined.

Keywords: antivirus, Kampo medicine

17th International Congress of Virology 266

Abstract Submission

Influenza virus PO713

NOVEL PROTECTIVE HUMAN MONOCLONAL ANTI-HA STEM ANTIBODIES AGAINST INFLUENZA A VIRUS INFECTION

S. Yamayoshi 1,*, M. Ito 1, R. Uraki 1, M. Kiso 1, T. Sasaki 2, K. Ikuta 2, Y. Kawaoka 1 3 4 5 1Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, 2Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 3Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States, 4Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, 5ERATO Infection-Induced Host Responses Project, JST, Saitama, Japan

Objectives: Numerous anti-influenza agents are under development because antiviral treatment decreases the burden of seasonal influenza and provides protection against pandemic influenza before vaccines are available. Broadly reactive anti- HA antibodies could fill the need for novel broad spectrum anti-influenza virus agents. Such antibodies would prevent infection with several subtypes of influenza A virus. Many human monoclonal antibodies against the HA stem that meet this criterion have been reported. Here, we established novel broadly reactive human anti-HA stem monoclonal antibodies that protected mice from lethal infection.

Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of two volunteers who were infected with H3N2 virus 1 week earlier during the 2014–2015 influenza season. The isolated PBMCs were fused with fusion partner SPYMEG cells. The resulting hybridomas were tested for anti-HA antibody production by using an ELISA with recombinant H1-, H3-, H5-, H7-, and type B-HAs as the antigen. The acquired clones were assessed by examining their reactivity with 18 subtypes of HA, identifying their epitopes, measuring their neutralization potency, and evaluating their in vivo protective efficacy.

Results: After screening and cloning, we ultimately acquired 3 human monoclonal antibody clones that each recognized at least 2 subtypes of the recombinant HA antigens tested. Clones A and B used VH genes that were not used by previously reported anti-stem antibodies. Clone A recognized several group 1 HAs and all group 2 HAs, and neutralized some of these viruses in vitro; clone C reacted with several group 1 HAs and all group 2 HAs but failed to neutralize them. Clones B reacted with all group 2 HAs and neutralized H3N2 virus. Mutant H3N2 viruses that escaped from clone A or B possessed defining amino acid substitutions in their HA stem. A competitive binding assay revealed that both clones A and B targeted the HA stem. Prophylactic treatment with either clone A or B protected mice from lethal infection with H3N2 virus.

Conclusion: We obtained novel human anti-HA stem monoclonal antibodies that exhibit protective efficacy both in vitro and in vivo.

Keywords: broad-spectrum antibodies, Human monoclonal antibody

17th International Congress of Virology 267

Abstract Submission

Influenza virus PO714

PHYLOGENETIC RELATIONSHIPS OF THE HA AND NA GENES BETWEEN VACCINE AND SEASONAL INFLUENZA A(H3N2) STRAINS IN KOREA

J. I. Kim 1, S. Park 1, I. Lee 1, H. J. Cheong 2, J. Y. Noh 2, K. W. Hong 3, J.-W. Song 1, S.-H. Kee 1, K.-J. Song 1, M.-S. Park 1,* 1Microbiology, Korea University College of Medicine, 2Infectious Diseases, Korea University College of Medicine Guro Hospital, Seoul, 3Infectious Diseases, Hallym University College of Medicine, Chuncheon, Korea, Republic Of

Objectives: Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season.

Methods: To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes.

Results: In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non- vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other.

Conclusion: Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness.

Keywords: influenza H3N2 virus, phylogenetic analysis, surface glycoproteins

17th International Congress of Virology 268

Abstract Submission

Influenza virus PO715

POLYMERIC NANOPARTICLE-ENCAPSULATED CURCUMIN INHIBITS INflUENZA VIRUS INFECTION

H.-M. Chang 1,*, P.-S. Lai 2, W.-L. Hsu 1 1Graduate Institute of Microbiology and Public Health, 2Department of Chemistry, National Chung Hsing University, Taichung, Taiwan

Objectives: Curcumin (Cur), a natural compound and an ingredient in curry, has anti-inflammatory, anti-oxidant, anti- carcinogenic and anti-viral properties. Previously, we reported that Cur inhibits type A influenza virus (IAV) infection by blocking viral haemagglutination (HA) activity and via competing the cellular receptor binding site as evident with HA inhibition assay (HI ) and the molecular docking simulation, respectively. Despite these encouraging findings, application of curcumin is severely limited by its intrinsic properties such as low aqueous solubility and poor stability, leading to poor bioavailability. In this study, the anti-influenza activity of novel aqueous formulation of polymeric encapsulated curcumin (Cur-c) was evaluated.

Methods: Cytotoxicity of the Cur-c was estimated by the survival rate of treated Madin-Darby canine kidney (MDCK) cells. The Cur-c anti-influenza activity was initially evaluated based on viral plaque formation ability and the yield of viral progenies using Time-of-drug addition protocol. Furthermore, effect of Cur-c on viral RNA replication and cellular signaling pathways required for IAV infection were also examined.

Results: Our results demonstrated that treatment with 30 μM Cur-c reduced the yield of virus by over 90% in MDCK cells. Noticeably, addition of Cur-c after viral entry completely abrogates plaque formation ability of IAV, while curcumin exerts a mild inhibition effect, indicating Cur-c inhibits IAV production via non-identical mechanisms with Curcumin. Aside from viral entry, it is likely Cur-c also affects other key stage(s) of viral replication. A series of experiments were then set out to monitor the effect of Cur-c on viral RNA replication, protein expression, and the cell signaling pathways required for IAV propagation. Results of signaling reporter cells and western blot analyses revealed that Cur-c significantly repressed the activation of Akt and NF-kB signaling pathways. Furthermore, Cur-c inhibits viral RNA replication to a higher extent than curcumin.

Conclusion: Study herein described novel mechanisms by which curcumin inhibits IAV. Furthermore, polymeric encapsulation formulation significantly improves the aqueous dispersion and that possibly enhances intracellular uptake of curcumin.

Keywords: Anti-influenza activity, Curcumin, Cell signaling pathways, Polymeric nanoparticle

17th International Congress of Virology 269

Abstract Submission

Influenza virus PO716

POST-EXPOSURE ADMINISTRATION OF WHOLE INACTIVATED H5N1 INFLUENZA VACCINE PROTECTS MICE FROM LETHAL HOMOLOGOUS INFECTION

M. Hagan 1 2,*, C. Ranadheera 2, J. Audet 1 2, J. Morin 3, A. Leung 2, D. Kobasa 1 2 1Medical Microbiology, University of Manitoba, 2Special Pathogens, Public Health Agency of Canada, Winnipeg, 3Département des Technologies de laboratoire, Cégep de Sherbrooke, Sherbrooke, Canada

Objectives: Highly pathogenic avian influenza viruses, such as H5N1, are found throughout wild aquatic birds and domestic poultry, with certain strains that are able to cause significant disease in humans. Although these avian influenza viruses do not readily infect humans, their ability to mutate quickly and reassort genes with other influenza strains may allow these viruses to adapt to better infect humans. Recently, H5 viruses originating from Eurasia have appeared for the first time in North American wild and domestic bird populations. The wide range of H5 subtypes circulating globally makes it impractical to pre-vaccinate all individuals at high risk of exposure to these viruses. Instead, we were interested in evaluating the efficacy of a formalin-inactivated influenza virus, analogous to the conventional influenza vaccine, as a post-exposure vaccine therapy.

Methods: Here, we used a well characterized strain of H5N1, A/Vietnam/1203/2004, to generate a whole formalin- inactivated influenza vaccine for post-exposure treatment. Mice were infected with a lethal dose of H5N1 and treated with the whole inactivated vaccine up to 3 days post-infection. Protection was assessed based on weight loss and severity of clinical signs and symptoms. Levels of CD4+ and CD8+ T cell responses were measured by flow cytometry, while antibody responses were determined by ELISA and microneutralization assays. To improve post-exposure vaccine efficacy, adjuvants were also included in the inactivated vaccine preparations. Post-exposure protection against other strains of H5N1, including A/Indonesia/5/2005 and A/Hong Kong/483/97, was also evaluated.

Results: Immediate post-exposure treatment with a whole inactivated H5N1 vaccine afforded complete protection from lethal outcome following homologous virus infection. A delay in treatment up to 24h post-exposure remained highly efficacious, however, treatment after 24h post-exposure was no longer protective. By measurement of T cell and antibody responses, we show that whole inactivated H5N1 rapidly induced a protective immune response in mice after the infection. In vaccine treated mice, protection was associated with the generation of a more rapid and robust antibody response. We also observed that vaccination resulted in decreased activation of CD8+ T cells, suggesting a dampening of the Th1 response that is generally associated with pro-inflammatory immune responses, and may have contributed to the recovery of all mice that were treated at the optimal dose of vaccine. The addition of adjuvants to the inactivated vaccine did not increase vaccine efficacy. However, the inactivated vaccine provided cross-protection to the other strains of H5N1 viruses tested.

Conclusion: These results offer a new strategy of using inactivated vaccines as an effective post-exposure treatment against H5N1 viruses, and present another option to provide protection for front-line responders in the event that a new pandemic strain emerges.

Keywords: Influenza H5N1, post-exposure, Vaccine

17th International Congress of Virology 270

Abstract Submission

Influenza virus PO717

Reassortment H5N6 Highly Pathogenic Avian Influenza Viruses with H6-Like PB2 Genes in Quail, Vietnam

T. H. Dang 1 2,*, D. T. Nguyen 2, V.-K. Hung 2, W. Kim 1,* 1Dept. of Medical Microbiology, Chung Ang University, College of Medicine, Seoul, Korea, Republic Of, 2Central Vietnam Veterinary Institute, Nha Trang, Viet Nam

Objectives: Avian influenza A (H5N6) virus is the most highly contagious infectious disease that has been affecting domestic poultry and humans in South Asian countries. Vietnam might be an evolutionary hotspot and source of worldwide pandemic strains.

Methods: Sample collection RNA extraction and real-time RT-PCR to detect influenza A virus Full-length genome amplification, sequencing and phylogenetic analysis

Results: In 2015, novel reassortant H5N6 influenza viruses, designated A/ Quail /Vietnam/CVVI-01/2015 and A/ Quail /Vietnam/CVVI-03/2015, were isolated from quail during H5N6 outbreaks in Central Vietnam, and their whole genomes were analyzed. Genetic analysis indicated that hemagglutinin (HA), neuraminidase (NA), and polymerase basic protein 2 (PB2) genes of two H5N6 viruses were most closely related to H5N2 (A/chicken/Zhejiang/727079/2014) from China and H10N6 (A/chicken/Jiangxi/12782/2014) from China, and H6N6 (A/duck/Yamagata/061004/2014) from Japan viruses, respectively. The five other internal genes were homologous to influenza A H5N2 (A/chicken/Heilongjiang/S7/2014) strain isolated from China. Phylogenetic analysis revealed that HA gene of new strains belonged to H5 clade 2.3.4.4 detected in Vietnam, Laos and China

Conclusion: Data from this study supports that these two H5N6 strains might be novel reassortant viruses containing a H6N6-like PB2 gene. Our findings highlight the risks for prolonged co-circulation of avian influenza viruses and it is also useful for better understanding the genetic and antigenic evolution of H5 avian influenza viruses in Asian countries.

Keywords: avian influenza A virus, H5N6 virus, Vietnam

17th International Congress of Virology 271

Abstract Submission

Influenza virus PO718

REDUCTION OF HEMAGGLUTINATION INHIBITION ANTIBODY TITERS AGAINST RECENT H1N1 OUTBREAK STRAINS AMONG VACCINEES WITH 2015-2016 SEASONAL INFLUENZA VIRUS VACCINATION

J.-R. Wang*, C.-H. Tai 1, S.-W. Huang 2 1National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, 2Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan city, Taiwan

Objectives: Influenza A virus can cause mild or severe clinical symptoms and even death as circulating in human population. Recent influenza A H1N1 virus is a swine-origin triple reassortment strain that globally spread since April 2009. In Taiwan, a large outbreak of H1N1 virus occurred in early 2016 with dozens of severe or fatal cases. Rapid evolution of influenza RNA genes might result in viral antigenic drift or shift. These antigenic property changes may lead to large outbreaks or pandemics owing to the reduction of immunity in humans against reemerging variants.

Methods: To determine whether reduced protection efficacy among human population contributed to this large outbreak, we examined viral genetic evolution and humoral response of vaccinees against H1N1 strains circulating in Taiwan by phylogeny and hemagglutination inhibition (HI) antibody investigation.

Results: Our surveillance data indicated that emerging subclades 6B, 6B.1, and 6B.2 of H1N1 viruses continuously evolved and acquired various potential antigenic escape mutations in hemagglutinin gene. As further examining antigenic properties of circulating strains, we found that post-immunization antisera from vaccinees generally exhibited reduced HI antibody titers against recent outbreak strains in contrast to H1N1 vaccine strain CA/07/09, indicating the low protection efficacy among Taiwan vaccinees against outbreak strains.

Conclusion: Our study demonstrated that recent H1N1 outbreak strains isolated in Taiwan with new substitutions. These strains exhibited distinct antigenic properties that might escape antibody recognition of vaccinees’ immunity, resulted in a large outbreak in early 2016 in Taiwan. Continuous surveillance of viral evolution and vaccine efficacy are thus important issues for the disease prevention and control against emerging variants of influenza viruses.

Keywords: antibody response, Influenza virus

17th International Congress of Virology 272

Late breaking abstract

Influenza virus PO719

Role of transmembrane domain of influenza A virus M2 protein in determining association with membrane microdomains

R. Manzoor 1,*, R. Yoshida 1, A. Takada 1 1Research Center for Zoonosis Control, HOKKAIDO UNIVERSITY, Sapporo, Japan

Objectives: Influenza A virus expresses three envelop proteins i.e. hemagglutinin (HA), neuraminidase (NA), and M2. Of these, HA and NA proteins associate with lipid rafts. The transmembrane domains (TMDs) of HA and NA proteins are 27 and 29 amino acid residues long, respectively. Additionally, the HA cytoplasmic tail possesses two palmitoylation sites. In contrast, the M2 protein (TMD 19 amino acid residues), despite the presence of lipid raft targeting features i.e., palmitoylation at Cys50 and cholesterol recognition/interaction amino acid consensus (CRAC) motif, remains localized in the peri-raft regions. Does the relatively shorter M2-TMD prevent the association of M2 with lipid raft domains? So, the present study was conducted to determine the relation between M2-TMD length, and lipid raft association as well as virus replication.

Methods: Insertion mutations were introduced in the M2-TMD so that TMDs of mutant proteins were three, six or eight amino acid longer than that of wtM2-TMD. The mutant proteins were characterized by comparing their cell surface expression and cytotoxic potential, an ion channel dependent activity, with that of wtM2 protein. Lipid raft localization of wtM2 and M2-TMD mutants was assessed by TritonX-100 (TX100) solubility assay and immunofluorescence (IF) microscopy. The viruses with M2-TMD mutations were generated by reverse genetics and examined by transmission electron microscopy (TEM).

Results: The cell surface expression and cytotoxic potential of M2-TMD mutants were comparable to that of wtM2 protein. TritonX-100 solubility assay showed that M2-TMD mutants, like wtM2, were largely found in TX100 soluble fractions, the non-raft microdomains. IF staining of lipid rafts and M2 protein, and colocalization analysis also confirmed that increasing the TMD length did not affect their non-lipid raft association character. Then attempts were made to rescue the viruses with M2-TMD mutations using reverse genetics approach. Interestingly, only virus with three amino acid insertions could be rescued, although viral proteins and viral RNA were detected from the supernatants of cells transfected for rescue of wt- and M2-TMD mutant viruses. Electron microscopy of rgPR8/wtM2 and rgPR8/M2-3aa viruses revealed no morphology or size differences.

Conlusion: The M2 protein, besides increasing the TMD length and having two inherent lipid raft targeting features, did not associate with lipid rafts suggesting that the M2-TMD length is not the only non-lipid microdomain localization determinant. Furthermore, increasing the M2-TMD length did not seem to alter the M2 function, but somehow prevented virus rescue either by destabilizing the virus particles or by preventing the successful virus budding.

Keywords: influenza A virus, M2 protein

17th International Congress of Virology 273

Abstract Submission

Influenza virus PO720

THE ACTIVATION OF B CELLS ENHANCES DC-SIGN EXPRESSION AND INCREASE SUSCEPTIBILITY OF B CELLS TO AVIAN INFLUENZA H5N1 INFECTION

A. Thitithanyanont 1,*, P. Na-Ek, J. Thewsoongnoen 1, M. Thanunchai 1Microbiology, Faculty of Science, Mahidol University, Ratchathewi, Thailand

Objectives: The interplay between highly pathogenic avian influenza (HPAI) H5N1 virus and immune cells has been extensively studied for years, as host immune components are thought to play significant roles in promoting the systemic spread of the virus and in the induction of cytokine storm. In this study, the relationship between the HPAI H5N1 virus, activated B cells, and DC-SIGN expression were explored.

Methods: Human B cells were isolated out of peripheral blood mononuclear cells (PBMCs) and then stimulated B cells with CD40L and IL-4 for 24 hours in order to mimic the natural activation of B cells via membrane-bound stimulatory ligands, while untreated B cells were cultured for the same amount of time without any additives. Following stimulation, both treated and untreated B cells were incubated with HPAI H5N1 virus for another 12 hours, then cells in both conditions were double- stained with anti-DC-SIGN and anti-CD86. The co-expression of DC-SIGN, CD86 or nuclear protein (NP) on treated or untreated B cells were observed.

Results: We demonstrated that B lymphocytes obtained from two different environments; (i) present in PBMC mixture (ii) isolate out of PBMCs prior to stimulation with CD40L and IL-4, were induced to an activated state following H5N1 infection. We could determine that activated B cells expressed DC-SIGN, which had the ability to mediate HPAI H5N1 infection, while non-activated B cells showed minimal changes in DC-SIGN expression and infection susceptibility.

Conclusion: Our findings uncover the interplay between H5N1 virus and B cells and provide important information in understanding how the virus overcomes our immune system, contributing to its unusual immunopathogenesis.

Keywords: B cell, DC-SIGN, H5N1

17th International Congress of Virology 274

Late breaking abstract

Influenza virus PO721

The research of screening anti-H7N9 high-affinity neutralizing monoclonal antibodyby single B cell cloning from immunized Chinese macaque L. Jia*

Objectives: The influenza virus is currently one of the main factors threatening human health. Influenza virus happened in recent years, such as H7N9，H5N1 and H1N1 avian influenza,can cause patient morbidity, high mortality, and it has serious impact on human health, economic development and social stabilityit. Now the main way to deal with the influenza virus are vaccines and drugs. Vaccine is one of the most effective way to prevent influenza virus infection, but in the influenza large- scale outbreak period, the best way to control disease is drug therapy. But the vaccine cannot control the new subtype influenza strains. Antiviral drugs has treatment effectbefore the new virus resistant strainsappear, but the treatment effect is not obvious in late infection. The anti- influenza drug M2 ion channel protein inhibitor and neuraminidase (NA) inhibitor, Such as Oseltamivir and Amantadine can effectively control the condition. But with medication, it prones to drug-resistant strains, and medication must also be in the period of effective drugs ,or treatment effect was significantly reduced. There was the use of recovered patient’s sera to severe influenza clinical treatment . But getting high-affinity neutralizing antibody from recovered patient’s blood is difficult to deal with outbreak of influenza after all, and acquired high antibody titer.

Methods: This research uses rhesus monkeys , who has high degree of homology in the genetic with human. Rhesus monkeys will produce specific neutralizing antibodies after immune to influenza virus vaccine several times. Through multiple immune to stimulate B cells, it promotes the generation of high affinity antibodies.Using FACS to sort B cellmarking CD3-/CD19-/CD27-/CD80++. Then using the single cell PCR technology to clone antibody gene , and express in 293T cells. So we can get antibody gene, whose variable region of heavy chain and light chain can naturally match.Using single cell PCR to clone antibody gene is mainly used in plasma cell and memory B cell clone.Compared with the traditional method of antibody production, single cell PCR technology have more advantages in high efficiency and total anthropogenic, genetic diversity . Finally, using ELISA method to detect antigen-antibody affinity, BLI technology for quantitiveaffinity analysis, and virus micro-neutralizationexperiment for testing neutralization titer of antibody and so on .

Results: This research screened a strainof high affinity neutralizing antibody named 2 G11.using the experimental method above. Results in vitro analysis show that: ELISA and western blot results show that, 2G11 can combine H7N9-Anhui strain. And compared with the reported broadly neutralizing antibody named FI6 , the binding activity of 2G11 is 1000 times than FI6. virus micro-neutralizationexperiment also prove that 2 G11 can neutralize H7N9 influenza virus well. The experimental data show that, 77 μg/ml supernatant of antibody can still neutralize 90% of the virus after diluted in proportion of 1:320. And its neutralizing activity is much higher than reported antibody FI6.

Conlusion: This research screened a high -affinity neutralizing antibody named 2G11 using single cell PCR technology from immune rhesus monkeys,which can specifically neutralize H7N9 virus. It will provide effective basis and thinking to clinical treatment of highly pathogenic influenza virus.

Keywords: None

17th International Congress of Virology 275

Abstract Submission

Influenza virus PO722

THE SPATIOTEMPORAL ANALYSIS OF HEMAGGLUTININ SEGMENT OF AVIAN INFLUENZA A VIRUSES BY A NEWLY DEVELOPED STRAIN-BASED ALIGNMENT SYSTEM

C.-R. Yang 1,*, C.-C. King 2, C.-C. Ku 1 1The Graduate Institute of Immunology, College of Medicine, 2Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan

Objectives: The re-emergence of highly pathogenic avian influenza A viruses (HPAIV) has become enzootic in poultry farms and wide-spread infections in wild birds, causing a threat to public health. There have been several sporadic HPAIV outbreaks predominantly caused by H5N2 virus in Taiwan but the largest attack in the avian farms in 2015 was caused by a clade 2.3.4.4 H5N8 virus. Notably, several H5 subtypes of AIV including H5N2, H5N3, H5N8 were found co-circulating in Taiwan. Whether they could continue to evolve through the genetic recombination with low pathogenic viruses locally and bring threat to human pandemic infection is greatly concerned. The presence of R-X-K/R-R amino acid motif at the cleavage site in the Hemagglutinin (HA) of AIV is found associated with viral virulence in avian hosts. A better understanding on how the pathogenicity-related motif is emerged and distributed from a global view is important for developing the control strategies against AIV infections.

Methods: In this study, we have established a strain-based alignment system to analyze 1123 available H5N2 AIVs during 1972-2016 from NCBI and GISAID sequence database. To understand when the HA motif of HPAI was first emerged and how the HPAI virus was transmitted worldwide, in the first attempt, all of the HA segments were divided into high pathogenic (RKKR and RRKR) and low pathogenic (IETR and RETR) group based on the type of R-X-K/R-R motif at the cleavage site. The spatiotemporal distribution of each type of HA motif was thus analyzed.

Results: Dynamic graph showed that each type of R-X-K/R-R motif in the HA of H5N2 viruses has different transmission map. For instance, RKKR was first reported in Italy from A/Italy/312/1997/H5N2/chicken and subsequently occurred in USA (2004), South Africa (2006), Taiwan (2008) and Mexico (2012). The last report on RKKR AIV in Taiwan was in 2013. On the other hand, RRKR was first originated from A/hebei/0908/2009/H5N2/duck in China (2009) followed by Taiwan (2012), Vietnam (2012) and spread to USA and the Europe (2015). Our analysis also showed that the RRKR motif has become the dominant and replaced RKKR in H5N2 HA segment in 2015. Moreover, all of the 40 strains of novel H5N8 AIV isolated from chickens and the waterfowls in Taiwan in 2015 also contained RRKR motif, suggesting that such motif may play an important role in infecting a wide range of avian hosts.

Conclusion: Our results demonstrated that HAPI H5N2 containing pathogenic HA motif is transmitted globally by a time sequential pattern. Mutation or variation in the amino acid residue within the motif can also occur under certain epidemiological conditions. How co-circulation of different H5 subtypes causes the impact on the emergence of novel AIVs with pandemic potential and what makes it to happen require further investigations.

Keywords: Avian Influenza Viruses, HA Cleavage Motif, Highly Pathogenic

17th International Congress of Virology 276

Abstract Submission

Influenza virus PO723

Transient expression and purification of recombinant hemagglutinin protein from H7N7 Influenza virus

W. Y. Leong 1,*, S. S. Paul 1, Y.-J. Tan 1 1Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Objectives: Over the past decade, novel avian influenza A virus (IAV) subtypes such as H7N7, H7N9 and H5N6 has emerged and demonstrated human infection, underscoring the need for new therapeutic agents against these subtypes. Recently, antibodies specific for the hemagglutinin (HA) of IAV have shown promising results that urge further research in this field. To assess the efficacy of these antibodies, it is essential to develop methods to generate purified HA that can be used in different serological assays. Various studies have presented methods to express HA using baculovirus-insect cells and mammalian cells which are capable of conserving the antigenic structure of the protein. In this study, we present an optimised method to transiently express and purify recombinant H7N7-HA protein using human embryonic kidney (HEK) 293FT cells.

Methods: A recombinant plasmid vector encoding H7N7-HA which lacks the transmembrane and endodomain in the C- terminal was constructed. These domains were replaced with a trimeric T4 fibritin Foldon sequence, an Avitag sequence and a hexahistidine tag. The recombinant plasmid was transfected into HEK-293FT cells and the supernatant was collected on different days. The supernatant was purified using NTA-Ni agarose beads and a step-gradient elution process. The presence and purity of the recombinant H7N7-HA protein was validated using western blot and SDS-page, respectively.

Results: Western blot of the supernatant was conducted using anti-hexahistadine antibodies and the results showed secretion of H7N7-HA protein at 24 hours-post-transfection, which continued to increase up to 6 days-post-transfection. We were also able to purify H7N7-HA from the supernatant using the NTA-Ni agarose beads, as verified by the band at approximately 80 kDa in the SDS-page gel. Additionally, only one isolated band was observed upon staining with coomassie blue, suggesting that the final product is very pure.

Conclusion: The results here have presented a reliable method to transiently express recombinant HA with high purity. With this method, future works in our laboratory aim to express the HA protein from different IAV subtypes which will be used to pan for high affinity clones in a phage-display library containing HA-specific single-chain variable fragments (scFvs).

Keywords: Hemagglutinin (HA), Influenza A

17th International Congress of Virology 277

Abstract Submission

Influenza virus

PO724

Using protein-protein interaction networks for functional inference in the influenza A virus (IAV)-host interactome

M. P. Dobay 1,*, S. Stertz 2, M. Delorenzi 1

1Bioinformatics Core Facility, SIB Swiss Institute of Bioinformatics, Lausanne, 2Institute of Medical Virology, University of Zurich, Zurich, Switzerland

Objectives: Genome-wide siRNA screens and proteome-wide assays have significantly advanced the identification of host factors that are necessary, or that promote viral infection. Nonetheless, standard protocols for predicting the processes in which these host factors are involved in the context of the viral life cycle have yet to be established. Here we discuss our recommendations on the use of protein-protein interaction networks (PPIs) in functional inference for influenza A virus (IAV) host factors.

Methods: Functional inference using guilt-by-association methods based on PPIs (STRING, v.9.0 and v.10.0; HIPPIE, v.1.7 and v.1.8) were applied on IAV host factors (n=2226 candidates out of 21505 screened factors) derived from a meta-analysis of eight genome-wide siRNA screens. Briefly, a query protein q is assigned a set of putative functions F based on the functions reported for its network neighborhood.

Results: We and others have recently demonstrated that the choice and parameters used in manipulating PPIs result in degenerate functional assignments. Furthermore, we have shown that the network neighborhood contains as much as 60% of nodes that were reported in a biological context not relevant to the query. We have thus explored the use of context filtering, which takes advantage of textmining evidence and curated literature linked to both PPI vertices and edges, to select subgraphs in a PPI based on biological processes of interest (e.g. restriction to all proteins that have been implicated in viral uptake, intracellular transport, replication, and egress), prior to functional assignment. We show that context filtering improves the probability that the retained network neighborhood is restricted to other host factors, and that the functions of these neighbors could be used to infer the stage of the viral life cycle in which a particular query protein is involved. We also show that current textmining-sourced literature linked to PPI edges are incomplete, and may benefit from an orthogonal textmining of PubMed via webservices (rentrez). For instance, using the context-filtered network neighborhood of all putative IAV host factors derived from the union of STRING v.10.0 and HIPPIE v.1.8, released in January 2015 and that do not contain information on PEPD and UBR4, we are able to infer its respective involvement in entry and transport, among other precosses, from its network neighborhood (Figure 1).

Conclusion: Context filtering improves the utility of PPIs in identifying the functions and facilitating interaction partners of IAV host factors. The results of this study also have wider-reaching implications, as other methods such as kinase prediction protocols, extensively use information from PPIs.

17th International Congress of Virology 278

Image:

Keywords: functional inference, host factor, Influenza A virus

17th International Congress of Virology 279

Abstract Submission

Influenza virus PO876

Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia

B. T. Beyene 1 2 3,*, C. Hui 4, M. Z. Myaing 2, B. H. Tan 5, S. Maurer-Stroh 1 6 7, R. Sugrue 2 1Bioinformatics Institute, A*STAR, 2Division of Molecular Genetics and Cell Biology, School of Biological Science, Nanyang Technological University, Singapore, Singapore, 3Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Ethiopia, Addis Ababa, Ethiopia, 4Genome Institute of Singapore, A*STAR, 5Detection and Diagnostics Laboratory, Defence Science Organisation National Laboratories, 6National Public Health Laboratory, Ministry of Health, 7Department of Biological Sciences, National University of Singapore, Singapore, Singapore

Objectives: Influenza A virus (IAV) is a major public health problem responsible for the death of half a million people every year. Zoonotic transmissions of the virus from swine and avian origin have occurred and can in the worst case result in pandemics. Pulmonary macrophages are the first immune cells that fight influenza infection; however, they are also implicated in the pathogenesis. Understanding the adaptation of avian influenza viruses to mammalian host and the response of macrophages to their infection provides insights for therapeutic and prevention strategies.

Methods: In this study, we used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) in responses to infections with H1N1/WSN and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. PMФ were isolated from BALB/c mice lungs and infected with the three IAV isolates. After 24-hour post infection, the level of virus infectivity and transcriptome responses were assessed using immunofluorescence staining and Affymetrix mRNA microarray, respectively. The expressions of selected cytokines were validated by biochemical cytokine protein assay.

Results: The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ similarly well as the mouse-adapted H1N1/WSN. The global transcriptome response indicated the number and extent of differentially expressed genes were highest in H5N3 infections compared to H1N1/WSN and H5N2 viruses. Pathway and gene ontology (biological process) analysis of the differentially expressed genes revealed induction of interferon responses in H1N1/WSN and H5N3 infections. However, inflammatory responses were prominent in H5N3 infections. Increased gene expression extent of inflammatory chemokines and cytokines in H5N3 infections was consistent in the biochemical proteins assay.

Conclusion: The LPAI H5N2 and H5N3 are able to infect mammalian host cells and together with increased expression of inflammatory mediators particularly in H5N3 could impose threats to human health in the future.

Keywords: Macrophages, Influenza virus, inflammatory response

17th International Congress of Virology 280

Abstract Submission Influenza virus PO887

Rapid virulence shift of an H5N2 avian influenza virus during a single passage in mice J.-H. Nam*, S.-M. Shim 1, E.-J. Song 1, E. Españo 1, S.-H. Jung 1, J.-K. Kim 1 1Department of Pharmacy, Korea University, College of Pharmacy, Sejong, Korea, Republic Of

Objectives: We isolated low-Pathogenic avian influenza (LPAI) H5N2 virus, A/Aquatic Bird/Korea/CN2/2009, which was a prevalent subtype in South Korea in 2009 and serially passaged in mice to evaluate its potential to become highly pathogenic Methods: Serial lung-to-lung passages of the LPAI H5N2 isolate from bird to mice were performed using lung homogenates from mice infected with the wild- type virus and we characterized the phylogenetic and molecular analyses of the A/AB/Kor/CN2/09 (H5N2) and its variants Results: Unexpectedly, the virus became highly pathogenic in mice after a single lung-to-lung passage, resulting in 100% lethality with a mean death time (MDT) of 6.1 days post-infection (DPI). Moreover, pathogenicity gradually increased after subsequent in vivo passages with MDTs of 5.2 and 4.2 DPI after the second and third passages, respectively. Our molecular analysis revealed that two amino acid changes in the polymerase complex (a glutamate-to-lysine substitution at position 627 of PB2 and a threonine-to-isoleucine substitution at position 97 of PA) were associated with the increased pathogenicity Conclusion: We were able to identify two amino acid substitutions located in the polymerase subunits that are responsible for the acquisition of mouse virulence in a common LPAI H5N2 isolate after a single passage. Studies investigating the combined effect of these mutations are needed to further understand the role they play in mammalian adaptation of avian influenza viruses. Finally, the alarmingly rapid ability of our H5N2 virus to adapt and cause disease in mice highlights the possibility that currently circulating avian influenza viruses can cross the species barrier

Keywords: H5N2, phylogenetic and molecular analyses, virus adaptation

17th International Congress of Virology 281

Innate Defense Against Viruses

17th International Congress of Virology 282

Abstract Submission

Innate defense against viruses

PO725

Antiviral immunity of emerging viruses host: studies of bat innate immunity

P. Zhou 1,*, Z.-L. Shi 1

1Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

Objectives: Bats have been identified as natural reservoir for a variety of viral diseases agents including Ebola and SARS- CoV. Interestingly, while these viruses cause diseases in human and other mammals, they appear to be harmless to bats. We then ask whether bats have special innate immunity. Interferon system serves as the first line defence against viruses. Our bat group aim to answer the question why bats appear to be special by characterizing bat interferon system.

Methods: Bat cells including professional immune cells were made for bat innate immunity studies. Next generation sequencing was also applied to sequence the type I IFN locus as well as differential analysis of gene expression upon stimulation compared to human. Furthermore, a direct comparison of the cellular responses between human and Rhinolophus Sinicus (reservior host of WIV1 CoV) upon WIV1 infection was performed.

Results: We found the contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats, which may explain the ability of bats to coexist with viruses. We also found evidences to support a likely dampened DNA sensing in bat. In addition, while WIV1 infection induced similar IFN responses in two cells, it also behaves differently in modulating host cell cycle responses.

Conclusion: These studies provided primary evidences why bats can long-term carry coronaviruses and other deadly viruses, which in turn help us to understand the mechanisms of how viral replications can be controlled. Future works on various other pathways of innate immune signaling, for example the single stranded RNA sensing interferon production pathway will be launched.

Keywords: bats, emerging viruses, innate immunity

17th International Congress of Virology 283

Abstract Submission

Innate defense against viruses

PO726

ANTIVIRAL MECHANISM OF ZAP AGAINST JAPANESE ENCEPHALITIS VIRUS

H.-P. Chiu 1,*, R.-J. Lin 2, Y.-L. Lin 3

1Department of Microbiology, National Taiwan University, 2National Institute of Infectious Diseases and Vaccinology, National Health Research Institute, 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Objectives: Zinc-finger antiviral protein (ZAP), also known as Poly(ADP-ribose) polymerase-13 (PARP13), is a host antiviral factor. Two isoforms of ZAP (ZAP-L and ZAP-S) expressing by alternative splicing can target specific viral RNA and recruit mRNA degradation machinery for their antiviral effect. ZAP-S also promotes RIG-I signaling, leading to amplification of the antiviral innate immune response. Up to now, no flaviviruses have been reported to be sensitive to human ZAP proteins. We study the antiviral potential of human ZAP-L and ZAP-S against flavivirus such as Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DENV-2).

Methods: A549 and 293T cells overexpressing ZAP-L and ZAP-S were used to study the antiviral mechanism against JEV and DENV-2. We also used wild type and stable ZAPs knockdown cells to investigate the physiological role of ZAP proteins.

Results: Overexpression of ZAP-L and ZAP-S restricted JEV especially at early stage of infection, but had no effect on DENV-2. The zinc-finger motifs of ZAPs were essential for the viral RNA binding and the ZAP-responsive element (ZRE) on JEV genome was mapped to multiple fragments. Knockdown of EXOSC5, a component of RNA exosome complex, but not XRN1, a 5’-3’ exoribonuclease, rescued JEV replication in cells with ZAPs overexpression, indicating that 3’-5’ RNA decay pathway was involved in the antiviral effect of ZAPs. Moreover, ZAP-L and ZAP-S enhanced IRF3 activation and the expression of downstream genes in JEV infected cells through RIG-I but not MDA5 signaling. Endogenous ZAP-L and ZAP- S levels were increased upon JEV infection, and knockdown of ZAPs enhanced the level of JEV RNA.

Conclusion: In summary, ZAPs can control JEV infection via targeting and degrading viral RNA and also by improving the RIG-I mediated immune response.

Keywords: Flavivirus, host defense, zinc-finger antiviral protein

17th International Congress of Virology 284

Abstract Submission

Innate defense against viruses

PO727

Effect of Apolipoprotein AI on Dengue Non Structural Protein 1(NS1) In Dengue Disease Pathogenesis

D. Jayathilaka*, L. Gomes 1, G. Ogg 2, N. Malavige 1

1Microbiology, Center for Dengue Research, Nugegoda, Sri Lanka, 2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Objectives: Apolipoprotein AI is the main lipoprotein component of HDL in plasma. Significantly decreased levels of HDL have been observed in severe forms of dengue infection compared to milder forms of the disease. Apolipoprotein AI (Apo AI) is known to increase the infectivity of dengue viral particles by facilitating entry of the virus into the cells through SR-BI receptor which is the major cell receptor for HDL. Since NS1 is known to bind to plasma lipids, we sought to investigate if Apo AI potentiates the actions of dengue NS1 by increasing production of inflammatory cytokines.

Methods: Primary monocytes of 5 healthy individuals were co-cultured with recombinant NS1 from human derived cell lines(1µg/ml), lipopolysaccharide (LPS) (500pg/ml) and two concentrations of Apo AI (65µg/ml and 1.68µg/ml) alone for 16 and 24 hours, in duplicates. IL-6 levels were assessed in culture supernatants by quantitative ELISA.

Results: Dengue NS1 alone (median 290.4 pg/ml) and both concentrations of Apo AI (median 317.4 and 274.8 pg/ml) did not result in an increase in IL-6 production, when compared to the untreated cells (median 206.4). Co-culture of NS1 and Apo-AI together was associated with production of high levels of IL-6 when compared to monocytes treated with NS1 alone (p=0.03). IL-6 production was higher at Apo AI concentrations of 1.68μg/ml (median=3160) when compared Apo AI concentrations of 65 μg/ml (median=198.2) at 16 hours.

Conclusion: Since lower Apo AI concentrations were associated with increased production of IL-6 when co-cultured with NS1, it is possible that Apo AI contributes to disease pathogenesis in part by potentiating the actions of dengue NS1 at specific molar ratios.

Keywords: Apolipoprotein AI, IL-6, NS1

17th International Congress of Virology 285

Abstract Submission

Innate defense against viruses

PO728

FAS-associated factor-1 positively regulates Type I interferon response to RNA virus infection by targeting NLRX1

J.-S. Lee*

Objectives: Type I interferon-mediated antiviral response is critical for controlling virus infections. However, interferon- mediated immune responses need to be tightly regulated to maintain host immune homeostasis. Recently, molecules involved in regulating interferon-mediated innate immune response are the subject of much research. FAF1 is a component of the death-inducing signaling complex in Fas-mediated apoptosis. FAF1 containing multi-ubiquitin related domains has been reported to potentially interact with a diverse proteins and function as negative and/or positive regulator in variety of cellular possesses. However, biological roles of FAF1 are still poorly understand in antiviral innate immunity.

Methods: Here we checked whether FAF1 modulate Type I interferon pathway as a positive regulator. FAF1 gt/gt mice, which are deficient in FAF1 and FAF1 knockdown immune or epithelial cells were tested with RNA viruses and shows levels of inflammatory cytokines and type I interferon production in mRNA level and protein level. We also confirmed the molecular mechanism of FAF1 with diverse molecular biological analysis.

Results: FAF1 gt/gt mice, which are deficient in FAF1 and FAF1 knockdown immune markedly enhances susceptibility to RNA virus infection and shows lower level of inflammatory cytokines and type I interferon production. As a molecular mechanism, FAF1 binds competitively to NLRX1, a negative regulator of type I IFN signaling by modulation of MAVS-RIG- I interaction, and prevents interaction between NLRX1 and MAVS.

Conclusion: Our findings here not only demonstrate that FAF1 is a positive regulator of antiviral signaling, but also provide novel insight in the underlying mechanisms by which this positive regulation occurs. These findings are novel and provide a major conceptual advance in the field of host antiviral immunity.

Keywords: FAF1, IFNs, NLRX1

17th International Congress of Virology 286

Abstract Submission

Innate defense against viruses

PO729

HDAC6 Regulates Viral RNA Sensing by Deacetylation of RIG-I

J.-S. Lee*

Objectives: RIG-I is a key cytosolic sensor that detects RNA viruses through its C-terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While post-translational modification has been demonstrated to regulate RIG-I signaling activity, its significance for the sensing of viral RNAs remains unclear. In this study, we have unveiled a novel mechanism that activates RIG-I and identified the protein deacetylase HDAC6 as a modulator of innate immunity specifically against RNA viral infection.

Methods: Here we checked whether HDAC6 modulate Type I interferon pathway as a positive regulator. Knockdown or knockout of endogenous HDAC6 in immune cells were tested with viruses and shows levels of inflammatory cytokines and type I interferon production in mRNA level and protein level. In HDAC6 knockout mice, morbidity of mice against lethal VSV challenge observed. We also confirmed the molecular mechanism of HDAC6 with diverse molecular biological analysis.

Results:

In this study, we first show that the RIG-I C-terminal region undergoes deacetylation to regulate its viral RNA sensing activity and that the HDAC6-mediated deacetylation of RIG-I is critical for viral RNA detection. HDAC6 transiently bound to RIG-I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG- I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild-type mice.

Conclusion: Consequently, our results indicate that deacetylation of RIG-I by HDAC6 is critical for regulating RNA virus sensing and innate immune responses. These findings are novel and provide a major conceptual advance in the field of host antiviral immunity. [This research was supported by the Ministry for Food, Agriculture, Forestry and Fisheries (Grant No. 315044031), Republic of Korea]

Keywords: Deacetylation, HDAC6, RIG-I

17th International Congress of Virology 287

Abstract Submission

Innate defense against viruses

PO730

HUMAN PARECHOVIRUSE TYPE 3 MODULATES HOST ANTIVIRAL INNATE IMMUNITY

T.-H. Chang 1,*, J.-T. Chang 2, M.-W. Jan 1, M.-H. Kung 1, B.-C. Chen 3, Y.-S. Chen 4

1Department of Medical Education and Research, 2Department of Pediatrics, 3Department of Microbiology, 4Department of Infectious Diseases, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Objectives: Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. HPeV infection has been a health threat to infants and young children. However, the pathogenic mechanism of HPeV remains unclear.

Methods: To understand the pathogenic mechanism of HPeVs, 3 new contemporary strains of HPeVs, HPeV1-KVP6, HPeV3-VGHKS 2007 (strain1) and HPeV3-VGHKS-2013 (strain 2), were isolated and propagated, and their full viral genome was sequenced (accession #KC769584, #KM986873, #KX068679). In addition, a specific antibody against HPeV VP1 protein was generated to detect HPeV infection. Because HPeV3 infection causes server neurological diseases in young patients, thus, the host antiviral activity was analyzed in human glioblastoma (HGB) cells with HPeVs infection.

Results: The immunoblotting showed that HPeV3 but not HPeV1 inhibited host antiviral signaling activation, such as IRF3, RIG-I, TBK1, IκBα and STAT1 in HGB cells. These effects subsequently led a fail-induction of type I interferon (IFN). Moreover, HPeV3 strain 2 displayed a higher level of infectivity and cytopathic effect than HPeV3 strain 1.

Conclusion: Our study revealed that HPeV3 might modulate the host antiviral activity to cause a pathogenic infection in HGB cells. And the level of inhibition might be various among different strain of HPeVs. The further investigation of different strain of HPeVs infection is required to describe the clinical and biological implications.

Keywords: Human Parechovirus, Innate immunity, Type I Interferon

17th International Congress of Virology 288

Abstract Submission

Innate defense against viruses

PO731

Rubicon modulates antiviral type I IFN signaling by targeting IRF3 dimerization

J.-S. Lee*

Objectives: Rubicon is a part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon TLR stimulation, and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon responses, remains largely unknown.

Methods: Here, we tested whether Rubicon acts as a negative regulator for virus-triggered IFN signaling. First. we check that knockdown or overexpression of Rubicon affects antiviral responses. For the virus infection experiments, GFP- expressing H1N1 influenza virus (PR8-GFP) or GFP-expressing vesicular stomatitis virus (VSV-GFP) and other ligands were used. And we confirmed phosphorylation of molecules (IRF3, p65, STAT1, p38, and TBK1) and transcription of IFN- related genes involved in signaling cascades triggered by virus infection. For the molecular mechanism of Rubicon, we performed the diverse molecular biological analysis.

Results: Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon has the opposite effect. Rubicon specifically interacts with the IRF association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response.

Conclusion: Thus, our findings suggest that the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral response, providing a novel cellular mechanism of IRF3 inhibition. The results of this study will increase our understanding of the role of negative feedback mechanisms that regulate type I IFN signaling and maintain immune homeostasis. [This research was supported by the National Research Foundation of Korea (Grant no. 2015020957)]

Keywords: Interferon response, IRF3 regulation, Rubicon

17th International Congress of Virology 289

Abstract Submission

Innate defense against viruses

PO732

VP3 of FMDV activates chicken immune responses through chicken Toll like receptor 2-2

Y.-T. Lin*, Y.-C. Chang, C.-L. Wang, S.-M. Liang

Objectives: Avian influenza virus (AIV) is the etiologic pathogen of bird flu which frequently causes high mortality in birds resulting in major financial losses in the poultry industry. The high mutation rates in AIVs may even cause pandemic influenza infection in humans. Since prevention of viral diseases particularly influenza is usually via vaccine inoculation, search for better adjuvant(s) to enhance host immune responses and facilitate vaccine development for protecting poultry from AIVs is urgently needed.

Methods: To search for better adjuvants for better AIV vaccine development, we undertook the following approaches in this study: (1) Cloned chicken Toll-like receptor 2 (chTLR2-2) and chTLR16 into expression vector and established a reporter assay with NF-kB promoter region for screening potential adjuvant(s). (2) Determined the ability of TLR agonists to induce cell mediated immune responses in chicken splenocytes by measuring mRNA expression levels of interferon-γ (IFN-γ) using qPCR. (3) Isolated chicken CD8+ cells and B cells from chicken splenocytes and dendritic cells from bone marrow (BMDCs) and analyzed the effects of TLR agonists on the production and mRNA expression of IFN-γ in these chicken immune cells by enzyme-linked immunosorbent assay (ELISA) and qPCR analysis respectively.

Results: We screened novel adjuvants by reporter assay and found that capsid protein VP3 of foot-and-mouth disease virus (FMDV) stimulates chTLR2-2 and chTLR16 to activate NF-kB signaling. VP3 also enhanced the mRNA expression of cytokines, including IFN-γ, interleukin-4 (IL-4), and IL-17 in chicken splenocytes. In addition, VP3 increased production and mRNA expression of IFN-γ in chicken CD8+ cells, B cells, and BMDCs.

Conclusion: Our results demonstrated that VP3 is a novel chTLR2-2/16 agonist and a potential adjuvant to potentiate chicken immunity for AIV vaccine development.

Keywords: Avian influenza virus (AIV), chTLR16, chTLR2-2

17th International Congress of Virology 290

Abstract Submission

Innate defense against viruses

PO871

QUANTITATIVE ANALYSES AND COMPARISON OF RHINOVIRUS VERSUS INFLUENZA INFECTIONS IN MULTIPLE- INDIVIDUALLY DERIVED HUMAN NASAL EPITHELIAL CELLS: A MODEL FOR UNDERSTANDING RHINOVIRUS IMMUNE ACTIVATION IN THE NASAL EPITHELIUM

H. H. Ong 1,*, K. S. Tan 1, Y. Yan 1, J. Liu 1, C. Li 1, H. W. Choi 2, D. Y. Wang 1, T. K. V. Chow 3

1Otolaryngology, Yong Loo Lin School of Medicine, 2Saw Swee Hock School of Public Health, 3Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Objectives: Human rhinoviruses (HRVs) are the commonest cause of the common cold which results in mild and self- limiting symptoms. While HRV infection causes less cytopathology compared to other respiratory virus infections, it is frequently associated with exacerbation of chronic pulmonary diseases such as asthma. Currently, there are limited in vitro models on HRV infection of upper airway epithelial cells compared to lower airway cells despite the nasal epithelium being the first contact site of the virus.

Methods: Hence, using the model of HRV infection of in vitro differentiated human nasal epithelial cells (hNECs), we systematically investigated the viral replication kinetics, host responses, and their association with HRV pathogenesis and disease progression.

Results: By infecting in vitro differentiated hNECs from multiple individuals using air-liquid interface culture, we found that individual-derived hNECs were susceptible to HRV infection, and that HRV mainly targeted ciliated cells of hNECs. HRV preferentially induced the TLR7/IRF7/IFN-β and IFN-λ1 pathways for antiviral responses in hNECs. Quantitative PCR analysis of chemokine genes suggested predominant signaling for recruitment of T-helper 1 (TH1) cells, and in the progression from innate to adaptive immunity, CXCL9, IP-10, CXCL11 and RANTES were identified as the likely initiators of immune responses in the lower airway following infection. Comparison of HRV infection versus influenza infection in the same model highlighted key differences that may be informative for HRV-specific pathogenesis. From this comparison, we further identified 11 genes that may be responsible in conferring the different pathogenic features between HRV and influenza.

Conclusion: Overall, this in vitro hNEC model of HRV infection provides a feasible platform for repeated, controlled studies of HRV infection in the nasal epithelium in multiple individuals, for elucidating novel insights into HRV replication dynamics and HRV-induced pathologies in the human nasal epithelium.

Keywords: Host Immune Responses, Human nasal epithelial model, rhinovirus

17th International Congress of Virology 291

Speaker Abstract Submission

Innate defense against viruses

PO873

NEGATIVE REGULATION FOR INNATE SENSOR-INDUCED INTERFERON RESPONSE

A. Takaoka 1,*, T. Yamada 1

1Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan

Please specify what speaker category are you in?: Keynote

Objectives: During viral infection, nucleic acid sensors are important to recognize virus-derived nucleic acids to trigger innate immune responses such as type I interferons (IFNs). However, it remains to be fully clarified how these innate sensor- mediated signalings are regulated in a constitutive manner. On the other hand, aryl hydrocarbon receptor (AHR) is a receptor for certain chemical compounds such as dioxins, but this receptor is activated by its endogenous ligands such as tryptophan metabolites including kynurenine (Kyn) under physiological conditions. In this study, we examined the role of endogenously activated AHR in innate sensor-triggered type I IFN response during viral infection to show a novel link between AHR signaling and innate signaling.

Methods: In this study, we first analyzed AHR-deficient mice to examine its role in type I IFN induction and antiviral response upon infection with various types of virus such as vesicular stomatitis virus, influenza virus, Newcastle disease virus, Sendai virus, encephalomyocarditis virus, and herpes simplex virus-1. In order to show the role of constitutively activated AHR by endogenous ligands, we next used an AHR-specific antagonist, CH-223191 and 680C91, a specific inhibitor of TDO that catalyzes the first step in the formation of Kyn from tryptophan. We further examined TIPARP knockdown mouse embryonic fibroblasts to show its involvement in the AHR-mediated negative regulation of type I IFN production upon viral infection. Enzymatic assay of ADP-ribosylation was conducted to prove that TIPARP post-translationally modifies TBK1. TIPARP- deficient mice treated with CH-223191 were also analyzed to show that IFN-mediated antiviral response is dependent on TIPARP expression through constitutive AHR signaling mediated by endogenous ligands.

Results: Our analyses with AHR-deficient cells showed that the AHR was a key regulator of type I IFN production activated by nucleic acid sensor-mediated signaling during infection with RNA and DNA viruses that were tested. We also showed that virally-induced IFN response was negatively regulated by constitutive AHR signaling mediated by endogenous ligands such as Kyn. Further investigation revealed that an ADP-ribosyltransferase TIPARP, one of the AHR-inducible gene products, is a critical mediator to suppress IFN response. We additionally found that TIPARP ADP-ribosylates TBK1, which is known as an essential kinase to activate IRF-3 for the induction of type I IFN genes, and inhibits its activity. Finally, we showed the indispensable role of TIPARP in vivo in the negative regulation mediated by constitutive AHR signaling.

Conclusion: In this study, we found a novel physiological role of AHR-mediated signaling in antiviral IFN response. Constitutive activation of the AHR signaling by endogenous ligands such as tryptophan metabolites has a restrictive effect on type I IFN-dependent host antiviral defense system. In this presentation, we will show our recently obtained data showing that modification of AHR signaling would possibly serve as a promising approach for the control of viral infection in liver tissues, where constitutive AHR signaling appears to be relatively upregulated. In addition, the present results also may partially explain the previously reported, inhibitory effects of AHR signaling by xenobiotic AHR ligands such as dioxins on host defense against viral infection. We would like to also show some supporting evidence regarding the effect of xenobiotic AHR ligands on IFN response.

Keywords: innate immune response, interferon response, Virus-host interaction

17th International Congress of Virology 292

Abstract Submission

Innate defense against viruses

PO878

Dengue virus infection and limited immune response in pteropus bats

A. T. Irving*, K. Pui San 1, K. Luko 1, L. Wang 1

1Emerging Infectious Diseases, Duke-NUS Medical School, singapore, Singapore

Objectives: Natural reservoir hosts can maintain low-level infection of pathogens without succumbing to severe disease. Multiple bat species are now known to host a plethora of pathogenic viruses without significant affect. Here we detail infection of Pteropus bat primary cells, immune cells and various cell lines with Dengue Virus.

Methods: Dengue virus antibodies and RNA has been found in wild-caught bats previously yet their ability to sustain dengue infection is questionable. We characterise viral entry, replication by qPCR/IF, egress by titration of supernatant and activation of immune response through cell assays, western blot, NGS and proteomics.

Results: We show infection in vitro and show the ability to infect, producing high titres of virus with only limited cellular responses. In addition, at low MOI’s (0.1-1) there is no noticeable induction of the interferon (IFN) response. Once increased to higher MOI’s (2-10) a typical IFN induction can be observed leading to a decrease in viral yields, with some strain/serotype variability.

Conclusion: The limited response to infection is of concern as immune response is often used as a measure of infection. The ability of multiple cell types to support Dengue virus replication and production raises the possibility of bats as an intermediate host in the life cycle of Dengue and other similar flaviviruses. The need for further investigation with in vivo models and the role bats may play could be critical in understanding the true dynamics of flaviviruses in various animal hosts.

Keywords: bat, dengue, innate immue response

17th International Congress of Virology 293

Abstract Submission

Innate defense against viruses

PO883

Virology

Evasion of innate immunity by mosquito-borne flaviviruses. Z. He 1,*, Y. Hu 2, J. Chen 2,*, S. An 2, X. Dong 2, J. Wu 2, X. Zhu 2, M. Li 2 1School of Public Health, 2Department of Microbiology, Zhongshan School of Medicine, SUN YAT-SEN UNIVERSITY, Guangzhou, China

Please specify what speaker category are you in?: Workshop speaker Objectives: Mosquito-borne flavivirus belongs to the family Flaviviridae. It includes dengue virus (DENV), Zika virus (ZIKV), and several other viruses which lead to extensive morbidity and mortality in humans, and are currently serious global health challenges. To establish infection and replication in its host cells, these flaviviruses must subvert the host innate immunity, particularly the production and/or antiviral effects of interferons (IFNs). The aim of this study is to understand the mechanisms by which DENV and ZIKV suppress the innate antiviral responses. Methods: Here we report that employment of the mammalian two-hybrid system to identify molecular targets of DENV or ZIKV proteins in RIG-I-like receptors (RLRs) signaling. Immunofluorescence assay was used to determine the co-localization of viral proteins and host proteins. Co-immunoprecipitation assay and Biacore analysis were exploited to investigate the interaction between viral proteins and host proteins. IFN-β luciferase reporter assay was performed to measure whether viral proteins could repress the activation of IFN-β promoter. The protein and mRNA levels of interferon were measured by ELISA and real-time PCR respectively. Western blotting analysis and real-time PCR was employed to demonstrate the inhibition effect of IFNs signaling by viral proteins. Results: We found that DENV NS4A protein co-localized with MAVS in the mitochondria-associated endoplasmic reticulum membranes (MAM). In addition, DENV NS4A plays an important role in suppressing interferon production through binding MAVS and disrupting the RIG-I-MAVS interaction. Moreover, we found that ZIKV strongly blocks both type I and III IFNs-induced signal transduction cascade by interfering with STAT1 function. Further study demonstrated that expression of NS2A or NS2B results in inhibition of type I and III IFNs signaling by repression of the STAT1 phosphorylation. Conclusion: Our study reveals a novel antagonistic system employing flaviviruses nonstructural proteins in restricting the innate antiviral responses.

Keywords: Flavivirus , Innate immunity, Interferon response

17th International Congress of Virology 294

Insect Viruses

17th International Congress of Virology 295

Abstract Submission

Insect viruses PO733

MONOCLONAL ANTIBODIES TO AUSTRALIAN MESONIVIRUSES ALLOW ANTIGENIC AND FUNCTIONAL ANALYSIS OF THE STRUCTURAL PROTEINS.

N. Newton 1,*, A. Colmant 1, C. O'Brien 1, D. Watterson 1, H. Bielefeldt-Ohmann 1, R. Hall 1, J. Hobson-Peters 1 1The School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Australia

Objectives: The Mesoniviridae (order Nidovirales) is a new family of insect-specific, RNA virus isolated from mosquitoes. Current species demarcation of the family is primarily based on bioinformatic analysis with further ecological, antigenic and structural information still required. This study reports structural and antigenic analysis of two Australian mesoniviruses, Casuarina virus (CASV) and Nam Dinh virus (NDiV), which represent the Alphamesonivirus-4 and Alphamesonivirus-1 species, respectively.

Methods: A panel of monoclonal antibodies (mAbs) generated to CASV, a new species of mesonivirus discovered in Darwin, Australia, were characterised using a combination of ELISA, western blot, immunoprecipitation, IFA and microneutralisation assays. Virion morphology was also compared between CASV and NDiV by transmission-electron microscopy (TEM).

Results: Epitopes of all three structural proteins (spike, membrane & nucleocapsid) as well as mAbs specific for spike subunits (S1 & S2) were identified during mAb characterisation. Three mAbs specific for the membrane protein were also cross-reactive to the Australian isolates of NDiV, a mesonivirus originally isolated in Vietnam. The importance of the spike protein in cell attachment and membrane fusion, as reported for other nidoviruses, was shown by the ability of anti-spike mAbs to neutralise CASV in vitro. No significant differences were observed for either the virion size or spike protein size between CASV and NDiV. However, a notably higher frequency of spike protein was seen on NDiV virions (>5 per virion) compared to CASV (<5 per virion).

Conclusion: Our panel of mesonivirus-reactive mAbs have proved to be valuable tools to elucidate the antigenic structure and function of mesonivirus proteins and provided useful data to consolidate the taxonomy of this new virus family. These mAbs have also enabled us to confirm significant antigenic differences between different mesoniviruses, consistent with the demarcation of these virus species.

Keywords: Insect-specific virus, Mesonivirus, Monoclonal antibodies

17th International Congress of Virology 296

Abstract Submission

Insect viruses PO734

PROTEIN-PROTEIN INTERACTIONS OF ME53 FROM AUTOGRAPHA CALIFORNICA NUCLEOPOLYHEDROVIRUS (ACMNPV)

E. Ozsahin 1, Y. Liu 1, P. Krell* 1Molecular and Cellular Biology, University of Guelph, Guelph, Canada

Objectives: We sought to identify the viral and host cell partner proteins which bind to AcMNPV ME53 in order to better understand the molecular mechanism of ME53 intracellular translocation and its function.

Background: Autographa californica nucleopolyhedrovirus is the type species of alphabaculoviruses that infect Lepidopteron insects and which are used as a biological pesticide and eukaryotic protein expression vector. Baculoviruses also have potential for use as a gene therapy vector. AcMNPV - me53 is a conserved gene between alpha and beta baculoviruses. ME53 is a structural protein of the nucleocapsid, but not the envelope, of both budded viruses and occlusion derived viruses. The me53 gene is transcribed both early and late post-infection. Although it is not essential for DNA replication, infectious budded virus production of me53 knockout virus dropped 10,000 fold compared to wild type virus. ME53 localizes in the cytoplasm early post infection and at late time post infection localizes with GP64 to the infected cell membrane and, through a nuclear translocation sequence, to the nucleus via virus and/or host-related proteins.

Methods: Two approaches were used to identify ME53 protein binding partners, one using the yeast two hybrid system for a broader search, and immunoprecipitation assays to confirm binding with select viral proteins. For the yeast two hybrid system, we constructed ME53-expressing bait plasmid for mating to prey cDNA libraries from AcMNPV-infected or uninfected Sf9 cells. Reciprocal yeast two hybrid was then done using ME53 as prey and the putative binding partners as bait. For immunoprecipitation assays HA-tagged ME53, expressed in recombinant AcMNPV bacmid-transfected cells was immunoprecipitated with anti-HA and binding proteins were detected through silver staining and identified by Western immunoblots using protein-specific antibodies.

Results: Through the yeast two hybrid system, cellular receptor for activated protein kinase C (RACK) was determined as a ME53 protein interaction partner from a cDNA library constructed from uninfected cells. Two AcMNPV proteins, VP80 and p6.9, were determined as ME53 protein interaction partners from a cDNA library constructed from AcMNPV-infected cells. These three proteins (RACK, VP80 and p6.9) and several targeted AcMNPV ORFs including Exon0, VP39, GP64 and parts of ME53, were cloned as reciprocal baits and preys and tested for protein-protein interaction. Immunoprecipitation assays for ME53:HA showed 6 silver-stained bands, one at about 50 kDa representing ME53:HA. By Western immunoblots, bands representing Exon0, VP39 and GP64 were also noted suggesting interactions of ME53 with these specific viral proteins. Conclusion: One host protein, RACK, and two viral proteins VP80 (a nucleocapsid protein which interacts with nuclear actin) and p6.9 (a major basic DNA-binding protein in the capsid) were identified by yeast two hybrid assays as putative ME53 binding partners. Immunoprecipitation assays suggested the interaction of ME53 with Exon 0, VP39 and GP64

Keywords: baculovirus, ME53-protein interaction, yeast two hybrid

17th International Congress of Virology 297

Abstract Submission

Insect Viruses

PO830

BINJARI VIRUS: A NEW INSECT- SPECIFIC FLAVIVIRUS ISOLATED FROM AEDES NORMANENSIS MOSQUITOES IN NORTHERN AUSTRALIA

J. Harrison 1,*, N. Newton 1, D. Warrilow 2, T. Piyasena 1, C. O'Brien 1, S. Davis 3, R. Hall 1, J. Hobson-Peters 1

1The University of Queensland, 2Public Health Virology Laboratory, Queensland Health, Brisbane, 3Department of Primary Industries and Fisheries, Berrimah Veterinary Laboratory, Dawin, Australia

Objectives: Mosquito-borne flaviviruses are responsible for significant diseases, such as dengue, Japanese encephalitis, West Nile, and Zika. However, a subgroup of flaviviruses known as insect-specific flavivirus (ISFs) are only found in mosquitoes and do not infect or replicate in vertebrates. ISFs are of particular interest due to their intriguing evolutionary origins and growing evidence that they can suppress the transmission of some flavivirus pathogens. In this project, we isolated and characterised a new species of ISF from mosquitoes in northern Australia.

Methods: The biodiversity of insect-specific flaviviruses in northern Australia was investigated using a novel broad- spectrum, sequence-independent virus screening system, based on the detection of viral double-stranded RNA intermediates in inoculated mosquito cell cultures, and a pan-flavivirus RT-PCR. New flaviviruses were further characterised by deep sequencing of the viral genomes and phylogenetic analyses, infection studies in a range of vertebrate and mosquito cell lines and antigenic profiling using an extensive panel of flavivirus-reactive monoclonal antibodies.

Results: A novel flavivirus, tentatively named Binjari virus (BinJV), was detected and isolated from a pool of Aedes normanensis mosquitoes collected 300 kms from Darwin in the Northern Territory of Australia. Analysis of the full-length genome sequence of the new virus revealed it was phylogenetically distinct from all other flaviviruses reported to date, but was most closely related to the dual-host affiliated ISFs, Chaoyang and Lammi viruses. Monoclonal antibody binding profiles further revealed that BinJV was antigenically quite distinct and lacked a well characterised epitope that is conserved in the fusion domain of the envelope protein of all vertebrate-infecting flaviviruses and dual-host affiliated ISFs tested to date.

Conclusion: The findings from our genetic and antigenic analyses of BinJV suggest it is a new flavivirus species and represents the first dual-host affiliated ISF detected in Australia. Confirmation of its inability to infect vertebrate cells using additional vertebrate cells lines of mammalian, avian and reptilian origin and elucidation of the mechanism(s) of restriction will provide valuable clues to the evolutionary origin of this interesting virus.

Keywords: Aedes normanensis, Insect-specific flavivirus, MAVRIC

17th International Congress of Virology 298

Negative Strand RNA Viruses

17th International Congress of Virology 299

Abstract Submission

Negative strand RNA Viruses

PO677

MULTIPLEX PCR-BASED NEXT-GENERATION SEQUENCING EXHIBITS THE MOST LIKELY INFECTION SITE AND GENETIC EXCHANGES OF HANTAAN VIRUS FROM HEMORRHAGIC FEVER WITH RENAL SYNDROME (HFRS) PATIENTS

W.-K. Kim 1,*, J.-A. Kim 1, D. H. Song 2, D. Lee 2, Y. C. Kim 3, M. Wiley 4, G. Palacios 4, H.-C. Kim 5, T. A. Klein 6, J.-W. Song 1

1Microbiology, Korea University School of Medicine, Seoul, 25th R&D Institute, Agency for Defense Development, Daejeon, 3The Armed Forces Medical Center, Seongnam, Korea, Republic Of, 4The Center for Genome Science, U.S. Army Medical Research Institute of Infectious Disease, Fort Detrick, 55th Medical Detachment, 65th Medical Brigade, APO AP 96205- 5247, 6MEDDAC-Korea, 65th Medical Brigade, APO AP 96205, United States

Objectives: Outbreaks of zoonotic RNA viruses pose a worldwide critical public health threat due to extensive rates of mutation and transmission in the lack of effective antivirals. Next-generation sequencing (NGS) is a robust strategy to delineate genomic sequences and characteristics of the emerging viruses, allowing the detection and tracking of the outbreak of viral infections. Endemic infection of hantaviruses from rodents to humans causes hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in Americas, with annually 200,000 clinical cases and fatality rates ranging from 1 to 35%. In combination with whole genome sequences of Hantaan virus (HTNV) from HFRS patients and natural rodent hosts (Apodemus agrarius) in highly endemic areas, Republic of Korea (ROK), phylogeographic analysis will provide significant insights for the surveillance and disease risk reduction of HFRS incidents. However, it is an obstacle to obtain the genomic sequence of viruses from clinical specimens due to the low copy of viral RNA. To enrich viral genomic sequences in HFRS patient specimens, Multiplex PCR-based NGS was developed in this study.

Methods: Sera and lung tissues from HFRS patients (ROKA13-8, ROKA14-11, US8A14-2, and US8A15-1) and HTNV- positive A. agrarius from endemic areas were collected, respectively. Multiplex PCR-based NGS applied to the genomic sequencing of HTNV from the patient specimens, while conventional RT-PCR was performed to gather whole genome sequences of HTNV from A. agrarius. Using Maximum Likelihood (ML) method, the acquired genomes were phylogenetically analyzed to describe the epidemiological links between HFRS cases and natural sources. Recombination and reassortment analyses were performed using Recombination Detection Program 4.

Results: Multiplex PCR-based NGS applied to genomic sequencing of HTNV RNA covered by 85.9-97.2%, 98.2-100%, and 100% for L, M, and S segments, respectively. Phylogenetic analyses of the HTNV tripartite genomes recovered from HFRS patients demonstrated geographic clustering of the strains with the HTNV circulating in rodents on each of their exercising location, suggesting the most likely infection site. A HFRS Patient, ROKA13-8, seemed to be infected with HTNV in Cheorwon, a military training site. ROKA14-11 was most likely infected with HTNV in Paju, a highly HFRS endemic area. US8A14-2 and US8A15-1 developed HFRS most likely due to the infection with HTNV at Dagmar North (Paju) and Multi- Purpose Range Complex (Pocheon), respectively. The genomic characteristics of HTNV from the clinical specimens suggested that a genome organization of US8A15-1 was compatible with recombination of HTNV S segment. Also, the configuration of L segment reassortment was observed from US8A14-2 strain identified in a HFRS patient.

Conclusion: In conclusion, multiplex PCR-based NGS is a potent and novel tool to recover the low copy of HTNV RNA from HFRS specimens. The most likely infection site was suggested in phylogeographic analyses using HTNV strains from patients and natural hosts. The genomic sequences of HTNV tripartite RNA revealed the natural occurrence of genetic exchanges, recombination and reassortment. The recombinant and reassortant were found to enable their transmission to humans, followed by the development of HFRS. Thus, this study will provide not only a robust tool to define viral genomic

17th International Congress of Virology 300

sequences from clinical specimens but also better understandings for the genetic diversity of HTNV and strategies for the disease risk assessment and mitigation against hantavirus outbreaks.

Keywords: HFRS patients, Multiplex PCR-based Next-generation sequencing, Phylogeographic analysis

17th International Congress of Virology 301

Abstract Submission

Negative strand RNA Viruses

PO684

EFFICACY OF EBOLA SPECIFIC EQUINE POLYCLONAL ANTIBODY BASED PRODUCT- EIG AGAINST LETHAL EBOLA VIRUS INFECTION IN ANIMAL MODELS

D. Kobasa*

Objectives: Filoviruses are emerging zoonotic pathogens representing a significant public health concern with high outbreak potential as well as a potential bioterrorism-related threat. To develop a therapeutic capacity that may be rapidly developed and deployed, we have assessed the efficacy of an equine-derived, despeciated Ebola polyclonal antibody (EIG) for the treatment of Ebola virus disease, developed by Emergent BioSolutions, Integrated BioTherapeutics, Inc, and Auburn University. The efficacy of this polyclonal therapeutic against a broad range of Ebola virus strains and protective capacity for lethal disease in rodent models of infection was examined.

Methods: Equine polyclonal antibodies against Zaire ebolavirus (EBOV) were generated in horses immunized with a Virus- Like Particle (VLP) vaccine and boosted with GPΔmuc (EBOV glycoprotein with deleted mucin-like domain) to enhance the antibody response. Plasma was collected by plasmapheresis and manufactured using a commercially validated process. The despeciated (proteolytically digested to remove Fc) F(ab′)2/Fab antibody fragment product, termed EIG, was then evaluated in the mouse and guinea pig models of EBOV infection. Animals were infected with 1000 infectious units of the species specific adapted EBOV (strain Mayinga) followed by treatment with EIG.

Results: Mice were challenged with 1000 PFU of mouse-adapted EBOV (Mayinga strain) and treated with EBOV EIG at 150mg/kg starting on day 0 or day 2 post-challenge up to day 4, resulting in 100% and 60% survival, respectively. Guinea pigs were challenged with 1000 PFU of guinea pig-adapted EBOV (Mayinga strain) and were treated twice daily for 6 days starting 24 hours after challenge with a dose range (20-100 mg/kg) of EBOV EIG that resulted in survival ranging from 83 to 100%. EIG was also assessed for neutralization activity against related filoviruses including EBOV strains Mayinga, Kikwit and Makona, and the species Bundibugyo and Taï Forest ebolaviruses. The results demonstrated a comparable neutralization activity (range1: 512- 1: 896) of EIG against all tested strains suggesting the potential for cross-protection with polyclonal antibody therapeutic.

Conclusion: EIG is a highly effective polyclonal antibody product for the treatment of Ebola virus disease in animal models and the outcome warrants further evaluation in the nonhuman primate model of disease. Furthermore, this study supports the utility of the equine antibody platform for the rapid production of a therapeutic product in the event of an outbreak by a zoonotic pathogen.

Keywords: Antibody, Ebola virus, treatment

17th International Congress of Virology 302

Abstract Submission

Negative strand RNA Viruses

PO735

ADAPTED LETHALITY: WHAT WE CAN LEARN FROM GUINEA-PIG ADAPTED EBOLA VIRUS INFECTION MODEL

A. Chepurnov*, S. Cheresiz, E. Semenova

Objectives: Establishment of small animal models of Ebola virus (EBOV) infection is important both for the study of genetic determinants involved in the complex pathology of EBOV disease, as well as for the preliminary screening of antivirals, production of therapeutic heterologic immunoglobulins, and experimental vaccine development.

Methods: Serial passage of different viruses in animals or cell cultures can result in the selection of mutants with different reproduction characteristics and either higher or lower virulence, as compared to the original wild type virus. Adult guinea pigs inoculated with the wild-type EBOV isolated from primates typically develop a non-lethal mild febrile or even asymptomatic disease. However, it has been previously shown that young guinea pigs may occasionally develop a lethal EBOV infection, and the serial passage of EBOV causes a substantial increase in lethality in these animals. Here, we report the establishment of four independent adaptation series in guinea pigs using the original stocks of EBOV subtype Zaire, strain Mayinga differing in their passage history.

Results: Here, we provide an overview of several our adaptation series in guinea pigs, which resulted in the establishment of guinea pig-adapted EBOV (GPA-EBOV) variants different in their characteristics, while uniformly lethal for the infected animals, and compare the virologic, genetic, pathomorphologic, and immunologic findings with those obtained in the adaptation experiments of the other research groups.

Conclusion: The mutations in EBOV VP24 protein to be the most important determinants of the virus virulence and lethality. The differences in the dynamics of hemolytic complement activity found in the infections of guinea pigs by the wild-type and adapted EBOV strains allowed us to be able to predict the outcome of EBOV infection in the first hours p/i.

Also, our studies indicate at a strong supression of neutrophilic phagocytosis in the infections of guinea pigs with GPA- EBOV strain, while only its mild activation on day 5 of wild-type EBOV infection was observed. This suggested that the reproduction of EBOV in macrophages, on the one side, with a concomitant supression of neutrophils, on the other, results in the disturbance of neutrophil/macrophage interaction as a driving force of a lethal EBOV disease.

Another interesting discovery was the presence of young thrombocytes in GPA-EBOV-infected animals. The phenomenon, which was lacking in the wild-type EBOV-infected animals, together with the increase of megacaryocyte counts in the bone marrow, can be the evidence of the activation of megacaryocytic hematopoesis and the exit of non-mature platelets into the circulation.

Previously, the elevated levels of proinflammatory cytokines have been reported to occur in the course of EBOV disease and contribute to the infection lethality in humans and NHPs, due to its role in the development of EBOV coagulopathies. However, in guinea pigs we, paradoxically, found higher (while comparable) TNF-a levels in the animals infected with a non-lethal wild-type, than in those infected with lethal adapted EBOV, thus, demonstrating lack of TNF involvement in GPA- EBOV pathogenesis.

Keywords: Ebola, animal model

17th International Congress of Virology 303

Abstract Submission

Negative strand RNA Viruses

PO736

ANALYSIS OF ENTRY MECHANISMS OF BAS-CONGO VIRUS USING A PSEUDOTYPE VSV

E.-S. Park 1,*, H. Tani 2, Y. Kaku 1, M. Suzuki 1, M. Kimura 1, K. Imaoka 1, S. Morikawa 1

1Department of veterinary science, National Institute of Infectious Diseases, Tokyo, 2Department of Virology, University of Toyama, Toyama, Japan

Objectives: A novel rhabdovirus, Bas-Congo virus (BASV), was identified from viral hemorrhagic fever patients in Bas- Congo province of Democratic Republic of the Congo in 2009. Two out of 3 patients in the outbreak died. BASV was not isolated but 98.2% of the genome sequence was determined. To develop antiviral strategies to BASV infection, it is important to understand virus-host cell interactions, especially focused on BASV cell entry into the host cells. In the present study, a pseudotype vesicular stomatitis virus (VSV) bearing BASV-G protein was employed to analyze the entry of the BASV into the host cells.

Methods: Pseudotype VSV bearing BASV-G protein (BASVpv) was constructed using BASV-G expression plasmid (pCAG- BASV-G) and either VSV-∆G-GFP or VSV-∆G- Luc. Infectivity of BASpv on a variety of mammalian cells were compared. To examine the pH-dependent endocytosis of the BASV, the effect of lysosomotropic agents, ammonium chloride and bafilomycin A1 on BASVpv entry was examined. To examine the involvement of cholesterol transporter protein (Niemann- Pick C1; NPC1) in BASVpv infection, the effect of cholesterol transport inhibitor, imipramine and U18666A, was also examined.

Results: A majority of cell lines so far tested were susceptible to BASVpv infection. The infection of BASVpv to Huh7 cells was inhibited by anti-BASV-G rabbit serum in a dose-dependent manner. Cell fusion upon transfection of pCAG-BASV-G was initiated by a transient treatment of the cells with a low pH buffer. BASVpv infection was inhibited by ammonium chloride, bafilomycin A1, imipramine and U18666A, respectively, in a dose-dependent manner.

Conclusion: BASVpv infected various mammalian cells and its infection was mediated by BASV-G protein. As for the other rhabdoviruses, low-pH-induced membrane fusion in the endosome by BASV-G is crucial for the viral entry to the cells. In addition, the cholesterol transporter protein is thought to be necessary for the entry of BASV. The result is considered to be valuable for the development of antiviral strategies against BASV.

Keywords: Bas-Congo virus, Pseudotype VSV, Viral entry

17th International Congress of Virology 304

Abstract Submission

Negative strand RNA Viruses

PO737

Molecular epidemiology of arenavirsues in South African small mammals

N. Ithete 1,*, L. Richards 2, A. Halajian 3, S. Matthee 4, M. C. Schoeman 5, W. Preiser 1

1Medical Virology, Pathology, Stellenbosch University, Cape Town, 2Durban Natural Science Museum, Durban, 3Department of Biodiversity, University of Limpopo, Sovenga, 4Conservation Ecology, Stellenbosch University, Stellenbosch, 5School of Life Sciences, University of KwaZulu-Natal, Durban, South Africa

Objectives: Arenaviruses are hosted by Murid rodents in the Murinae and Sigmondontinae subfamilies. They are divided into two serocomplexes; Tacaribe containing viruses hosted by New World rodents, and the Lassa-Lymphocytic choriomeningitis complex containing various arenaviruses hosted by African rodents, the ubiquitous LCMV and Dandenong virus from Australia. While non-pathogenic in rodents, some arenaviruses are highly pathogenic for human beings: In Africa, these are Lassa fever virus hosted by Mastomys natalensis and endemic to West Africa and Lujo virus with an unknown host that emerged in Zambia and caused a nosocomial outbreak in South Africa. THe aim of this study was to identify and characterise novel arenaviruses in South African small mammal species

Methods: South African rodents were screened for arenaviral RNA sequences. Small mammals representing 16 different genera were trapped at various locations in 8 provinces in South Africa between 2008 and 2016. RNA extracted from lung and kidney tissue was tested using broadly reactive RT-PCR assays targeting the L gene. The positive samples were further amplified using PCR assays targeting the GPC and NP genes, which were then used for phylogenetic analysis.

Results: novel arenavirus sequences were detected in four different rodent species indigenous to Southern Africa. Phylogenetic analysis showed that these arenaviruses belong to different clades; 1 clade rooting the Lassa-Mopeia clade and the other forming a monophyletic clade with Merino Walk virus, recently described from Myotomys unisulcatus from the Eastern Cape.

Conclusion: A surprisingly high diversity of arenaviruses was detected in this study and further characterisation efforts are ongoing. The identification of arenaviruses by our group and others in Southern Africa suggests that they are more widely distributed than previously thought. The distribution and pathogenicity (or not) for humans is yet to be defined.

Keywords: arenavirus, emerging viruses, rodent-borne

17th International Congress of Virology 305

Late breaking abstract

Negative strand RNA Viruses

PO738

Protection against all pathogenic ebolaviruses by targeting two conserved sites of vulnerability in the glycoprotein

K. Howell 1, X. Zhao 2, J. Brannan 3, S. He 4, A. Wec 5, E. Davidson 6, H. Turner 7, B. Doranz 6, A. Ward 7, X. Qiu 4, K. Chandran 5, J. Dye 3, Y. Li 2, M. J. Aman*

1INTEGRATED BIOTHERAPEUTICS, 2University of Maryland, Rockville, 3US Army Medical Research Institute of Infectious Diseases, Frederick, United States, 4PHA Canada, Winnipeg, Canada, 5Albert Einstein College of Medicine, New York, 6Integral Molecular, Philadelphia, 7The Scripps Research Institute, San Diego, United States

Objectives: Filoviruses including the Ebolavirus and Marburgvirus are among the deadliest human viruses. The 2014 West Africa outbreak of Ebola virus Disease (EVD) led to over 28000 cases and 11,000 deaths, highlighting the need for effective vaccines and therapeutics for these viruses. While ZMappTM, a cocktail of three monoclonal antibodies (mAb), is highly effective against the Zaire Ebola virus it does not protect against other pathogenic ebolavirus species such as Sudan (SUDV) and Bundibugyo (BDBV) viruses. The objective of this study was to develop broadly protective antibodies against all pathogenic ebolaviruses.

Methods: We immunized macaques with a cocktail of filovirus glycoproteins engineered to drive cross reactive antibody responses as immunogen. Memory B cells were isolated and IgG positive cells cross reactive to multiple ebolavirus glycoproteins were identified and sorted by flow cytometry. Antibodies were identified by single cell PCR amplification and sequencing of the heavy and light chain variable domains. Identified antibodies were expressed in mammalian cells as macaque-human chimeric antibodies and used for in vitro charcterization and efficacy studies in mice guinea pigs and ferrets.

Results: We isolated several antibodies with broadly neutralizing activity towards EBOV, SUDV, and BDBV. Using alanine scanning mutagenesis and single molecule electron microscopy we demonstrate that one of these mAbs, CA45, binds to a novel, shared epitope within the glycoprotein (GP) internal fusion loop (IFL) and another mAb (FVM04) binds to the receptor binding site. CA45 provided significant post-exposure protection against EBOV in guinea pigs and mice and fully protected against SUDV exposure in guinea pigs. FVM04 alone fully protected against EBOV and SUDV in mice. In guinea pigs FVM04 was fully protective against SUDV and partially protective against EBOV. A combination of CA45 and FVM04 showed superior activity with 100% protective efficacy against EBOV and SUDV in mice and guinea pigs and against BDBV in ferret when delivered post exposure at the peak of viremia. Furthermore, we identified a remodeled EBOV glycoproteins that binds with higher affinity to broadly neutralizing antibodies representing a potential pan-ebolavirus vaccine.

Conlusion: These data provide the first report of a cocktail of monoclonal antibodies effective against all three pathogenic ebolaviruses and provide a pathway for development of highly effective pan-ebolavirus immunotherapeutics as well as broadly protective vaccines.

Keywords: None

17th International Congress of Virology 306

Abstract Submission

Negative strand RNA Viruses

PO739

STUDIES ON THE INTRACELLULAR TRAFFICKING OF SFTSV STRUCTURAL PROTEINS

T. Lundu 1,*, K. Shimizu 2, Y. Tsuda 2, S. Kobayashi 1, K. Yoshii 1, K. Yoshimatsu 2, J. Arikawa 2, H. Kariwa 1

1Laboratory of Public Health, Graduate School of Veterinary Medicine, 2Department of Microbiology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan

Objectives: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged phlebovirus, member of the Bunyaviridae family that was identified in China, Japan and South Korea. SFTSV causes a severe systemic infection in humans characterised by fever, gastrointestinal disturbances, thrombocytopenia, multiple organ failure and a case fatality rate of 2-15%. The SFTSV genome consists of three negative-sense RNA segments. The proteins encoded by the large, medium, and small segments are the RNA dependent RNA polymerase (L), glycoproteins (GP) and the nucleocapsid protein (NP) and Nonstructural protein (NSs), respectively. Development of therapeutics against SFTSV is hindered by limited knowledge on the viral replication cycle. Bunyaviruses replicate in the cytoplasm; after the translation of viral proteins, virions assemble at the Golgi. However, the exact mechanisms through which the viral proteins and encapsidated genome of SFTSV are recruited to the Golgi apparatus are still unknown. This work focuses on describing the subcellular distribution of SFTSV structural proteins during viral replication.

Methods: We utilised a pCAGGs plasmid expression system for SFTSV proteins to show that the SFTSV GP, NP and L, employ different mechanisms to traffic through the endoplasmic reticulum (ER), the ER Golgi intermediate compartment (ERGIC) and the Golgi. Vero E6 cells, either infected with SFTSV or transfected with plasmids containing SFTSV proteins were examined by immunofluorescence microscopy.

Results: In SFTSV infected Vero E6 cells, GP localised to the ER, ERGIC and Golgi. NP was detected in the ERGIC and the Golgi whereas L had a diffuse cytoplasmic distribution and showed colocalisation with the ER, ERGIC and Golgi markers. When expressed in the absence of other SFTSV proteins, GP had the same distribution as in infected cells. Differences were observed in NP and L distribution. The L protein and NP did not colocalise with the markers of the ER, ERGIC or Golgi. Co-expression of NP with GP, L or NSs did not affect the localisation of NP. Conversely, L localised to the ERGIC and Golgi when expressed with GP.

Conclusion: These results suggest that the localisation of NP to the ERGIC and Golgi shows a requirement for interaction with other viral factors as well as GP. In this study, we describe for the first time the critical associations among the structural proteins as well as the steps that lead to the assembly and maturation of SFTSV.

Keywords: Golgi, Localisation, SFTSV

17th International Congress of Virology 307

Paramyxoviruses

17th International Congress of Virology 308

Abstract Submission

Paramyxoviruses PO741

ANALYSIS OF TRANSCRIPTIONAL REGULATORY NETWORK TRIGGERED BY MORBILLIVIRUS INFECTION.

H. Sato 1,*, M. Yoneda 1, T. Nakamura 1, F. Ikeda, J. Kawai 2, Y. Hayashizaki 2, C. Kai 1 1Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 2Omics Science Center, RIKEN , Kanagawa, Japan

Objectives: Morbillivirus infection causes severe and common syndromes including transient strong immunosuppression. The various pathogenic forms of morbillivirus are a result of the interaction between the host and the virus. We previously performed microarray analysis after morbillivirus infection, and found that the infections induced comprehensive downregulation of housekeeping genes. However, consequence of the unique host response were still remain unclear.

Methods: To clarify this phenomenon we employed combination of the CAGE (cap analysis of gene expression) with a next generation sequencer, which identifies active transcription starting site and quantifies their activities by sequencing mRNA 5’-ends in a high-throughput way, and the MARA (motif activity response analysis) which quantifies the motif activities in promoter caused by alteration of the activities of each transcription factors (TFs), over the infection time course. To validate obtained results, we utilized the motif-containing promoter assay with luciferase gene.

Results: We found that only limited set of TFs were activated or inactivated at early stage of infection, followed by sporadic change of activities of several peripheral TFs, and then resulted in comprehensive alteration of TF activities at late phage of infection. Based on the information, we reconstituted the transcriptional regulatory network after morbillivirus infection. As a result, we identified that two TFs act as master TFs which locate at upstream of the transcriptional cascade, and the change of their activities transduced to the mediator TFs, and then propagated to the nine terminal TFs which conduct the expression of downstream peripheral genes. To confirm the reconstructed network, we performed promoter assay. As expected, the infection modulated the motif activities of master TFs, and proceeding of the infection caused significant downregulation of the motif activities of terminal TFs. Furthermore, artificially-activated master TFs suppressed the virus growth, suggesting that the transcriptional network is a host defense system against morbillivirus infection.

Conclusion: We revealed that comprehensive downregulation of housekeeping genes is an intrinsic host system to counteract morbillivirus infection. These high-throughput experiments by combination of CAGE and MARA uncover a novel host response against virus infection, and will be useful for understanding the whole picture of the complexity of the virus infection in general.

Keywords: morbillivirus

17th International Congress of Virology 309

Late breaking abstract

Paramyxoviruses PO742

Canine distemper virus human SLAM (hSLAM) adaptation strain can replicate in lymphatic cells and organs of hSLAM transgenic mice

J. Zhao 1 2,*, A. Alcos 3, S. Delpeut 1, G. Sisson 1, C. Richardson 1, X. Yan 2 1Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada, 2Division of veterinary medicine, INSTITUTE OF SPECIAL ANIMALS AND PLANTS, CHINESE ACADEMY OF AGRICULTURAL SCIENCE, Changchun, China, 3Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada

Objectives: Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. The described cell receptor of CDV is SLAM (CD150). In order to characterize the risks of CDV cross the species barrier to humans and emerge as a new human pathogen, we got a hSLAM adapted CDV strain (5804Pe-VHS) and characterized its pathogenesis in hSLAM-transgenic mouse.

Methods: We adapted the wild type 5804Pe strain of CDV to use the human SLAM receptor in Vero-hSLAM cultured cells by serial passage. The hSLAM-transgenic mice were inoculated intranasal by CDV 5804Pe-VHS and 5804Pe strain with 6 the dose of 10 TCID50. The virus loads of assayed tissues and isolated PBMCs from the infected transgenic mouse at 7 days post-inoculation (dpi) were determined by virus titers assay, confocal fluorescence microscopy, and RT-PCR.

4 Results: CDV 5804Pe replicated only to moderate titers (10 TCID50/ml) in Vero-hSLAM at the first passage. However, after 6.5 three passages using these cells virus was adapted to hSLAM and replicated to high titers (10 TCID50/ml).The adaptive mutation was verified by DNA sequencing to be D540G in the H receptor binding protein of the virus. Antibodies against hSLAM were shown to block infections by this virus. All the spleens of 5804Pe-VHS inoculated mice were swollen and heavier than 5804Pe inoculated group. Virus was found in PMBCs, spleen, lymph nodes, thymus, lung, and kidney in 5804Pe-VHS group but not the 5804Pe group.

Conlusion: CDV can adapt to use hSLAM with only one amino acid mutations the hemagglutinin protein at position Asp540Gly (D540G). The adapted CDV, like wild type measles virus, can replicate in lymphatic cells and organs of hSLAM transgenic mice.

Keywords: Canine distemper virus, human SLAM, Mice

17th International Congress of Virology 310

Abstract Submission

Paramyxoviruses PO743

CHARACTERISATION OF NOVEL PARAMYXOVIRUSES ISOLATED FROM PTEROPID BAT URINE

R. Johnson 1 2,*, I. Smith 1, H. Netter 2, G. A. Marsh 1 1Australian Animal Health Laboratory, CSIRO Health and Biosecurity, Geelong, 2Department of Microbiology, Monash University, Melbourne, Australia

Objectives: The role of bats as reservoir hosts for multiple zoonotic pathogens makes the surveillance of bats and the discovery of novel pathogens necessary to prepare for emerging infectious diseases. Isolation and characterisation of novel bat-borne viruses improves our understanding of their zoonotic potential and prevents virus spillover from having a significant public health and economic impact.

Methods: Previous surveillance of Australian fruit bats has resulted in the identification of multiple viruses from the Paramyxoviridae family. During the winter of 2011 when an unprecedented number of Hendra virus outbreaks occurred, pteropid bat urine samples were collected in Alstonville in northern New South Wales, Australia to be analysed for the presence of novel viruses. Viruses were isolated from these urine samples and then were further analysed by whole genome sequencing, cross-reactivity and neutralisation assays, serological assays and animal challenge experiments to better understand their potential to cause disease.

Results: From these bat urine samples, multiple adenoviruses and paramyxoviruses were isolated, including Teviot paramyxovirus (TevPV) and the novel Alston paramyxovirus (AlsPV). Sequencing results show that these two viruses belong to different branches of the genus Rubulavirus. AlsPV is most closely related to Parainfluenza virus 5 (PIV5), the causative agent of respiratory disease in dogs, with the amino acid sequence identities for PIV5 and AlsPV proteins ranging between 81-93%. In comparison, TevPV is related to Tioman virus with which it shares a number of genetic features such as gene boundary sequences and intergenic sequences. Experimental infection of mice and ferrets indicates that ferrets can be infected with both viruses and produce neutralizing antibodies, however neither virus causes clinical symptoms.

Conclusion: The isolation of these viruses provides a greater understanding of the viral diversity present in Australian pteropid bats. Further characterisation of these novel viruses will be conducted to fully determine their zoonotic potential.

Keywords: bat, Paramyxoviridae, Rubulavirus

17th International Congress of Virology 311

Abstract Submission

Paramyxoviruses PO744

COMMON COLD TO SEVERE BRONCHIOLITIS AND PNEUMONIA IN CHILDREN CAUSED BY HUMAN METAPNEUMOVIRUS INFECTION IN SRI LANKA

J. Jayaweera*, F. Noordeen 1, A. Morel 2, S. Kothalawala 1, F. Pitchai 1 1Microbiology, Faculty of Medicine, University of Peradeniya, 2Pediactrics, Teaching Hospital Gampola, Kandy, Sri Lanka

Objectives: Acute respiratory tract infection (ARTI) is one of the most common acute illnesses in childhood. Viruses are the major causative agents of ARTI in childhood and human metapneumovirus (hMPV) is one of the emerging viruses causing ARTI. hMPV, is the first member of a new genus Metapneumovirus of the Paramyxoviridae family that infect humans. we do not know much about the occurrence of hMPV in the tropical countries including Sri Lanka. The present study was undertaken to elucidate the occurrence, seasonality, spectrum of illness and disease burden following hMPV associated childhood ARTI in 2 teaching hospitals in the wet and dry zones of Sri Lanka.

Methods: Nasopharyngeal aspirates (NPA) of hospitalized patients (1 month - 5 years) with ARTI were collected in Teaching Hospital, Gampola (THG) and Teaching Hospital, Anuradhapura (THA) from March 2013 to August 2014. After screening the NPA with indirect immunofluorescence assay (IFA), DAKO IMAGEN™ (UK) specific viral aetiology was detected using the direct immunofluorescence assay.Reverse transcriptase-polymerase chain reaction (RT-PCR) of hMPV was done in RSV positive children to detect viral co-infection and in IFA negative children to see the presence of hMPV monoinfections. The descriptive statistics was expressed using measures of central tendency.

Results: Of the 443 and 418 NPAs tested, viral aetiology was detected in 165 (37.2 %) and 165 (39.4 %) in THG and THA cohorts, respectively. Of the 165 children with virus detected ARTI, hMPV was detected in 16 children (9.6 %) in THA and in 9 children (5.4 %) in THG. In the dry zone (THA), the hMPV incidence was detected in December-April while in the wet zone (THG) from April-August.It was also noted that the hMPV distribution coincided the peaks of RSV infections.hMPV caused mild upper ARTI to severe life threatening lower ARTIs including severe bronchiolitis and pneumonia in the current study cohort. Period prevalence of viral ARTI was 8.25% and the hMPV incidence was 1.31 /100000 person years for ≤ 5- year-old children. The case fatality ratio was 8% in RSV/hMPV co-infections following severe bronchiolitis. Disability adjusted life years (DALYs) for the study cohort was 671.9 but for the RSV/hMPV co-infection it was 241.8.

Conclusion: hMPV associated in childhood ARTI was detected in the first time in Sri Lanka. Spectrum of respiratory disease in hMPV infection is similar to that of RSV infection but the hMPV incidence is low. Children with RSV and hMPV co- infections had severe respiratory disease. Viral diagnostic tests need to be available to detect viral ARTI and minimize irrational the use of antibiotics for viral ARTIs.

Keywords: Burden, hMPV, Seasonality

17th International Congress of Virology 312

Abstract Submission

Paramyxoviruses PO745

EFFECTS OF SERINE PROTEASE INHIBITORS ON REPLICATION OF TYPE 3 PARAINFLUENZA VIRUS IN PRIMARY CULTURES OF HUMAN TRACHEAL EPITHELIAL CELLS

Y. Iino*, A. Ide, K. Sato, O. Watanabe, S. Omiya, E. Isogai, M. Yamaya, H. Nishimura

Objectives: Type 3 parainfluenza virus (PIV3) causes acute respiratory infection among young children and sometimes causes severe pneumonia in immunocompromised patients. The cleavage of the fusion protein (F) by host proteases is essential for the replication. The proteases responsible for the cleavage are not characterized well except for TMPRSS2 so far. In this study, we investigated whether some kinds of protease inhibitors (PIs) inhibit replication of PIV3 in the actual epithelial cells of human respiratory tract or not.

Methods: Three clinical isolates of PIV3, 2910-Sendai-2003, 909-Sendai-2014 and 926-Sendai-2012 strains were used. These virus strains were infected to primary cultures of human tracheal epithelial cells (HTE) at a low multiplicity of infection and incubated in the presence of serine PIs, camostat, nafamostat, aprotinin, gabexate, sivelestat, as well as a furin inhibitor, and the viral growth was investigated. The inhibition of F protein cleavage was investigated with the western blotting technique.

Results: The inhibitory effects of the PIs on PIV3 replication depended on the viral strains. The growth of 909-Sendai-2014 and 926-Sendai-2012 strains was greatly inhibited with nafamostat and camostat at the concentration 0.1μg/ml and 40μg/ml (approximately 290nM and 80μM), respectively. The western blotting analyses revealed that F protein cleavage was reduced in the PI concentration-dependent manner. However, for 2910-Sendai-2003 strain, PIs did not show any inhibitory effect on the viral growth. The deduced amino acid sequence on the cleavage site of PIV3 F protein had the motif of furin protease susceptibility. The replication of the strain was inhibited partially by the furin inhibitor, and perfectly by additional inhibition by camostat.

Conclusion: Some PI drugs showed inhibitory effect on PIV3 infection in HTE cells, in association with the reduction of F protein cleavage. These findings suggested the possibility of PIs for treatment of PIV3 infection, as well as for characterization of the host proteases used by the virus for its replication.

Keywords: Airway epithelial cell, Protease inhibitor, Type 3 parainfluenza virus

17th International Congress of Virology 313

Abstract Submission

Paramyxoviruses PO746

GENETIC CHARACTERIZATION OF MEASLES VIRUSES ASSOCIATED WITH AN OUTBREAK IN MONGOLIA, 2015- 2016

R. Anderson*

Objectives: A large measles outbreak occurred in Mongolia during 2015 and continued into 2016, with a total of 49,226 cases. Since Mongolia declared elimnination of endemic measles in 2014, sequence analysis was used to identify the source of the imported virus.

Methods: A total of 80 specimens, collected from 9 different provinces between March 2015 and May 2016, were tested, and the measles genotype was determined based on the sequence of the 450 nucleotides (nt) coding for the carboxyl- terminal 150 amino acids of the nucleoprotein (N-450). To improve the low level of sequence diversity using only the N- 450 region, which is consistent with reintroduction of measles virus, the non-coding region between the matrix and fusion proteins of measles (MF-NCR) was obtained. The MF-NCR region (1018 nt) is more variable than the N-450 and can provide approximately the same resolution as whole genome sequencing.

Results: The N-450 sequence was obtained from 70/80 specimens, all sequences were assigned to genotype H1, which is the endemic genotype in China and frequently detected in measles outbreaks in Asian countries. The most closely related sequence in the global measles sequence database, MeaNS, was the named strained, MV/Hong Kong.CHN/49.12. The N-450 sequences differed from each other by a maximum of 3 nt, and the sequences clustered into 2 temporally associated groups which differed by 1 nt. The MF-NCR sequence was derived from 26/70 samples from Mongolia. There was a maximum difference of 6 nt in the MF-NCR sequences, with no distinciton between the sequences from 2015 or 2016.

Conclusion: While there is an increased number of substitutions in the MF-NCR sequences, the sequence analysis suggested that only one lineage of measles virus was circulating during the outbreak.

Keywords: 2015-2016, measles virus, Mongolia

17th International Congress of Virology 314

Abstract Submission

Paramyxoviruses PO747

NIPAH VIRUS C PROTEIN INHIBITS INFLAMMATORY CYTOKINE INDUCTION BY INTERACTING WITH PROTEIN PHOSPHATASE 2A INHIBITOR.

R. Horie, S. Uchida, H. Sato, C. Kai, M. Yoneda*

Objectives: Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. NiV is a zoonotic pathogen of significant concern to human health and has emerged from wildlife reservoir in the last 15 years to cause often fatal disease in broad range mammalian hosts. NiV causes severe encephalitis in human with high mortality. We had previously reported that the NiV nonstructural C protein (NiV-C) plays a key role in its severe pathogenecity. Recently, it is becoming clear that NiV-C regulates proinflammatory response, however the mechanism is still unclarified.

Methods: In present study, we isolated human proteins binding to the NiV-C using an affinity purification method. Further, we investeigated the role of the interaction in expression of proinflammatory cytokines by luciferase reporter assay and quantitative RT-PCR.

Results: Inhibitor of serine/threonine protein phosphatase 2A (I2PP2A) was identified as a NiV-C binding protein among them. It has been reported that PP2A interacts with numerous kinases including the MAPK proteins, which related pathways regulate proinflammatory cytokine production. When NiV-C was stably expressed in cells, total cellular PP2A activity was significantly increased and induction of proinflammatory cytokines, IL-8 and CXCL2, by poly(I:C) treatment were suppressed. And NiV-C also inhibited AP-1 and NF-κB dependent transcription and phosphorylation of p38 MAPK.

Conclusion: These results suggest that interaction between NiV-C and I2PP2A results in increase of PP2A activity and inhibition of proinflammatory cytokine induction through MAPK pathway.

Keywords: C protein, nipah virus

17th International Congress of Virology 315

Abstract Submission

Paramyxoviruses PO748

NUCLEOCAPSID PROTEIN OF NIPAH VIRUS DOWNREGULATES INTERFERON STIMULATED GENES BY INHIBITING THE NUCLEAR ACCUMULATION OF STATS

A. Sugai 1,*, H. Sato 1, I. Takayama 1, M. Yoneda 1, C. Kai 1 1Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Objectives: Nipah virus (NiV), a member of the Henipavirus genus in the Paramyxoviridae family, is a highly pathogenic zoonotic agent. NiV are well known for suppressing interferon (IFN)-mediated innate immunity with their phosphoprotein (P) gene products. In this study, we demonstrated that the nucleocapsid protein (N) of NiV also prevents the host IFN signaling pathway.

Methods: We measured the IFN antagonism of NiV-N protein through ISRE (IFN-stimulated response element)- and GAS (gamma-IFN activation site)-driven reporter gene expression. Complex formation of the STATs was examined by native PAGE analysis in the presence or absence of the NiV-N protein after IFN stimulation. Localization of STATs was investigated using Cos7 or 293T cells by indirect immunofluorescence assay. ISG (interferon-stimulated gene) expression was measured by quantitative RT-PCR.

Results: Reporter assays revealed that NiV N protein suppressed both type-I and type-II IFN responses, and the inhibitory effect was mediated by its core-domain. STATs (signal transducer and activator of transcription) are known to be activated via phosphorylation to form active complexes before nuclear translocation. We demonstrated that STATs were able to be phosphorylated while efficient formation of the complexes were obstructed in the N protein expressing cells. The reduction of nuclear accumulation of STAT1 and STAT2 after IFN stimulation in the presence of N protein caused downregulation of ISGs.

Conclusion: Thus, N protein suppressed IFN responses via different mechanisms from P protein. These results provide a new insight that N protein is involved in NiV pathogenicity by the IFN antagonism.

Keywords: Nipah virus, Nucleocapsid protein, STAT

17th International Congress of Virology 316

Abstract Submission

Paramyxoviruses PO749

PERSISTENT FELINE MORBILLIVIRUS INFECTION IN CRFK CELLS.

S. Sakaguchi 1,*, R. Koide 2, K. Shindo 2, T. Noda 2, T. Mizutani 1, T. Miyazawa 2 1Research and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Tokyo, 2Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan

Objectives: The feline morbillivirus (FeMV) found in 2012 belongs to the genus Morbillivirus of the family Paramyxoviridae. This virus is said to be involved in chronic nephritis of cats and is expected to become a clue to the resolution of renal failure that is a leading cause of death in older cats. The mechanism by which this virus causes diseases in the host has not yet been elucidated. In general, viruses of the genus morbillivirus establish persistent infection, and this is considered to be involved in their pathogenicity. It is reported that FeMV persistently infects cats and the virus is continuously excreted in the urine. Therefore, we attempted to establish persistently infected cells as a chronic infection model of FeMV in vitro.

Methods: Urine specimens of cats infected with FeMV were collected, centrifuged and filtered. These urine samples were inoculated into CRFK cells, which is derived from cat kidney, in the presence of trypsin. The cells were cultured while passaging, and virus infection was confirmed by observing cytopathic effect (CPE) by optical microscope. We isolated three strains of FeMV. Cells infected with these three strains were kept cultured, and cells infected with FeMV for more than one year were defined as persistently infected cells. The properties of these persistently infected cells were determined by titration of the virus produced from these cells and morphological analysis by microscopy.

Results: Virus was detected in both cells and supernatant in persistently infected cells of FeMV. As a result of measuring titer, these cells continued to release infectious virus particles into the supernatant. In addition, infected cells contained more infectious virus particles than in the supernatant. Aggregation of virus N protein occurred in the cytoplasm of these infected cells, and it was observed as a high density mass under the electron microscope. In addition, it was found that aggregation of this N protein shows a different shape from the initial stage of infection by indirect fluorescent assay.

Conclusion: We established persistently infected cells of FeMV. Propagation of infectious virus particles occurs in these infected cells, and sustained supply of stock viruses to be used for future research can be expected. On the other hand, since the localization of N protein was changed, it was suggested that some change was caused by persistent infection of FeMV, which may possibly be related to pathogenicity.

Keywords: cat, electron microscopy, persistent infection

17th International Congress of Virology 317

Abstract Submission

Paramyxoviruses PO750

THE C-TERMINAL REGION OF THE RESPIRATORY SYNCYTIAL VIRUS PHOSPHOPROTEIN HAS AN INHIBITORY EFFECT ON THE POLYMERASE ACTIVITY

K. Yaita*, K. Hara, T. Kashiwagi, N. Hamada, H. Watanabe

Objectives: Respiratory syncytial virus (RSV) can lead to serious lower respiratory tract illness, especially for infants and older adults. However, an effective antiviral therapy or vaccine has not been developed. Here, we tested whether the overexpression of viral ribonucleoprotein (RNP) components, nucleoprotein (N), M2-1, and phosphoprotein (P), could inhibit the viral polymerase activity.

Methods: BHK-21 (hamster) derived BSR-T7/5 cells, stably expressing T7 RNA polymerase (T7 pol), were used for the reconstitution of RNP by transfecting plasmids expressing L, P, N, M2-1 and vRNA-like luciferase RNA (gifted from Dr. Jean-François Eléouët). Concurrently, a plasmid expressing a full-length or truncated form of N, M2-1, and P was transfected to test the inhibitory effect on the RNA polymerase activity. Cells were harvested and lysed 24 h post-transfection and the polymerase activity was evaluated by measuring luciferase activity.

Results: The full-length or truncated form of N and M2-1 did not show a significant inhibitory effect, although the N-terminal region of M2-1 (aa 1-76) slightly reduced the polymerase activity compared with that of the full-length (58%). In contrast, the truncated form of P containing the C-terminal half (aa 121-241) remarkably reduced the polymerase activity to 12% as compared with the full-length. Further truncation analysis indicated that the oligomerization (aa 120-150), N-binding (aa 160-180), and L binding (aa 212-241) domains in the C-terminal region were critical for the inhibitory effect.

Conclusion: The P protein acts as an essential cofactor of the viral polymerase (L) by recruiting the L polymerase to the nucleocapsid. Here, we conclude that the C-terminal region of P has a capability to interfere with the full-length P and inhibits the polymerase activity, suggesting a significant potential to treat RSV infection.

Keywords: phosphoprotein, respiratory syncytial virus, RNA polymerase

17th International Congress of Virology 318

Abstract Submission

Paramyxoviruses PO751

THE MOLECULAR DETERMINANTS OF NEUROINVASION OF THE HENIPAVIRUSES IN THE MOUSE MODEL

S. Edwards 1,*, B. Clayton 1, G. Marsh 1 1CSIRO Australian Animal Health Laboratory, East Geelong, Australia

Objectives: Hendra virus (HeV) and Nipah virus (NiV) are henipaviruses that are highly pathogenic to a wide range of animals including humans, and are some of the few viruses from the Paramyxoviridae family with zoonotic ability. Neuroinvasion leading to fatal encephalitis is a common outcome for human infections. Recently, we reported that when mice are exposed to HeV via the intranasal route, they consistently develop fatal encephalitis in addition to a transient lung infection. However, when challenged with NiV, only the transient lung infection develops without encephalitis. This was an unexpected finding, as both HeV and NiV use the same host cell receptors, ephrin B2 and B3, for infection.

Methods: To elucidate the mechanisms for this difference, reverse genetics is utilized to construct recombinant HeV with NiV protein counterparts. Within the biosafety level-4 (BSL-4) containment laboratory, these viruses are then assessed for infection dynamics in murine cortical neuron cultures and in their ability to cause disease in the mouse model. Interestingly, when mice are infected, we are unable to re-isolate virus from the brain. To further characterize the mechanisms of neuronal spread, we will test a range of drugs known to inhibit important pathways (e.g. microtubule assembly, cell entry inhibitors, nerve signal inhibitor). The role of these different pathways/interactions in HeV spread will then be assessed.

Results: Our full-length reverse genetics system has resulted in the highly efficient rescue of recombinant HeV. Recombinant virus generated will be used to infect mice via the olfactory pathway (results pending).

Conclusion: Mechanisms important for the neuronal spread of HeV in the mouse could be determined by systematically comparing differences between HeV and NiV in the mouse model. The addition of drug inhibitors to HeV and NiV in neuron cell culture will allow us to further understand which mechanisms give rise to the neuronal phenotype. Identifying the molecular determinants that contribute to henipavirus infection within the host is important, as understanding these factors could potentially contribute to the development of vaccines and therapeutic treatments for henipavirus infection.

Keywords: Henipavirus, molecular modification, neuroinvasion

17th International Congress of Virology 319

Abstract Submission

Paramyxoviruses PO752

UTILIZATION OF HUMANIZED MICE AS A SMALL ANIMAL MODEL FOR MEASLES VIRUS INFECTION T. Nakanishi 1 2 3,*, M. Yoneda 2, T. Fujiyuki 2, Y. Amagai 2, I. Saito 1, C. Kai 2 1Laboratory of Molecular Genetics, The Institute of Medical Science, 2Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 3Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan

Objectives: Natural hosts of measles virus (MV) are only humans and no other animals except nonhuman primates have susceptibility to MV infection. To study the mechanisms of MV pathogenicity, we previously established nonhuman primate models for the infection. They are susceptible to MV infection and develop symptoms that are similar to those of human patients such as efficient infection of the virus to lymphocytes, leukopenia, and rushes. However, experiments in monkeys are expensive and cumbersome. Recently, mouse lines expressing MV receptor human SLAM were reported, but they showed only limited MV replication in lymphoid tissues only when crossed with mice lacking IFN-a/b receptor and no other typical symptoms of measles were observed. In this study, we aimed to establish a small animal model for MV infection by utilizing humanized mice.

Methods: NSG immunodeficient mice were transplanted with human CD34-positive cord blood cells to produce humanized mice. Tissue samples from the humanized mice were subjected to flowcytometry and immunohistochemical analyses after the mice were inoculated with a recombinant wild-type MV expressing EGFP.

Results: Expression of SLAM was observed in human lymphocytes developed in the humanized mice. When we challenged the humanized mice with a recombinant wild-type MV expressing EGFP, a strong EGFP fluorescence was found in spleen, lymph nodes, and bone marrow. The fluorescence was confirmed to associate with MV infection of human T and B cells. The number of human lymphocytes were decreased and subsequently recovered. Moreover, green fluorescent spots similar to skin rashes were observed in the abdominal skin. The spots were found to be due to accumulation of human lymphocytes to the hair follicles.

Conclusion: These results indicate that our small animal model would be useful for elucidating the mechanism of MV infection and its pathogenicity.

Keywords: EGFP, humanized mice, measles virus

17th International Congress of Virology 320

Abstract Submission

Paramyxoviruses

PO861

MODELLING THE ABORTIVE REPLICATION AND REACTIVATION NATURE OF RESPIRATORY SYNCYTIAL VIRUS (RSV) INFECTION IN PALIVIZUMAB-TREATED HELA AND A549 CELLS

M. W.-P. Poh 1,*, A. Fonceca 1 2, A. Kicic 1 3 4 5, M. Everard 1 2

1School of Paediatrics and Child Health, University of Western Australia, 2Department of Health, Princess Margaret Hospital , 3Airway Epithelial Research, Telethon Kids Institute, 4Department of Respiratory Medicine , Princess Margaret Hospital, Subiaco, 5Center for Cell Therapy and Regenerative Medicine, University of Western Australia, Crawley, Australia

Objectives: Abortive replication of Respiratory Syncytial Virus (RSV) has been described both in in vitro cell lines and in animal models in the presence of the RSV monoclonal antibody Palivizumab as well as in latent RSV infection and reactivation in human dendritic cells. Naturally occurring RSV antibodies are known to wane very rapidly giving rise to the possibility that reactivation of viral replication may occur as circulating antibody levels fall. The aim of this study was to determine whether latent RSV might be reactivated in a cell culture model by the removal of Palivizumab.

Methods: HeLa or A549 cells (2.5 x 104) were seeded into a 96-well flat-bottom plate. The following day, the 80%>confluent cells were infected with fluorescent-tagged RSV (A2 laboratory strain) (MOI 3). The appearance of fluorescence indicates the production of a fluorescent protein and is a marker of replication. All cells were cultured in the presence of the Palivizumab (1600ug/ml). Cell culture medium was replenished every five days (with or without fresh Palivizumab). The fluorescence emitted intracellularly by infectious RSV virions was tracked and analysed using the Incucyte® Zoom System (Essen BioScience).

Results: When cultured over 4-weeks, HeLa and A549 cells cultured with RSV had maximum fluorescence levels, whereas levels in HeLa or A549+RSV cultures continuously cultured in the presence of Palivizumab were significantly lower (p<0.05) compared to those cultured in absence of Palivizumab.

Conclusion: The results confirm the suggestion that Palivizamab does not prevent infection but results in abortive replication. The reactivation of replication once antibody levels fall is a novel finding that is likely to be of relevance to transmission of the virus in humans.

Keywords: abortive replication, Palivizumab Synagis, respiratory syncytial virus

17th International Congress of Virology 321

Abstract Submission

Paramyxoviruses

PO685

HIGHLY EFFICIENT SENDAI VIRUS MEDIATED CRISPR/CAS9 GENE EDITING IN HEMATOPOIETIC STEM CELLS A. Park 1, R. Watkinson 1, O. Pernet 2, P. Hong 1, P. Thibault 1, D. S. An 2, B. Lee 1,* 1Microbiology, ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, New York, 2School of Nursing, University of California, Los Angeles, Los Angeles , United States

Objectives: A highly efficient CRISPR/Cas9 delivery system is critical for hematopoietic stem cell (HSC) based gene therapy for many diseases. Current, non-viral and viral methods (lentivirus and adeno-associated virus serotype 6) in common use for delivery of CRISPR/Cas9 have several drawbacks including limited efficiencies in hematopoietic stem cells (HSCs). Persistence of DNA based viral vectors may also lead to greater off-target effects in the long-term. Methods: In previous work, we repurposed Sendai virus (SeV), as a delivery system for highly efficient Cas9-mediated gene editing (75-98% without selection) in human cells, including primary human macrophages, with minimal off-target effects. SeV is a non- pathogenic paramyxovirus in humans, has an established safety record, and has been extensively studied and modified for gene therapy applications. SeV is an RNA virus that replicates solely in the cytoplasm and has no viral DNA phase, thereby eliminating any potential for undesired random integration into the host genome. We placed an EGFP-P2A-Cas9 and a gRNA cassette as additional transcriptional units between the N and P and P and M genes, respectively. Flanking the gRNA cassette with self-cleaving ribozymes allowed this RNA vector to produce the precise ends of the gRNA-tracrRNA scaffold required for gRNA function, and we showed for the first time that self-cleaving ribozymes were tolerated within the paramyxovirus genome. Results: Here, we show further safety improvements in our SeV CRISPR/Cas9 delivery system by introducing mutations to confer temperature sensitivity (ts) that allows removal of the virus at non-permissive temperatures (37-38°C) once editing at a permissive temperature (34°C) has occurred. This non-cytopathic ts rSeV-Cas9 vector transduced human fetal liver and peripheral blood mobilized CD34+ HSCs at ~90% and resulted in ~80% mutagenesis of the targeted locus (ccr5) within 2 days. Shifting to 37°C resulted in rapid loss of the rSeV-Cas9 vector. Introducing ts mutations into replication-deficient SeV vectors (e.g. by removing the fusion protein essential for virus entry) will likely synergistically enhance the safety of our SeV-Cas9 vectors for clinical applications requiring highly efficient gene editing in HSCs. Conclusion: Our study shows that SeV can be engineered as a delivery system for highly efficient Cas9-mediated gene editing in HSCs. The general prinicple can be applied to other paramyxoviruses being developed for geen therapy purposes such as Measles virus and Newcastle disease virus. Additional applications of our rSeV-Cas9 delivery system will be discussed.

Keywords: CRISPR/Cas9 gene editing, hematopoietic stem cells, sendai virus

17th International Congress of Virology 322

Plant Virus

Abstract Submission

Plant viruses

PO753

17th International Congress of Virology 323

A Beta-satellite DNA component of tomato Ageratum yellow vein virus (Malaysia) is an important pathogenic determinant of the virus in tomato plants.

R. Y. Othman*, C. H. Teo 1, M. Omar 1 1CEBAR, University of Malaya, Kuala Lumpur, Malaysia

Objectives: Ageratum yellow vein virus-Malaysia [Malaysia-Tomato Leaf curl-2011] (AYVV-MY [MY-Leaf Curl-11]) is a newly described begomovirus strain isolated from naturally infected tomatoes with severe leaf curl symptom and is among viruses that cause global yield losses in the tomato industry. Its monopartite genome is associated with a beta satellite which is suspected to be linked to disease severity.Strategies to control the virus include the development of transgenics containing RNAi components which have been found to be effective againts natural infection. However the effect of beta satellites remain unverified, This work investigated the possibile relationship of the satellite compnent with disease severity .

Methods: To investigate the role of the components of its genome, infectious clones of both DNA-A and its associated satellite molecule DNA β were individually constructed and tested in tomato (Solanum lycopersicum) plants using agro- infiltration inoculation. Virus replication was monitored using PCR and symptoms were observed for at least 30 days dpi.

Results: All plants inoculated with either DNA-A alone or with both DNA-A and DNA β developed systemic symptoms of downward leaf curl and yellowing after 15 days of post inoculation (dpi), which confirmed the infectivity of these clones. However the single DNA-A only inoculated plants showed delay and/or undeveloped disease symptoms compared to the plants co-inoculated with the beta satellite. These showed severity of symptoms greatly enhanced after 15 dpi. Viral sequences for both DNA components could be detected in the symptomatic plants as early as 3 dpi using viral gene- specific PCR.

Conclusion: This suggests that the DNA β is an important pathogenic determinant for induction of systemic leaf curl disease symptoms in S. lycopersicum. The implication to transgenic strategies will be discussed.

Keywords: beta satellite, Geminiviruses, tomato

Abstract Submission

Plant viruses

PO754

17th International Congress of Virology 324

A NOVEL RNA2-ENCODED PROTEASE IS REQUIRED IN ADDITION TO THE RNA1-ENCODED 3C-LIKE PROTEASE FOR PROCESSING OF STRAWBERRY MOTTLE VIRUS POLYPROTEINS

K. S. Mann 1, M. Walker 1, H. Sanfaçon 1,* 1Summerland Research and Development Centre, Agriculture and Agri-Food Canada, Summerland, Canada

Objectives: Strawberry decline disease has emerged as a significant problem for strawberry production in Eastern Canada and is likely caused by the synergistic effects of mixed virus infections. Strawberry mottle virus (SMoV, family Secoviridae) is one of the prevalent viruses found in association with the disease. Similar to other members of the family Secoviridae (secovirids), the SMoV genome consists of two positive sense RNAs, each encoding a single large polyprotein. In all characterized secovirids, the two polyproteins are processed in cis (RNA1 polyprotein) and in trans (RNA2 polyprotein) by a single cysteine protease (PRO), which is related to the 3C PRO of picornaviruses. While RNA1 encodes replication proteins and the 3C-like PRO, the RNA2 polyprotein normally contain domains for the movement and capsid proteins (MP and CP). The RNA2 polyprotein of SMoV Canadian isolates also includes an extended C-terminal region with a coding capacity of 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we sought to characterize the proteolytic processing of SMoV polyproteins and define functional protein domains.

Methods: We used a rabbit reticulocyte in vitro processing assay and full-length or truncated SMoV polyproteins to characterize proteolytic cleavage events. Cleavage sites and conserved amino acids were predicted using amino acid sequence alignments derived from a collection of SMoV isolates and related viruses and were analyzed by site-directed mutagenesis (point mutation or deletion).

Results: As reported for some secovirids (i.e., nepoviruses), we identified five cleavage sites in the RNA1 polyprotein that are recognized in cis by the 3C-like protease. A primary cleavage occurred between the putative helicase and VPg domains, while other cleavages were cleaved less efficiently at least in vitro. Mutation of the predicted cysteine of the 3C- like protease catalytic triad abolished processing of the RNA1 polyprotein. A consensus cleavage site sequence was determined to be AxEQ/G. The consensus sequence is not present in the RNA2 polyprotein, although a related sequence (AxEE/G) between the predicted MP and CP domains was found to be cleaved by intermediate RNA1-encoded polyproteins that contained the PRO domain (e.g. VPg-PRO). An additional cleavage event was detected in the RNA2 polyprotein downstream of the CP domain, but was not dependent on the RNA1-encoded 3C-like PRO. C-terminal truncation of the RNA2 polyprotein or point mutation of a conserved glutamic acid (E1305) to alanine prevented the cleavage, suggesting the presence of a second viral protease in the C-terminal region of the RNA2 polyprotein. Cleavage was also reduced after addition of a broad-spectrum protease inhibitor mix. Systematic mutagenesis narrowed down the cleavage event to a LDVKPAFPF sequence, located immediately downstream of the predicted CP domain. Cleavage at this site was a rapid co-translational event that occurred only in cis.

Conclusion: In contrast with other characterized secovirids, we show that processing of SMoV polyproteins require two viral proteases, a 3C-like cysteine protease encoded by RNA1 and a second novel protease encoded by RNA2.

Keywords: Proteolytic processing, Secoviridae, Viral protease

Abstract Submission

Plant viruses

PO755

17th International Congress of Virology 325

A role of reactive oxygen species during the replication of a plant RNA virus

K. Hyodo 1,*, N. Suzuki 1, T. Okuno 2 1Institute of Plant Science and Resources, Okayama University, Kurashiki, 2Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Japan

- Objectives: Reactive oxygen species (ROS), including superoxide anion (O2 ), hydrogen peroxide (H2O2), or hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. A respiratory burst - oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2 production to inhibit pathogen infection - during plant innate immunity. RBOH-derived O2 can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA virus, hijacks the host’s ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, at present, what types of ROS are required for viral replication is unknown.

Methods: We investigated the effects of several types of ROS scavengers on RCNMV replication in N. benthamiana protoplasts.

Results: Here, we found that RCNMV replication was sensitive to cytosolic ROS scavenger ascorbic acid but insensitive - to lipid ROS scavenger α-tocopherol. Furthermore, O2 scavenger Tiron, but not H2O2 scavenger DMTU, inhibited RCNMV - replication, suggesting that O2 is required for RCNMV replication. Using affinity purification and LC-MS/MS analysis, we identified that several methionine residues of RCNMV replication proteins were oxidezed in infected leaves. Site-directed mutagenesis of one such methionine residue to leucine or isoleucine (mimicking unoxidized methionine) inhibited the accumulation of the 480-kDa viral replication complex (VRC) and viral replication, without affecting the accumulations of replication proteins.

- Conclusion: Our findings suggest that NbRBOHB-derived O2 seems to induce oxidation of viral replication proteins, leading to the proper assembly of the functional VRC.

Keywords: oxidation, reactive oxygen species, viral replication complex

Abstract Submission

Plant viruses

PO756

17th International Congress of Virology 326

ASSESSMENT OF RESISTANCE OF TRANSGENIC CHRYSANTHEMUM PLANTS TO INFECTION BY CHRYSANTHEMUM STUNT VIROID

B.-N. Chung 1, S.-K. Choi 1, J.-Y. Yoon 1, S.-J. Kwon*, I.-S. Cho 1 1Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Wanju, Korea, Republic Of

Objectives: Chrysanthemum stunt viroid (CSVd) is the most important viroid in chrysanthemum worldwide. It affects plant height, leaf size and flower quality in chrysanthemum plants . Methods: Transgenic chrysanthemum plants cultivar ‘Sinma’ were generated that express 299-bp segments derived from CSVd, as inverted repeat double stranded RNAs, separated by an intron. The orientation of the expressed double stranded RNAs was antisense – intron – sense RNAs and the double stranded RNAs were processed to small RNAs. Transgenic lines were assessed for resistance to CSVd infection by both mechanical inoculation of CSVd RNA transcripts and by grafting on the top of the CSVd-infected chrysanthemum. For in vitro transcription of CSVd, CSVd-SK1 clone plasmid DNA was linearized with EcoRI enzyme and reacted with SP6 RNA polymerase. Transgenic lines challenged with CSVd were monitored for 30 days, and were assessed for infection with CSVd by reverse-transcription PCR (RT-PCR).

Results: RNAs extracted from the foliage of the transgenic lines were assessed by northern blot hybridization for the presence of siRNAs against the CSVd used in the generation of the constructs. In this study, efficient methods for selection of resistance lines to viroid infection in transgenic chrysanthemum plants will be discussed. Previously, transgenic plants were generated that expressed fused viral segments from a single promoter and showed resistance to CMV and tomato leaf curl virus in, tomato spotted wilt virus and tomato leaf curl Taiwan virus in tomato, and CMV and watermelon mosaic virus in watermelon.

Conclusion: The strategy of expressing inverted repeat sequences from a transformation vector of multiple viruses in potato plants showed a higher percentage of resistance lines. Our study is the first report trying to make transgenic plants revealing resistance to viroid infection using strategy of expressing inverted repeat sequences in floriculture crops.

Keywords: None

Abstract Submission

Plant viruses

17th International Congress of Virology 327

PO757

ASSESSMENT OF THE INFECTIVITY OF BANANA STREAK MY VIRUS IN A BROAD RANGE OF BANANA GENOTYPES

A. James*, M. Onsarigo 1, R. Harding 1, J. Dale 1 1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia

Objectives: Banana streak disease, caused by a range of banana streak viruses, occurs in most banana producing countries worldwide. Several studies have reported the field assessment of banana genotypes following infection with one or more BSV species, with significant differences in virus incidence and symptom expression between and within genotypes. Field studies have used a severity index, based on symptom expression, to rate the symptom severity. However this system does not necessarily reflect the level of viral DNA accumulation (i.e. the viral load) within infected plants. Although BSVs are known to infect almost all bananas, there are no reports on the level of viral DNA accumulation in different banana cultivars or Musa sp.

Methods: We have developed an infectious clone of one BSV species, Banana streak MY virus (BSMYV), and have used this to study the levels of viral DNA accumulation in 24 genotypically diverse banana accessions, including both cultivated and wild bananas, under glasshouse conditions. Severity scores were recorded and the viral load was quantified using qPCR. Leaf emergence rate and plant height, as a measure of growth rate, were also recorded to identify if BSV infection results in reduced plant growth.

Results: Results indicate that (i) the majority of banana accessions are susceptible to infection with BSMYV; (ii) virus accumulation differs between genotypes, but also within plants of the same accession; and (iii) severity scores do not correlate well with the viral load as measured using qPCR.

Conclusion: n/a

Keywords: None

Abstract Submission

Plant viruses

17th International Congress of Virology 328

PO758

Bipartite genomic RNA segments of a plant virus can move from cell to cell in a different manner

K. Kitasaka 1, K. Mise 1, T. Okuno 2, Y. Takano 1, M. Kaido 1,* 1Graduate School of Agriculture, Kyoto University, Kyoto, 2Faculty of Agriculture, Ryukoku University, Otsu, Japan

Objectives: Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a bipartite genome and a member of the genus Dianthovirus in the family Tombusviridae. Genomic RNA1 encodes p27 auxiliary replication protein, p88 RNA-dependent RNA polymerase, and coat protein, while RNA2 encodes movement protein (MP). RCNMV forms small punctate structures on the cortical endoplasmic reticulum (cortical punctate structure; CPS) in infected cells during the early stages of infection. CPSs are the sites of viral RNA replication and the localization of MP to CPS is required for the efficient cell-to-cell movement of the virus. In this study, we established the system to evaluate the cell-to-cell movement of each of the genomic RNAs in the absence of the other. Using this system, we show that genomic RNA segments can be recognized by the MP in a different manner.

Methods: RCNMV protein(s) were expressed in Nicotiana benthamiana leaves via agroinfiltration method and the leaves were subsequently inoculated mechanically with in vitro transcripts of individual viral genomic RNA segment. Cell-to-cell movement of viral RNA was evaluated by determining the accumulation level of viral RNA by Northern blotting. Subcellular localization of MP derivatives fused with GFP was detected by confocal microscopy.

Results: RCNMV MP expressed via agroinfiltration supported the cell-to-cell movement of mechanically inoculated RNA1. p27 and p88 expressed via agroinfiltration supported the cell-to-cell movement of mechanically inoculated RNA2. On the other hand, the derivative of MP, MPdC70, lacking 70 amino acids from the C-terminus that is required for localization to CPS, failed to support the movement of RNA1. Similarly, MPdC70 supplied in trans together with p27 and p88 via agroinfiltration failed to support the efficient movement of an RNA2 derivative with a frameshift mutation abolishing in cis expression of MP. However, the RNA2 derivative encoding MPdC70 efficiently moved from cell to cell in the leaves expressing p27 and p88.

Conclusion: These results suggest that the cell-to-cell movement of RCNMV RNA1 requires the formation of CPS, while RNA2 could be transported to the neighboring cells by MP only supplied in cis, not through CPS.

Keywords: bipartite genome, cell-to-cell movement, movement protein

Abstract Submission

Plant viruses

17th International Congress of Virology 329

PO759

COMPLETE SEQUENCE ANALYSIS OF THE SEVEN OPEN READING FRAMES OF LYCHNIS RINGSPOT VIRUS-SN

Y. H. Bang 1,*, E. G. Song 1, S. H. Choi 1, J.-S. Hong 2, K. H. Ryu 1, Y. Eun Kwon 1Plant Virus GenBank, Seoul Women's University, Seoul, 2Department of Applied Biology, Kangwon National University, Chuncheon, Korea, Republic Of

Objectives: Lychnis ringspot virus (LRSV) is a member of the genus Hordeivirus in the family Virgaviridae. In previous studies, the three strains of LRSV showed distinguishable symptoms in host plants. The three strains were named as follows: LRSV-AS (asymptomatic), LRSV-SM (systemic mosaic) and LRSV-SN (severe necrosis). Especially, the symptoms caused by LRSV-SN showed more severe than other strains in Chenopodium amaranticolor, Datura stramonium, Dianthus caryophyllus, Lychnis coronaria, L. wilfordii and Nicotiana benthamiana. The objective of this study is to determine the characterization and genome sequence analysis of of LRSV-SN.

Methods: The host ranges of LRSV-SN were tested using several host plants, such as C. amaranticolor, D. stramonium, D. caryophyllus, L. coronaria, L. wilfordii and N. benthamiana. LRSV-SN was propagated systemically in N. benthamiana. Particularly, N. benthamiana were used for virus propagation and purification. The purified virions were observed under transmission electron microscope. The virions were used to produce antibodies against the LRSV-SN. The specificity and cross-reactivity of antibodies were confirmed by western blot analysis. The viral RNAs of LRSV-SN were isolated from the purified virions and were used for full-length cDNA library. The specific primers of LRSV-SN were designed based on the sequences of LRSV-SN obtained in this study. All nucleotide sequences obtained herein were analyzed by DNASTAR software (Wisconsin, USA) and phylogenetic tree analysis was done by DNAMAN software package (version 5.1; Lynnon Biosoft, Canada). The sequences of members of the genus Hordeivirus and other viral genera were obtained from the GenBank Database under the accession numbers (http://www.ncbi.nlm.nih.gov/).

Results: LRSV-SN showed ringspot symptoms on C. amaranticolor, D. stramonium and L. coronaria and systemic mosaic symptoms on L. wilfordii and N. benthamiana and systemic chlorosis on D. caryophyllus. The viral particles, known as virions, isolated from N. benthamiana were observed similar to those of other hordeiviruses under transmission electron microscope. The sequences of the replicase protein (αa and γa), the coat protein (βa), the movement protein (βb, βc and βd) and pathogenicity related protein (γb) of the LRSV-SN were determined in this study. The replicase protein consist of αa and γa was 129kDa (3,423bp) and 73kDa (1,884bp). The coat protein (βa) and movement protein consist of βb, βc and βd (triple gene block) were 21.7kDa (588bp) and 50kDa (1,361bp), 17kDa (464bp) and 13kDa (373bp), respectively. The pathogenicity related protein (γb) was 16kDa (435bp) in γb. The sequences of LRSV-SN were more closely grouped with the previously reported LRSV strain (GenBank accessions Z46640 and Z46353).

Conclusion: Consequently, the genome sequences of four proteins of LRSV-SN were determined in this study. In the phylogenetic tree, the sequences of LRSV-SN were closely clustered in the previously reported LRSV strain. In conclusion, this study suggests that new strain of LRSV, LRSV-SN, could be included in the LRSV species within the genus Hordeivirus.

Keywords: hordeivirus, LRSV, lychnis ringspot virus

Abstract Submission

Plant viruses

17th International Congress of Virology 330

PO760

Current status and spread of Rice yellow mottle virus in eastern Uganda

P. J. Odongo 1 2,*, N. Kojima 3, T. Tsuboi 4, G. Asea 2, K. T. Natsuaki 1 1International Agriculture development, Tokyo University of Agriculture, Tokyo, Japan, 2National Crops Resources Research Institute-Namulonge, Kampala, Uganda, 3Appropriate Agriculture International Co., Ltd, 4Japan International Cooperation Agency, Tokyo, Japan

Objectives: Rice yellow mottle virus (RYMV) endemic to Africa is a major rice (Oryza spp) virus in Uganda. A survey was carried to find out the current status and spread of the virus in eastern Uganda and additionally, the response of wild and cultivated rice cultivars to RYMV were assessed by artificial inoculation.

Methods: Field visits were conducted in 2015 and 2016 and farmers' cultural practices and field conditions were also assessed. As for the host reaction experiment, plants were mechanically inoculated at 21 days after sowing and were evaluated by enzyme-linked immunosorbent assay (ELISA) and reverse transcription -polymerase chain reaction (RT- PCR) for detecting the virus.

Results: The presence of Rice yellow mottle disease by RYMV was established in all the areas with recent introduction of rice cultivation and the farmers’ intensive practices may sustain and propagate the virus further. Oryza spp and O. sativa varied in resistance and we grouped them into resistant, moderately resistant and susceptible to RYMV. Wild rice species O.barthii, O.australiens, O.alta and O.rufipogon and most of the local and improved rice cultivars like SUPA were confirmed as susceptible to RYMV. We report for the first time O. glumaepatula (wild rice) as moderately resistant characterized by lack of symptoms but the virus was detectable by RT-PCR. Eragrotis tef was also evaluated as susceptible.

Conclusion: This study recommends the use of resistant wild rice species as potential sources of genetic resistance for breeding programs in Uganda. Information on genotype status, the spread and incidence of RYMV in Uganda are significant for designing control measures against RYMV.

Keywords: Detection, Rice yellow mottle virus , Spread

Abstract Submission

17th International Congress of Virology 331

Plant viruses PO761

Effects of plant-derived signal peptides on production of dimeric IFNγ glycoprotein in Nicotiana benthamiana plant by BaMV expression system

M.-C. Jiang*, Y.-H. Hsu 1 1Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan

Objectives: Human Interferon gamma (IFNγ) is a dimeric glycoprotein that is used as medical prescription to multiple diseases. To consider a safe and biologically active outcome, plant-based production system is an alternative way for current platform. Although plant platform exists a major challenge of recombinant protein in low productivity, the viral vectors could compensate this issue and greatly improve yields. According to our previous result truncation of signal peptide of IFNγ, named as mIFNγ, and replacement of P19 with BaMV triple-gene block, named as pKB△T19, were optimal expression sequence and viral vector, respectively. In this system, dimeric mIFNγ could be successfully produced in Nicotiana benthamiana plant. In order to increase mIFNγ shelf-life for therapeutic use, we screen different plant-derived signal peptides to lead target protein through intact pathway for decorated glycosylation.

Methods: To develop secretory expression cassettes, we used five different plant-derived signal peptides individually fused at N terminal of mIFNγ, named SSExt mIFNγ, SSRAmy3D mIFNγ, SSNbPr1 mIFNγ, SSVsp mIFNγ , and SSPDI mIFNγ. These expression cassettes were transformed into Agrobacterium and infiltrated into N. benthamina leaves. After 5 dpi, infiltrated leaves were collected and divided into intracellular (IC) and apoplast washing fluid (AWF) by vacuum procedure. Divided two protein samples were examined by western blot analysis and further quantified the concentration of SSmIFNγ by ELISA. To detect BaMV replication, northern blot hybridization with specific 3’UTR was performed.

Results: In western blot, these five species SS mIFNγ in IC were observed four distinct molecular weight of 18.3, 20.3, 22.3 and 36 to 44 kDa, compared to mIFNγ only presented two distinct molecular weight of 18.3 and 36.6 kDa. SS mIFNγ was also detected in AWF, but mIFNγ was almost no signal. In addition, ELISA quantifying result showed protein accumulation of SSExt mIFNγ was about 3 times higher than mIFNγ. We further examined the RNA accumulation of BaMV, the expression cassette of SSExt mIFNγ could produce more stable BaMV RNA than others.

Conclusion: In this study, we elucidated five plant-derived signal peptides had capacity to transport SSmIFNγ into secretory pathway and protein accumulated partly at apoplast space. As expected, SSmIFNγ formed monomeric and dimeric glycoprotein with 0, 1 or 2 N-glycan. By ELISA quantification, SSExtmIFNγ accumulated the highest level protein up to 292.7 μg/g fresh weight, corresponding to 3.5 % of total soluble protein content in infiltrated N. benthamiana leaves. These findings suggest the potential of BaMV-based vector as platform for producing dimeric IFNγ glycoprotein in N. benthamiana.

Keywords: Bamboo masaic virus (BaMV), glycoprotein, interferon gamma

Abstract Submission

17th International Congress of Virology 332

Plant viruses

PO762

EFFICACY OF STACKED RNAI CONSTRUCTS FOR BROAD SPECTRUM RESISTANCE TO CASSAVA BROWN STREAK DISEASE

B. Mware 1,*, C. Niblett 2, A. Bailey 2, L. Tripathi 1 1International Institute of Tropical Agriculture (IITA), PO Box 30709-00100, Nairobi , Kenya, 2Venganza Inc., St. Augustine, Florida, United States

Objectives: Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) (genus Ipomovirus, family Potiviridae), is one of the most important viral diseases of cassava (Manihot esculenta Crantz) (Mbazibwa et al., 2009 & Winter et al., 2010). The disease poses serious danger to food and economic security for farmers in East and Central Africa in addition to being a potential threat to cassava production in West Africa (Legg et al., 2014 & Patil et al., 2015). Conventional breeding has generated increased tolerance levels to CBSD, but has not provided effective complete resistance against CBSV. An RNA interference (RNAi) approach utlizing viral coat protein inverted repeat sequences has been used successfully to generate CBSD resistance in Uganda. However, evidence reveals increasing diversity in isolates of CBSV and UCBSV (Ndunguru et al., 2015), which presents a challenge to efforts aimed at development of effective CBSD resistance. The viral genome contains eight genes, but seven genes of important function in the virus life cycle have not been tested individually or in a stacked system for broad spectrum RNAi-based resistance. This study developed short hairpin RNAi constructs with potential to control all the groups of CBSV isolates and tested their efficacy by both transient and transgenic systems in Nicotiana benthamiana to select the best constructs for cassava transformation.

Methods: In collaboration with Venganza Inc. (USA), CBSV/UCBSV isolate sequences from public databases were aligned leading to three distinct virus isolate groups. The most conserved sequences of each gene across the three groups of isolates were selected and used to develop short hairpins. To target three genes by each construct, three short hairpins were stacked in a single plasmid that received the viral target genes by Gateway recombination. The constructs were Agro-infiltrated into 3-4 leaf stage Nicotiana benthamiana plants which were inoculated with CBSV/UCBSV 2 days post-infiltration for initial efficacy testing of constructs by transient expression assay. Further validation of constructs was performed by stable transformation, regeneration of Nicotiana benthamiana leaf discs before screening of 1st and 2nd generation seedlings for response to CBSV/UCBSV infection. Virus detection was based on visual assessment of symptoms and by PCR analysis.

Results: Based on transient assays, the different constructs showed varying levels of protection against CBSV/UCBSV infection. Preliminary results indicate that constructs containing CI (RNA helicase), VPg (Essential regulator of viral RNA synthesis and accumulation) and Nia (Protease) hairpin sequences provide strong protection against CBSV/UCBSV. Transgenic Nicotiana benthamiana have been generated and first generation seedlings planted for screening of their response to CBSV/UCBSV infection.

Conclusion: We report evidence of protection against virulent CBSV/UCBSV isolates by targeting conserved sequences of different genes across the entire spectrum of CBSV/UCBSV group of isolates. Preliminary results indicate that stacking of viral gene hairpins has the potential to provide a broad-spectrum RNAi-based CBSD management option.

Keywords: Cassava brown streak disease, Gene stacking, RNA interference

Abstract Submission

17th International Congress of Virology 333

Plant viruses

PO763

ENHANCED GENE SILENCING IN GRASSES USING AN MODIFIED BROME MOSAIC VIRUS-BASED VECTOR

X. S. Ding*, K. D. Cooper 1, R. S. Nelson 1 1Plant Biology, SAMUEL ROBERTS NOBLE FOUNDATION, Ardmore, United States

Objectives: Application of virus-induced gene silencing (VIGS) as a tool for functional genomics in plant has been reported numerous times by many laboratories in the world. Compared with RNA silencing using traditional stable transformation, VIGS is less labor-intensive and requires less time. We reported VIGS in rice, barley and maize using a Brome mosaic virus (BMV)-based VIGS vector in 2006 (Ding et al., 2006). Like other virus-based vectors (i.e., Barley stripe mosaic virus, Cucumber mosaic virus and Foxtail mosaic virus), BMV VIGS vector-mediated gene silencing in grasses is transient, lasting only 2 to 3 leaves. In 2010, a DNA-based BMV VIGS vector was reported for gene silencing in rice through an Agrobacterium-mediated infiltration procedure. Compared with the silencing induced by inoculation of BMV VIGS vector RNA transcripts, the Agrobacterium-infiltrated rice plants showed much stronger and more complete photobleaching phenotypes in leaves, and the photobleaching phenotypes lasted 4 to 5 leaves. Unfortunately, infection of other grasses through Agrobacterium-mediated infiltration was not successful.

Methods: To further improve gene silencing in grasses, we tried various approaches to increase the stability of the foreign gene insert within the BMV RNA3 vector sequence during VIGS via a computer-assisted approach.

Results: Several modified BMV RNA3 vectors were constructed and tested for their abilities to silence gene expression in maize. One modified BMV RNA3 vector carrying mutations in its coat protein ORF and the 3’ end untranslated region showed improved maintenance (5-6 days longer) of its 250 bp phytoene desaturase or heat shock protein 70 gene insert during virus infection in N. benthamiana leaves than did the original DNA-based VIGS vector with the same insert. Inoculation of maize seedlings with virion partially purified from Agrobacterium-infiltrated N. benthamiana leaves resulted in silencing phenotypes with greater photobleaching in 4-5 leaves than that caused by the original DNA-based BMV VIGS vector carrying the same insert (less photobleaching in 2-3 leaves). Together with a quick and low-cost inoculation procedure (Zhu et al., 2014), we now can achieve more reliable gene silencing in maize and other grasses, including foxtail millet (Setaria italic) and sorghum.

Conclusion: This easy-to-use RNAi approach should benefit researchers who are interested in studying gene function in grasses.

References Ding XS, Schneider WL, Chaluvadi SR, Mian MA, Nelson RS. 2006. Mol Plant-Microbe Interact 19:1229–1239 Zhu M, Chen Y, Ding XS, Webb SL, Zhou T, Nelson RS, Fan Z. 2014. New Phytologist 203: 1291-1304.

Keywords: None

Abstract Submission

17th International Congress of Virology 334

Plant viruses

PO764

Functional analysis of NbUbE3R1 and its effect on Bamboo mosaic virus replication C.-H. Tsai*

Objectives: Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus and belongs to Potexvirus genus of Alphaflexiviridae. To identify host factors involved in BaMV infection cycle, our lab used cDNA-amplified fragment length polymorphism technique to screen the differentially expressed gene of Nicotiana benthamiana after BaMV inoculation. One of upregulated gene ACGT2-1was further characterized in this study.

Methods: To reveal whether ACGT2-1 is involved in BaMV infection cycle, Tobacco rattle virus-based virus-induced gene silencing (loss-of-function) experiment is performed to inspect the influence on the accumulation of BaMV in plants. To clone the full-length gene of ACGT2-1, 5' and 3' rapid amplification of cDNA ends technique is used. The full-length of ACGT2-1 is expressed in plants (gain of function) to inspect the effect on BaMV accumulation.

Results: The expression level of ACGT2-1 is knocked down in N. benthamiana, the accumulation of BaMV coat protein in knockdown plants and protoplasts are increased significantly compared to those of the controls. Northern blot analysis is also indicated that the accumulation of BaMV RNA is increased when ACGT2-1 is silenced. These results suggest that ACGT2-1 plays a defense role in the replication step of BaMV infection cycle. The full-length cDNA of ACGT2-1 is obtained. The sequence encodes an open reading frame comprising a conserved RING-domain and a transmembrane domain. Based on these observations, this gene can be functioning as an ubiquitination E3 ligase, we designate this protein as NbUbE3R1. To confirm the defense role of NbUbE3R1 against BaMV, the wild type NbUbE3R1-OFP and mutant NbUbE3R1/△TM-OFP (no transmembrane domain) are transiently expressed in plants. The results indicate that NbUbE3R1 is mainly associated with plasma membrane, the accumulation of BaMV coat protein is decreased significantly when NbUbE3R1 is transiently expressed but not in NbUbE3R1/△TM-expressed plants.

Conclusion: In this study, we have identified an upregulated gene NbUbE3R1 after BaMV infection plays a role against BaMV replication. It is possible that the Ubiquitin E3 ligase NbUbE3R1 targets the viral replication-related proteins for degradation.

Keywords: defense, E3 ligase, RNA replication

Abstract Submission

17th International Congress of Virology 335

Plant viruses

PO765

GENERATION OF AN INFECTIOUS CLONE OF A RECENT KOREAN ISOLATE OF YOUCAI MOSAIC VIRUS (TOBAMOVIRUS) UNDER THE CONTROL OF T7 AND 35S PROMOTER

H.-K. Ju 1,*, B. Kim 1, G.-W. Choi 1, I.-H. Kim 1, J. Kim 1, E.-Y. Seo 1, C.-H. Park 1, J. Hammond 2, H.-S. Lim 1 1Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon, Korea, Republic Of, 2United States Department of Agriculture-Agrocultural Research Service, Floral and Nursery Plants Research Unit, Beltsville, United States

Objectives: Youcai mosaic virus (YoMV, also known as Chinese rape mosaic virus, or Oilseed rape mosaic virus) was first isolated from Brassica campestris in China in 1962. According to recent reports, YoMV has a wide host range, infecting plants from 11 families including Brassicaceae, Solanaceae, Asteraceae and Liliaceae in Korea, Japan, Germany, Spain, Switzerland and China. Recently we isolated a suspected YoMV isolate from a Chinese cabbage field in Korea.

Methods: In order to confirm YoMV sequence and symptoms we cloned YoMV into versatile binary vector pJY, which incorporates dual T7 and 35S promoters for in vitro transcription and Agroinfiltration respectively.

Results: The c.6.3 kb infectious clone produced the same symptoms of mottle/mosaic and stem necrosis, after inoculation by either transcripts or Agroinfiltration, as shown in N. benthamiana inoculated from virus-infected tissue. Full length sequence analysis was performed with other tobamovirus species and nine YoMV isolates from NCBI. Our new isolate showed 91.58-99.81% identities in amino acid sequence compared to other YoMV isolates and is most related to an isolate from Japan. Full length sequence analysis using DNAman predicted a 125 kDa protein with a 182 kDa protein produced by readthrough of 125 kDa. The Movement protein and Coat protein were predicted to be 30 kDa and 17.5 kDa respectively.

Conclusion: A new suspected YoMV isolate causing symptoms in a cabbage field in Korea was confirmed to be YoMV; we constructed a full length infectious clone which will be valuable to investigate YoMV pathogenesis and characterize the new isolate.

Keywords: Infectious clone, Phylogenetics, Youcai mosaic virus

Abstract Submission

17th International Congress of Virology 336

Plant viruses

PO766

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF PLANT VIRUSES IN HERBAL CROPS BASED ON NEXT-GENERATION SEQUENCING AND METAGENOMICS ANALYSIS

S.-J. Kwon*, I.-S. Cho 1, J.-Y. Yoon 1, B.-N. Chung 1 1Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Wanju, Korea, Republic Of

Objectives: Various herbal crops have been grown and consumed as traditional medicines in Korea. As the positive effects of herbal crops for human health and their major ingredients have been proved scientifically, their production and consumption have gradually increased in Korea. However, disease information (i.e. kinds, incidence, distribution, pathology and control methods, etc) in herbal crops is very restricted.

Methods: Thus, the identification of previously unreported pathogens, especially viruses, is quite difficult and sometimes takes long time. In this study, we employed next-generation sequencing (NGS) technologies for high-throughput identification of plant viruses and molecular characterization of their genome sequences.

Results: Using this approach, we analyzed various symptomatic herbal crop samples collected by field surveys. The analyzed herbal crops include Chinese foxglove (Rehmannia glutinosa), Chinese yam (Dioscorea opposita Thunb.), green tea (Camellia sinensis), achyranthes (Achyranthes bidentata Blume) and Chinese matrimoni vine (Lycium chinense Miller). The analyses resulted in identification of various viruses, including Broad bean wilt virus 2, Chinese yam necrotic mosaic virus, Plantago asiatica mosaic virus, Rehmannia mosaic virus, Youcai mosaic virus and Mint virus I.

Conclusion: Broad bean wilt virus 2 was isolated from various herbal crops, including Chinese foxglove, Chinese yam, achyranthes, and Chinese matrimoni vine. Whereas, Plantago asiatica mosaic virus, Rehmannia mosaic virus was only detected on Chinese foxglove. We also performed phylogenetic analyses of the identified viruses using their genomic sequences obtained by NGS to investigate their evolutionary positions.

Keywords: plant viruses, herbal crops, next generation sequencing

17th International Congress of Virology 337

Abstract Submission

Plant viruses

PO767

INTERACTION BETWEEN THE NEW WORLD BEGOMOVIRUS EUPHORBIA YELLOW MOSAIC VIRUS AND ITS ASSOCIATED ALPHASATELLITE

T. Mar 1, I. Mendes 1, D. Lau 2, E. Fiallo-Olivé 3, J. Navas-Castillo 3, M. Alves 1, F. Zerbini 1,* 1Dep. de Fitopatologia, UNIVERSIDADE FEDERAL DE VIÇOSA, Viçosa, 2Embrapa Trigo, Passo Fundo, RS, Brazil, 3CSIC, Málaga, Spain

Objectives: The majority of Old World monopartite begomoviruses (family Geminiviridae) are associated with satellite DNAs. Alphasatellites are capable of autonomous replication, but depend on the helper virus for movement, encapsidation and transmission by the insect vector. Recently, Euphorbia yellow mosaic alphasattelite (EuYMA) was found in association with Euphorbia yellow mosaic virus (EuYMV) infecting Euphorbia heterophylla plants in Brazil. The objective of this work was to study the interaction between EuYMV and its associated alphasatellite.

Methods: Samples of E. heterophylla plants showing typical symptoms of begomovirus infection (yellow mosaic, leaf curling, and stunting) were collected in locations in several Brazilian states from 2009 to 2014. The samples were stored in plastic bags and transported to the laboratory where they were press-mounted until DNA extraction. Viral genomes were amplified using rolling-circle amplification (RCA). Aliquots of the amplification products were subjected to cleavage with restriction enzymes to obtain fragments of approx. 2,600 or 1300 nucleotides (nt), corresponding to one genomic copy of virus and satellite, respectively. These fragments were cloned and completely sequenced. Multiple sequence alignments were performed using MUSCLE as implemented in MEGA 6. Phylogenetic reconstruction was performed with Bayesian inference. Tandem dimeric constructs of each genomic component of EuYMV and of the associated EuYMA were obtained by partial restriction digestion and were used in host range assays using biolistics. Virus and alphasatellite titers were determined by quantitative PCR. A silencing suppression assay for the alpha-Rep protein was performed in N. benthamiana plants. Transmission of EuYMV by B. tabaci MEAM1 was tested in the presence or absence of EuYMA.

Results: We assessed the geographical range of EuYMA in a representative sampling (n=165) of E. heterophylla plants collected in several states of Brazil from 2009 to 2014. Infectious clones were generated and used to assess the phenotype of viral infection in the presence or absence of the alphasatellite in tomato, E. heterophylla, Nicotiana benthamiana, Arabidopsis thaliana and Crotalaria juncea. Phenotypic differences of EuYMV infection in the presence or absence of EuYMA were observed in A. thaliana, N. benthamiana and E. heterophylla. Symptoms were more severe when EuYMV was inoculated in combination with EuYMA in N. benthamiana and E. heterophylla, and the presence of the alphasatellite was determinant for symptom development in A. thaliana. The quantification of EuYMV and EuYMA indicated that EuYMA affects the accumulation of EuYMV during infection on a host-dependent basis. Agroinfiltration assays indicated that the EuYMA alpha-Rep protein does not act as a post-transcriptional silencing suppressor in N. benthamiana. Transmission assays indicated that EuYMA negatively affects the transmission of EuYMV by Bemisia tabaci MEAM1.

Conclusion: Together, these results indicate that EuYMA is capable of modulating symptoms, viral accumulation and whitefly transmission of EuYMV, potentially interfering with virus dissemination in the field.

Keywords: Bemisia, DNA satellite, geminivirus

17th International Congress of Virology 338

Abstract Submission

Plant viruses

PO768

MOLECULAR CHARACTERISATION AND DIAGNOSIS OF BADNAVIRUSES INFECTING YAMS IN THE SOUTH PACIFIC

A. Sukal 1,*, D. Kidanemariam 1, J. Dale 1, A. James 1, R. Harding 1 1CTCB, Queensland University of Technology, Brisbane, Australia

Objectives: Yams (Dioscorea spp.) are economically important, annual or perennial tuber-bearing, tropical plants. Globally yam is ranked as the fourth most important root crop by production and provides a staple food for millions of people in Africa, South America, Asia and the Pacific. In Pacific Island countries (PICs), the production and utilization of yams is limited by several issues including diseases and the lack of genetic diversity. An important global in vitro collection of yam germplasm is conserved in tissue culture by the Secretariat of the Pacific Community (SPC) in Fiji. Evaluation of this germplasm and its distribution to PICs holds the key to improved production. Like other vegetatively propagated crops, yam has a tendency to accumulate and perpetuate tuber-borne fungal and viral diseases. Although tissue culture eliminates fungal pathogens, viruses remain an issue. As such, quarantine regulations prohibit the movement of the yam germplasm from the SPC germplasm collection to other countries due to the risks associated with movement of untested and/or virus-infected material. To comply with these standards, sensitive diagnostic tests are needed to enable the virus indexing of yam germplasm. Several different viruses are known to infect yams, but the circular dsDNA-containing badnaviruses remain the least studied and the most difficult to diagnose. The limited studies conducted on yam-infecting badnaviruses suggest they are prevalent in PICs and are highly diverse. This genetic variability hinders the development of reliable PCR-based diagnostic tests. This is further complicated by the fact that badnavirus sequences are integrated in the genomes of some yam cultivars leading to false positives using PCR-based tests.

Methods: Rolling circle amplification (RCA) is a technique that has been shown to selectively amplify circular viral DNA without amplification of the integrated counterpart. As such, RCA is being used to characterize the diversity of badnavirus infecting Pacific germplasm. A subset of the germplasm conserved at the SPC has been screened using RCA and amplified episomal badnavirus DNA has been sequenced to better understand the diversity of badnavirus infecting pacific yam germplasm.

Results: Full genomes of three species of yam infecting badnavirus have been sequenced from the Pacific. Other partial genomes have also been characterized.

Conclusion: The characterisation of badnaviruses infecting yams will assist in the development of reliable diagnostic techniques and furnish germplasm centers with the tools to safely mobilize their yam collections, so that food and nutritional security can be achieved in the Pacific and other regions.

Keywords: badnavirus, Dioscorea , rolling circle amplification

17th International Congress of Virology 339

Abstract Submission

Plant viruses PO769

MOLECULAR CHARACTERIZATION OF VIRUSES INFECTING TARO IN EAST AFRICA D. Kidanemariam 1 2,*, A. Sukal 1, A. Abraham 3, F. Stomeo 4, J. Dale 1, A. James 1, R. Harding 1 1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia, 2Holetta Biotechnology, Ethiopian Institute of Agricultural Research , 3College of Biological and Chemical Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia, 4Biosciences for east and central Africa , International Livestock Research Institute , Nairobi, Kenya

Objectives: Taro (Colocasia esculenta L.) is a widely cultivated edible member of the Araceae family. It is one of the staple food crops in sub-Saharan Africa, with both the foliage and corms utilised as a food source. Despite its substantial contribution to the food and income security for many small-scale farmers in east Africa, the average yields are below the potential of the crop due to various factors including diminishing soil fertility, lack of improved varieties and the presence of pests and diseases. Viruses are known to be one of the most important constraints to taro production, and can cause severe yield reduction. Since the production of taro in east Africa has declined significantly over time, the identification of viruses affecting taro in the region has become a research priority.

Methods: Leaf samples were collected from major taro growing areas in Ethiopia, Kenya, Tanzania and Uganda and nucleic acid was extracted. Initially, samples were screened for different RNA and DNA viruses by PCR using published virus- specific and/or degenerate primers. For RNA viruses, representative samples were also subjected to illumina MiSeq NGS sequencing and data validated using Sanger sequencing. For badnaviruses, the complete genome of representative samples was amplified using rolling circle amplification (RCA) and sequenced using a primer walking approach.

Results: To date, several viruses have been detected including Dasheen mosaic virus (DsMV), Taro bacilliform virus (TaBV) and Cucumber mosaic virus (CMV), with additional analysis ongoing.

Conclusion: Results of the surveys and virus incidence/distribution will be discussed.

Keywords: CMV, DsMV, TaBV, east Africa, taro, NGS, RCA

17th International Congress of Virology 340

Abstract Submission

Plant viruses PO770

PHYLOGENY AND MOLECULAR CHARACTERISTICS OF UNSEGMENTED AND SEGMENTED PLANT RHABDOVIRUSES R. G. Dietzgen 1,*, D. Zou 1, P. J. Walker 2 1QAAFI, 2School of Biological Sciences, The University of Queensland, St. Lucia, Australia

Objectives: Plant rhabdoviruses infect diverse monocots and dicots, and are transmitted by arthropods in a persistent propagative manner. The family Rhabdoviridae is classified taxonomically in the order Mononegavirales together with other families of negative-strand RNA viruses with unsegmented genomes. In a recent taxonomy update, two genera of viruses with segmented genomes (Dichorhavirus and Varicosavirus) were also included in the family. Further, an increasing number of plant rhabdovirus genomes are being reported due to improvements in next generation sequencing technologies that do not require prior virus purification. Phylogenetic analyses of unsegmented and bi-segmented plant rhabdoviruses reveal several new clades of viruses that are not assigned to currently recognized genera, indicating a need for revisions.

Methods: Complete plant rhabdovirus genomes and partial polymerase (L) gene sequences were downloaded from the National Center for Biotechnology Information (NCBI). Nucleotide and deduced amino acid sequences were analysed using Pairwise Sequence Comparison (PASC) and Sequence Demarcation Tool (SDT) software. Multiple sequence alignments and phylogenetic trees were generated in Geneious R9. Genome organisation, plant host and insect vector information and country of origin of virus isolates were obtained from NCBI and the published scientific literature.

Results: The taxonomic classification of unsegmented plant rhabdoviruses into the two genera Cytorhabdovirus and Nucleorhabdovirus based on their intracellular sites of replication and virion accumulation has been fully supported by genome sequence data thus far, but the naming using this characteristic is out of step with that of vertebrate rhabdovirus genera. With the increasing availability of rhabdovirus genome sequences and the inclusion of rhabdoviruses with bi- segmented genomes, phylogenetic analysis has revealed new sub-clades of viruses with similar genome organisation. These may warrant establishment of several new taxonomic groups to replace the current classification. Significantly, viruses assigned to the new genus Dichorhavirus (bi-segmented genomes) form a monophyletic group that lies within a clade of viruses with unsegmented genomes that are currently assigned to the genus Nucleorhabdovirus. Taking into account the phylogenetic groupings, overall genome organisation, location and number of accessory genes, plant hosts, insect vectors and geographic distribution, we propose alternative classifications of plant-infecting rhabdoviruses.

Conclusion: The emerging diversity and evolutionary relatedness of plant rhabdoviruses is not clearly represented in the current taxonomy. We propose a revision of taxonomic classification and establishment of several new genera to better reflect genome organisation and phylogeny of plant-infecting rhabdoviruses.

Keywords: Phylogeny, Rhabdoviruses, Taxonomy

17th International Congress of Virology 341

Abstract Submission

Plant viruses PO771

RNAI-MEDIATED RESISTANCE FOR CONTROL OF BANANA BUNCHY TOP VIRUS DISEASE B. Mware 1,*, J. Anthony 2, R. Harding 2, J. Dale 2 1International Institute of Tropical Agriculture (IITA), PO Box 30709-00100, Nairobi, Kenya, 2Centre for Tropical Crops and Biocommodities, Queensland University of Technology, 2 George St, Brisbane, Queensland, 4000, Australia

Objectives: Banana bunchy top disease (BBTD), caused by Banana bunchy top virus (BBTV) (genus Babuvirus, family Nanoviridae), is the most important viral disease of banana. The disease is widespread in the Asia-Pacific region with outbreak occurrences in sub-Saharan Africa. BBTV consists of at least six circular, single-stranded DNA components each about 1 kb. BBTV isolates are grouped into the ‘South Pacific’ subgroup comprising isolates from Australia, the Pacific Islands, India, Iran, Myanmar, Pakistan and Africa, and the ‘Asian’ subgroup isolates from Philippines, Vietnam, China (including Taiwan) and Indonesia. BBTV infection causes severe stunting and loss of fruit production with complete yield losses when infection occurs at early stages of plant development. Despite the fact that quarantine and phytosanitary measures can control bunchy top, this approach is not feasible in many countries. Further, the apparent lack of natural resistance in Musa germplasm precludes the use of conventional breeding for developing resistance. The main aim of this study, was to genetically modify Australian dessert banana varieties (both Dwarf Cavendish and Grand Nain) with a suite of RNAi constructs targeting three different BBTV genes, namely the Master Rep (DNA-R), the movement protein (DNA-M) and the nuclear shuttle protein (DNA-N), to generate immunity to BBTV and determine the most appropriate target for robust transgenic resistance.

Methods: Polymerase chain reaction and gene cloning techniques were applied to develop a total of 10 hairpin constructs containing one, two or three gene hairpin sequences based on an Australian isolate of BBTV. The hairpin constructs were used for agrobacterium-mediated transformation of banana cell suspensions which were regenerated to over 250 transgenic banana plants.

Results: Of the three genes targeted, hairpins based on the BBTV DNA-M sequence provided the most effective resistance to BBTV infection with 7 of 11 lines tested showing complete immunity 12 weeks post infection. The DNA-M hairpin was also effective when co-expressed with a hairpin targeting the DNA-R gene sequence. In this case, 62 % of lines containing both hairpins showed complete immunity to the virus. Importantly, this action correlated strongly with (i) low copy number integration events, where single copy transgenic plants showed higher levels of resistance to high copy number events and (ii) the production of gene-specific, small 21 nt dsRNA species, hallmarks of the RNAi process.

Conclusion: This study reports for the first time RNAi-mediated transgenic resistance in banana targeting BBTV movement protein gene. Based on the current results silencing movement protein gene, a silencing suppressor together with Master Rep gene may lead to a robust transgenic resistance for control of BBTD in banana.

Keywords: BBTV, Resistance, RNAi

17th International Congress of Virology 342

Abstract Submission

Plant viruses PO772

Silencing of the nuclear proteins, fibrillarin and coilin, reduces accumulation of potato virus Y A. Makhotenko 1 2, S. Makarova 1 2, J. Shaw 3, A. Love 3, N. Kalinina 1 2, M. Taliansky 2 3,* 1Lomonosov Moscow State University, Moscow, 2Pushchino Branch, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow Region, Russian Federation, 3The James Hutton Institute, Dundee, United Kingdom

Objectives: Potato virus Y (PVY) is an important plant virus and causes great yield and quality losses. An increasing number of reports reveal that in addition to their well-known roles in RNA metabolism and RNA-protein assembly, the nucleolus and Cajal bodies (CBs), both located within the cell nucleus, are involved in plant virus interactions. In particular, nucleolar (fibrillarin) and CB (coilin) proteins have been shown to interact with proteins encoded by several plant viruses. To examine whether fibrillarin and coilin play roles in PVY infection, expression of these proteins was suppressed in Nicotiana tabacum plants using transgenic (RNAi) silencing (knock-down, KD). Methods: Fibrillarin- and coilin KD transgenic plants were generated by transgenic expression of hairpin RNA constructs derived from fibrillarin and coilin N. tabacum genes respectively, which were designed to avoid “off-target” silencing. Results: In this work, we showed that fibrillarin silencing (60-80%) substantially attenuated PVY-induced symptoms and significantly reduced accumulation of PVY in inoculated leaves. However, systemic spread of PVY to upper non-inoculated leaves was unaffected by fibrillarin deficiency. Further accumulation of PVY in systemically infected leaves was also reduced in fibrillarin KD plants. The molecular mechanism of this reduction is unlikely to be accounted for by RNA silencing as the amounts of PVY- specific siRNAs detected in fibrillarin KD leaves were not increased in comparison to WT but were slightly reduced. Coilin silencing had a similar effect to fibrillarin silencing on PVY infection. Conclusion: In conclusion, the involvement of the nuclear proteins, fibrillarin and coilin in plant-virus (PVY) interactions represents novel phenomenon that expands knowledge of the biology of subnuclear bodies, the nucleolus and CBs, respectively. The possible molecular basis of these effects and practical implications for control of virus infections will be discussed. This study was supported by Research Grant No. 14.W03.31.0003 from the Government of the Russian Federation.

Keywords: coilin, fibrillarin, potato virus Y

17th International Congress of Virology 343

Abstract Submission

Plant viruses PO773

Subcellular localization of Cucumber Mosaic Virus 2b protein regulated via phosphorylation K. Nemes 1, Á. Gellért 2, A. Almási 1, P. Vági 1, K. Kádár 1, K. Salanki 1,* 1Plant Pathology, Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 2Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Budapest, Hungary

Objectives: The CMV 2b protein is multifunctional; it has a role in almost all steps of the viral cycle. It is a suppressor of antiviral RNA silencing, recognizing and binding not only RNA (siRNA, miRNA) but the protein components (AGO1 and AGO4) of the defense machinery. It has also roles in symptom induction, evasion of the defense mechanism mediated by salicylic and jasmonic acid, as well as in cell-to-cell movement. During viral infection, CMV 2b protein accumulates within the cytoplasm and in the cell nucleus as well. Phosphorylation site of 2b protein is conserved in all of the CMV isolates and its deletion and two point mutations were connected to nuclear localization, although the phosphorylated form of the 2b protein and the role of the actual phosphorylated state of the 2b protein were not analyzed previously.

Methods: To discern the impact of 2b protein phosphorylation, we examined the effect of the mutations located in putative phosphorylation site (40-42 SPS) in symptom induction, gene silencing suppressor activity as well as in subcellular localization.

Results: First we have directly demonstrated the phosphorylation state of 2b protein. Then we have created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. Infection with Rs-SPS/40-42/SPD and Rs-SPS/40-42/DPS induced mild symptoms: mild leaf distortion, mild systemic mosaic, and mild stunting, while Rs-SPS/40-42/APS, Rs-SPS/40-42/SAS and Rs-SPS/40-42/SPA exhibited severe symptoms, including systemic mosaic, leaf distortion, and chlorosis on N. benthamiana plants. However, symptoms induced by these mutants were never as severe as those induced by Rs-CMV.

Conclusion: Gene silencing suppressor activity of the mutant 2b proteins investigated in patch assays. We found that mutants 2b/40-42/SAS, 2b/40-42/APS, 2b/40-42/APS and 2b/40-42/SPA retained

Keywords: 2b protein, cucumber mosaic virus, nuclear trafficking

17th International Congress of Virology 344

Abstract Submission

Plant viruses PO774

The importance of a putative helix at C-terminal region of Watermelon silver mottle virus nonstructural NSs protein for self-interaction and RNA silencing suppression C.-H. Huang 1,*, M.-H. Foo 1, T.-T. Lin 1, S.-S. Lin 2, S.-D. Yeh 1 1Plant Pathology, National Chung Hsing University, Taichung, 2Biotechnology, National Taiwan University, Taipei, Taiwan

Objectives: Watermelon silver mottle virus (WSMoV), a member of the genus Tospovirus, encodes a viral suppressor NSs protein against host RNA silencing. This protein forms filamentous inclusion body in the cytoplasm of infected plant cells and functions as a pathogenicity determinant. Our previous study indicated that NSs protein has an ability of self-interaction as evidenced by yeast two hybrid (Y2H) assay. Here, we investigated the funtional motifs which are responsible for self- interaction of NSs protein.

Methods: In this study, in vitro co-imunoprecipitation (Co-IP) and in vivo bimolecular fluorescence complement (BiFC) assays were further used to analyze the functional motifs for self-interaction of NSs protein. Furthermore, mutated NSs proteins were expressed by Escherichia coli and the nucleic acid binding ability was tested with gel shift assay using synthesized and radiolabeled small and long dsRNAs.

Results: We identified a putative α-helix at C-terminal region of the NSs protein (aa. 338-360, denoted as H8) involves in its protein stability and self-interaction. Mutation at the three aa residues within the H8 helix, Y338/H350/F353 (denoted as triple-point-mutation, TPM), disrupted the stability of NSs protein and caused dysfunction in self-interaction. In addition, we found the mutated proteins of ΔH8 and TPM are unstable in agroinfiltrated cells of Nicotiana benthamiana plants and thus result in weaker RNA silencing suppression activity. Gel shift assay revealed that the TPM protein retains similar siRNA binding ability as the wild type NSs.

Conclusion: Taken together, our results suggest that monomeric NSs protein possesses RNA silencing suppression function. Presumably, self-interaction helps stabilize NSs protein and prevent its degradation in planta.

Keywords: RNA silencing suppressor, Self-interaction, tospovirus

17th International Congress of Virology 345

Abstract Submission

Plant viruses PO775

The major potyvirus causing passionfruit woodiness disease in Taiwan is reclassified as East Asian Passiflora virus Y. H. Chong*

Objectives: Passionfruit woodiness virus (PWV) is a potyvirus that causes serious damage on passionfruit. Based on filamentous particles, aphid transmission and symptoms of distorted and woody fruits, the devastating passionfruit woodiness disease (PWD) in Taiwan has been regarded as PWV for decades. However, molecular characterization is needed to clarify the classification status of the causal agent.

Methods: Four potyvirus isolates collected from different parts of Taiwan, originally designated as PWV-TW, PWV 0920-6, PWV-dpd and PWV-pt, were used for molecular characterization. Complementary DNA fragments corresponding to the different genomic parts of the four isolates were amplified by RT-PCR, sequenced and analyzed. Phylogenetic analyses with other potyviurses were conducted.

Results: The CP genes of four Taiwan isolates share 94.9% - 98.5% nt identities (95.9% and 97.6% aa identities) with Japan East asian passiflora virus (EAPV). However, their CP genes share only 67.5% - 69.4% nt identities (68.5% - 69.7% aa identities) with Australia PWV. The genomic sequences of the four Taiwan isolates also share high degrees of homology with that of EAPV-AO (>98%) and EAPV-IB (> 88%), but only 61.8% - 64.4% identities with PWV. These results coupled with phylogenetic analyses indicate that the four Taiwan potyviruses are strains of EAPV and not strains of PWV. A field survey with 175 samples from different areas of Taiwan revealed that the PWD is caused by EAPV, and not by PWV.

Conclusion: Our results of molecular characterization and field survey indicate that the major potyvirus causing PWD in Taiwan is EAPV.

Keywords: East Asian Passiflora virus, Passionfruit, Passionfruit woodiness disease

17th International Congress of Virology 346

Abstract Submission

Plant viruses PO776

TWO HOMOLOGOUS HOST PROTEINS INTERACT WITH POTATO VIRUS X RNAS AND CPS AND AFFECT VIRAL REPLICATION AND MOVEMENT

H. Choi 1 2,*, K. Widyasari 1 2, K.-H. Kim 1 2 1Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, 2Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea, Republic Of

Objectives: The viruses encode a limited number of proteins and thus specific interaction between viruses and hosts is a critical to complete virus infection cycles. In this study, we identified the function of two homologous proteins referred as NbCPIP2a and NbCPIP2b that are induced upon potato virus X (PVX) infection.

Methods: To identify the interaction trait of NbCPIP2a and NbCPIP2b, we performed electrophoretic mobility shift assay (EMSA) for protein-RNA interaction, and bimolecular fluorescence complementation assays (BiFC) for protein-protein interaction. Transient overexpression and silencing experiment mediated Agrobacterium were investigaed for gain-of- function and loss-of-function experiment.

Results: Interaction activity of both proteins with SL1 (+), SL1 (-) RNA, and coat protein (CP) of PVX was confirmed by EMSA and BiFC. Interaction studies with deletion and site-directed mutants introduced onto NbPCIP2 showed that five amino acid residues of DnaJ_C domain located in C-terminus of NbCPIP2 proteins were important for interaction with PVX CP. Overexpression of NbCPIP2a positively influenced systemic movement of PVX in N. benthamiana, whereas NbCPIP2b overexpression did not affect systemic movement of PVX. Transient overexpression and silencing experiments demonstrated NbCPIP2a and NbCPIP2b were positive regulators for the replication of PVX; however, NbCPIP2a showed greater effect on PVX replication than that of NbCPIP2b.

Conclusion: Taken together, these results indicate the NbCPIP2a and NbCPIP2b displaying five amino acid substitutions specifically bind PVX SL1 RNAs as well as CP and positively affect PVX replication.

Keywords: Host protein, Nicotiana benthamiana, Potato virus X

17th International Congress of Virology 347

Abstract Submission

Plant Viruses PO829

A putative new chrysovirus-like virus from resurrection fern N. Aboughanem-Sabanadzovic 1, A. Lawrence 2, J. Hill 3, S. Sabanadzovic 3,* 1Institute for Genomics, Biocomputing and Biotechnology, 2Institute for Imaging and Analytical Technologies , 3Departement of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, United States

Objectives: Objective of this work was to study viruses associated with the resurrection fern (Pleopeltis polypodioides; syn. Polypodium polypodioides). The Resurrection fern is an epiphyte native to the southern United States that is characterized by extreme resistance to drought.

Methods: In order to investigate viruses associated with resulrrection fern we employed dsRNA extraction and PAGE analysis, random-primed cloning and sequencing, 5'/3' RACE, etc.. Assembly, analyzes and comparion of sequences were performed with a suite of proper software.

Results: Few samples of resurrection fern collected from different locations contained virually identical dsRNA pattern consisting of three molecules. Cloning/sequencing experiments and pairwise sequence comparison indicated that samples contain the same putative new virus, designated as resurrection fern virus 1 (RFV1), as they shared 95-98% identical residues.Complete sequencing revealed tripartite genome with similarities with extant chrysoviruses. Phylogenetic analyses of viral RdRp showed close relationships of RFV1 with recently described putative tripartite chrysoviruses from Raphanus sativus and Brassica campestris. Partially purified preparations from dsRNA-containing ferns showed the presence of isometric particles assumed to be RFV1 virions. Virus-specific primers, designed in this study, identified the presence of RFV1 in several samples from different locations and hosts (oak, cedar and pecan). Initial attempts to identify a fungus as a possible primary host for this virus have failed, suggesting that RFV1 represent a true plant chrysovirus.

Conclusion: In this work we identified and characterized a putative new isometric virus with tripartite dsRNA genome from resurrrection fern with affinities with extant members of the genus Chrysovirus.The virus was provisionally named resurrection fern virus 1 (RFV1). Accoridng to initial results of our survey RFV1 seems rather widespread in population of resurrection fern in Mississippi.

Keywords: None

17th International Congress of Virology 348

Abstract Submission

Plant viruses PO882

THE TIME OF EMERGENCE AND DISSEMINATION OF TURNIP MOSAIC VIRUS R. Yasaka 1,*, S. Korkmaz 2, A. Golnaraghi 3, N. Katis 4, S. Ho 5, A. Gibbs 6, K. Ohshima 1 1Department of Applied Biological Sciences, Saga university, Saga, Japan, 2Department of Plant Protection, University of Canakkale Onsekiz Mart, Canakkale, Turkey, 3Department of Plant Protection, Islamic Azad University, Tehran, Iran, Islamic Republic Of, 4Plant Pathology Laboratory, Aristotle University of Thessaloniki, Thessaloniki, Greece, 5School of life and environmental sciences, University of Sydney, Sydney, 6Emeritus Faculty, Australian National University, Canberra, Australia

Objectives: Turnip mosaic virus (TuMV) is a member of the genus Potyvirus in the family Potyviridae that damages most domestic brassica crops in modern agriculture. This virus originated from wild orchids in Europe and then spread among species of wild and domestic Brassicaceae plants, from the Mediterranean region, including South-east Europe, the Middle East and Central Asia, to other parts of the world. However, the population of the virus in the Middle East and neighboring countries is largely unknown. This region is thought to be the center of emergence and spread of this virus, and the region in which it adapted to agricultural crops. Here we estimate the evolutionary rate and timescale of this virus and infer its phylodynamic history using a combined data set of novel and published genome sequences. Methods: We collected 179 TuMV isolates in Greece, Iran and Turkey between 1993 and 2012 and determined their full genomic sequences. Putative recombination breakpoints in all sequences were identified using the RDP4 package. The phylogenetic relationships were inferred using SPLITSTREE and PhyML. Synonymous sites from a total of 417 novel and published full genomic sequences were used in Bayesian coalescent analyses. Substitution rates and divergence times were estimated using BEAST after removing the recombinant sequences. Bayes factors were used identify the disseminations between pairs of locations. Results: Phylogenetic analysis showed the presence of six phylogenetic groups in TuMV: Orchis, basal-B, basal-BR, Asian-BR, world-B and new Iranian groups. The basal-B group further split into basal-B1 and B2 and the world-B group split into the world-B1, B2 and B3 subgroups. A Bayesian phylogenetic method was used to estimate the evolutionary rates and timescale for the complete major ORF (open reading frame, polyprotein), HC-Pro* (partial helper-component proteinase protein), P3* (partial protein 3) and NIb* (partial nuclear inclusion b protein) regions. The mean evolutionary rates were 0.82–1.65×10-3 substitutions per site per year for the four protein-coding regions. Moreover, the time to the most recent common ancestor (TMRCA) for these regions was also estimated. The TMRCA of the major ORF region was approximately 1570 years, and those of three shorter HC-Pro*, P3* and NIb* regions were 1059–1178 years. We found evidence that circulated not only within each country (Greece, Iran and Turkey) but also between these countries. All the results agreed that TuMV had entered the Middle East from the west and had progressively spread eastwards. Conclusion: TuMV probably originated from a virus of wild orchids in Germany and, while adapting to wild and domestic brassicas, spread via Southern Europe to Asia Minor no more than 700 years ago. The timescale of the dissemination of TuMV seems to correlate well with the establishment of agriculture in the Middle East and neighboring countries.

Keywords: Dissemination, Molecular evolution, Timescale

17th International Congress of Virology 349

Positive Strand RNA Viruses

17th International Congress of Virology 350

Abstract Submission

Positive Strand RNA Viruses PO676

DUAL SEASONAL PATTERNS AND INCREASED LATE SUMMER AND AUTUMN ACTIVITY OF HUMAN NOROVIRUSES IN HONG KONG DURING 2014-2016 M. Chan 1,*, T.-N. Hung 1, K. Kwok 1, L.-Y. Chan 1, P. Chan 1 2 1Department of Microbiology, 2Stanely Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong, China

Objectives: Human noroviruses are a diverse group of diarrheagenic RNA viruses that are leading causes of acute gastroenteritis and foodborne illnesses in all ages worldwide. These viruses generally have higher activities in winter months and are thus also known as “winter-vomiting viruses”. This study aimed at studying norovirus seasonality in Hong Kong which is a subtropical coastal city in southern China with a climate towards temperate region.

Methods: A hospital-based norovirus surveillance has been established in Hong Kong since March 2014. Laboratory- confirmed hospitalized norovirus infections in both children and adults were recruited. Stool samples were collected and noroviruses were genotyped by Sanger sequencing of viral protein 1 gene. Norovirus seasons were defined as three consecutive months showing highest local activities.

Results: From March 2014 through December 2016, a total of 995 cases were studied. Virus genotype was determined in 892 (89.6%) cases. Dual seasonal patterns, consisting of late summer/autumn and winter seasons, for noroviruses were observed. Late summer/autumn seasons peaked every year from August to October and they were attributed to GII.4 Sydney 2012. In sharp contrast, winter seasons peaked in January to February (2014/15 and 2015/16) and they were attributed to another genotype called GII.17 Kawasaki that emerged in Asia in late 2014. In the past three years, numbers of hospitalized norovirus infections in late summer/autumn seasons have more than doubled (111%) and that in winter seasons have nearly halved (48%). Preliminary molecular surveillance data suggested that the ongoing winter of 2016/17 may be predominated by another previously rare non-GII.4 genotype called GII.2. Conclusion: We provided evidence showing dual seasonal patterns for noroviruses caused by different genotypes in Hong Kong. Late summer and autumn activity of noroviruses was on the rise.

Acknowledgements: This study was supported in part by the Commissioned Health and Medical Research Fund (Phase 3) of Food and Health Bureau of the HKSAR Government (to MCWC; reference number CU-15-C2).

Keywords: Molecular epidemiology, Norovirus, Seasonality

17th International Congress of Virology 351

Late breaking abstract

Positive Strand RNA Viruses PO777

Cell Cycle Arrest at G1/S phase Induced by MERS Coronavirus Nucleocapsid Protein O.-W. Ng 1,*, J. O. Aboagye 1, Y.-J. Tan 1 2 1Department of Microbiology and Immunology, NATIONAL UNIVERSITY OF SINGAPORE, 2Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

Objectives: First emerged in 2012, the Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a human coronavirus capable of causing severe lower respiratory tract infections which can be fatal. The ongoing MERS epidemic in Middle East over the past years significantly highlights the continual worldwide threats of MERS-CoV. It is therefore crucial to understand the viral-host interactions involved in viral replication and pathogenesis for the development of antivirals against MERS-CoV, which are currently unavailable. The coronavirus nucleocapsid (N) protein is a multi-functional protein capable of interacting with numerous host factors, resulting in a range of cellular effects in host cells. Little is currently known about the functions of the MERS-CoV N (MERS- N) protein in the process of viral replication and pathogenesis. Here, we aim to define the role and cellular functions of MERS-N protein when expressed in host cells.

Methods: A549 cells stably expressing MERS-N protein were generated and used for this study. Flow cytometry was used to analyse cell cycle progression of the cells. A gene profiling assay focusing on cell cycle genes was carried out using the stable cells to identify genes that are deregulated by the expression of MERS-N.

Results: In current study, we investigated the effects of the MERS-CoV N protein on cell cycle of host cells. By flow cytometry analysis, it was found that the A549 stable cells expressing the MERS-N protein showed a slower progression through the G1 and/or S phase compared to control cells. To identify the genes responsible for this phenomenon, a gene profiling assay focusing on cell cycle genes was carried out using the stable cells. Here, we report the identification and validation of host genes related to cell cycle that were found to be deregulated by the expression of MERS-CoV N protein.

Conlusion: MERS-N protein is capable of inducing cell cycle arrest at G1/S phase when expressed in mammalian host cells. This study shed light into the functions of MERS-N protein, which currently remain largely unknown. This knowledge can be important and applicable in the development of antiviral strategies targeting the N protein for effective treatment of MERS.

Keywords: MERS, coronavirus, nucleocapsid

17th International Congress of Virology 352

Abstract Submission

Positive Strand RNA Viruses PO778

Clinical and Genetic Characteristics of Enterovirus D68 in Viet Nam (2009-2015) N. Nguyen*, T. A. Nguyen 1, T. H. Vu Thi 1, T. Tran Tan 1, N. Lam Anh 1, A. McBride 2, C. Anscombe 1 2, R. van Doorn 1 2 3, S. Baker 1 2 3 4, G. Thwaites 1 2 3 4 5, T. Le Van 1 1 2 3 4 5 1Emerging Ìnection, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam, 2Emerging Ìnection, Oxford University Clinical Research Unit, London, United Kingdom, 3Oucru Ha Noi, Oxford University Clinical Research Unit, Ha Noi City, 4Enteric, 5Oucru HCM, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam

Objectives: To describe the detection, associated clinical features and evolutionary process of enterovirus D68 (EV-D68) in swabs collected from children with respiratory infections enrolled in 3 different observational studies conducted at referral hospitals across central and southern Vietnam between 2009 and 2015

Methods: Enterovirus/rhinovirus PCR positive specimens from the above mentioned clinical studies were further screened for EV-D68 infection by a viral specific PCR. EV-D68 positive specimens were subjected to whole genome sequencing using a previously published in-house non-ribosomal random PCR and MiSeq based approach. Phylogenetic reconstruction and estimation of evolutionary rate were carried out in BEAST (Bayesian Evolutionary Analysis by Sampling Trees). Recombination detection was performed using Recombination Detection Program

Results: EV-D68 was detected in 21/786 (2.7%) enterovirus/rhinovirus PCR positive swabs. All the EV-68 patients were young children (median: 17.2 months, interquartile range: 11.8-24.5) and had mild respiratory infections. A total of 10 whole genome sequences of EV-D68 were generated. All the Vietnamese isolates clustered within strains that had previously been identified in Asian counties, of which 9 fell in the B1 clade, and the remaining sequence was identified within the A2 clade. The evolutionary rate of EV-D68 was estimated to be 5.12E-3 substitutions/site/year, while dating analysis indicated that the virus might have been imported into Vietnam in 2008. One recombination event was detected with the recombination being intra sub-clade, within B1.

Conclusion: We have demonstrated for the first time EV-D68 has been circulating at low levels in Vietnam since 2008, causing a mild acute respiratory infection in children in our cohort. EV-D68 in Vietnam is most closely related to other circulating Asian strains, and clusters separately from those associated with acute flaccid paralysis in the USA and Europe.

Keywords: EV-D68 in VN

17th International Congress of Virology 353

Abstract Submission

Positive Strand RNA Viruses PO779

IN VITRO AND IN VIVO FOLLOW-UP ON A RECOMBINANT MURINE NOROVIRUS GENERATED VIA CELL CO- INFECTION L. Ludwig 1, E. De Oliveira-Filho 1 2, E. Mathijs 3, E. Di Felice 1 4, F. Dal Pozzo 5, D. Thiry 6, C. Sagerman 5, A. Mauroy 1, E. Thiry 1,* 1Veterinary Virology and Animal Viral Diseases, FARAH centre, Faculty of Veterinary Medicine, University of Liege, Liège, Belgium, 2Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães, FIOCRUZ, Recife, Brazil, 3Department of Virology - Molecular Platform, Veterinary & Agrochemical Research Centre, Brussels, Belgium, 4Facoltà di Medicina Veterinaria, Università degli Studi di Teramo, Teramo, Italy, 5Research Unit of Epidemiology and risk analysis applied to veterinary sciences (UREAR), FARAH centre, Faculty of Veterinary Medicine, 6Bacteriology and Pathology of Bacterial Diseases, FARAH centre, Faculty of Veterinary Medicine, University of Liege, Liège, Belgium

Objectives: Noroviruses are recognised as an important cause of non-bacterial gastroenteritis in humans. Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Recombination can create considerable changes in viruses, allowing for antigenic shifts, host jumps and pathogenesis and fitness modifications. We recently isolated a viable recombinant murine norovirus (RecMNV) of reduced replicative fitness in vitro after coinfection of murine norovirus strains MNV1-CW1 and -WU20 in RAW 264.7 cells (Mathijs et al. 2010). Here, we (1) evaluate the infectivity of RecMNV in vivo and (2) follow its replicative and genetic adaptations over serial in vitro passages.

Methods: (1) In vivo infectivity of RecMNV was compared to its parental strains in Balb/cByJ mice via weight loss measurement and viral load estimation in faeces, tissues and organs 48 and 72 hours post-infection using plaque assay and RT-qPCR. (2) RecMNV progenies resulting from the first (RecE) and tenth (RecL) in vitro passages were compared and their sequences determined and evaluated against those of their parental strains.

Results: (1) Average body weights of RecMNV- infected mice were generally higher than those of MNV-1- or WU20-infected mice. Viral loads were detected in all examined organs, suggesting that, like its parental strains, RecMNV can disseminate beyond the digestive tract to produce a systemic infection. (2) RecL showed a significant increase of lysis plaque diameters and faster replication kinetics than RecE. Molecular analysis of RecE and both parental strains showed seven nucleotide changes in the RecE genome, comprising two non- silent mutations. In ORF3, a mutation at position 7245 introduced a stop codon, resulting in a 20 amino-acid shorter VP2 for both RecE and RecL. Comparison of RecE and RecL revealed twelve nucleotide changes, comprising five non-silent mutations.

Conclusion: Our data suggest that recombination occurring in vitro between two homologous murine norovirus strains can give rise to a chimeric strain, which shows similar biological properties to its parental strains and is capable of productive in vivo infection. In addition, we provide evidence of viral adaptation and in vitro replicative fitness regain after a recombination event. References Mathijs, E. et al., 2010. Experimental evidence of recombination in murine noroviruses. Journal of General Virology, 91(11), pp.2723–2733.

Keywords: Murine norovirus, Recombination, Replicative fitness

17th International Congress of Virology 354

Abstract Submission

Positive Strand RNA Viruses PO780

INTRA-HOST GENETIC DIVERSITY OF NOROVIRUS IN IMMUNOCOMPROMISED PATIENT WITH CHRONIC INFECTION M. Poljsak-Prijatelj 1,*, T. Konte 2, M. Kolenc 1, T. Naglič 1, M. Sagadin 1, D. Duh 3, A. Steyer 1 1Electron microscopy, UNI Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, 2Institute of Biochemistry, University of Ljubljana, Faculty of Medicine, Ljubljana, 3Department for Microbiological Research, 2000 Maribor, National Laboratory of Health, Environment and Food, Centre for Medical Microbiology, Maribor, Slovenia

Objectives: In immunocompromised patient acute norovirus gastroenteritis could led to the chronic infection with prolonged virus shedding for months or even years. In such patients noroviruses (NoVs) undergo significant evolutional changes.

Methods: We followed up a patient with deficient immune response resulting from immunosuppressive therapy and persistent norovirus shedding from May 2010 to June 2012 with. A total of 3 stool samples were chronologically selected for the intra-host variant analysis focusing on the partial ORF1 and full ORF2 region of norovirus genome. Amplicons were generated for NGS (MiSeq Illumina). Reads were de-novo assembled and capsid gene haplotypes were generated using ShoRAH program. Amino acids were analysed for conservation rates with Jalview and the capsid protein models were constructed to visualize the variability of surface residues with the SWISS-MODEL software.

Results: The mean amplicon coverage was 1000-2800 reads. Comparing to the initial sample, the generated haplotypes showed 96.56 – 97.76% and 94.07 – 95.56% identity for nucleotide and amino acid sequences, respectively. The most interesting change was identified at the HBGA binding site of the virus appearing in the second sampling point, with a deletion of three nucleotides, codon corresponding for the amino acid at position 393 of the VP1 protein. Nucleotide sequence alignment and maximum likelihood phylogenetic tree indicated a single strain evolution within the host, minimizing the possibility of reinfection of the patient with another NoV strain. Analysis of amino acid residues showed lower conservation rates for the surface residues at the predicted antibody-neutralization binding domains.

Conclusion: In this study using next generation sequencing we showed that the within-host population of NoV specific amino acid deletion could accumulate at one of the important virus-host interaction epitopes. Chronic NoV infections are possible source of new viral variants, potential pandemic viral strains. A long-term exposure of virus infection to the host immune system could select viral variants capable to escape human immune protection.

Keywords: norovirus, immunocompromised host, next generation sequencing

17th International Congress of Virology 355

Abstract Submission

Positive Strand RNA Viruses PO781

SYNTHESIS OF NONCANONICAL SUBGENOMIC MRNAS FROM NONSTRUCTURAL PROTEIN GENE IN CORONAVIRUSES C.-H. Lin*, C. Hsu, H.-Y. Wu

Objectives: Coronaviruses are single-stranded, positive-sense RNA viruses with the largest known viral RNA genome, 27- 32 Kb. The 5’-most two-thirds of the genome is non-structural protein gene and the 3’-most one-third of the genome contains mostly structural protein gene. Synthesis of subgenomic mRNAs (sgmRNAs) in coronaviruses is correlated to transcription regulatory sequences (TRS) from which a nested set of sgmRNAs are produced. It is known that sgmRNAs are derived from TRS located in the structural protein gene of genome; however, whether sgmRNAs can be synthesized from non- structural protein gene has not been probed. The aim of the study, therefore, is to identify sgmRNA species synthesized from non-structural protein gene in coronavirus- infected cells.

Methods: To test whether sgmRNAs can be synthesized from non-structural protein gene, TRS-like motifs were predicted based on the sequence in non-structural protein gene of porcine epidemic diarrhea virus (PEDV), bovine coronavirus (BCoV) and infectious bronchitis virus (IBV), and RT-PCR was performed followed by sequencing analysis to determine the synthesis of sgmRNAs from non-structural protein gene. Northern blot assay was also employed to determine the synthesis of such sgmRNAs.

Results: By RT-PCR and sequencing it was determined that sgmRNAs can be synthesized from non-structural protein gene of PEDV, BCoV and IBV, which belongs to α, β and γ coronaviruses, respectively, indicating this is a common feature among coronaviruses. The signals representing sgmRNAs synthesized from non-structural protein gene were also detected by Northern blot assay. In addition, the encoded proteins from such non-structural protein gene-derived sgmRNAs are predicted to have transmembrane or replicase-related activity.

Conclusion: This is the first study to demonstrate that, in addition to structural protein gene, sgmRNAs can also be synthesized from non-structural protein gene. The biological significance of such noncanonical synthesis of sgmRNA may be to increase the variety of viral proteins, which may play important roles in virus pathogenesis.

Keywords: coronavirus, subgenomic mRNA, transcription regulatory sequence

17th International Congress of Virology 356

Abstract Submission Positive Strand RNA Viruses PO782 THE POLY(A)-BINDING FEATURE OF CORONAVIRUS NUCLEOCAPSID PROTEIN MODULATES VIRAL TRANSLATION: IMPLICATION FOR THE REGULATION BETWEEN VIRAL TRANSLATION AND REPLICATION T.-L. Tsai*, H.-Y. Wu

Objectives: The genome of bovine coronavirus (BCoV) is a single-stranded, positive-sense RNA molecule of ~30 kilobases in length. During coronavirus infection, the positive-strand genome functions as a template for both the synthesis of viral proteins and replication of the genome; however the strategy to coordinate the use of coronavirus genome for translation or replication has not yet been determined. It has been shown in the previous study that BCoV nucleocapsid (N) protein specifically binds to poly(A) tail with high affinity and is able to compete with cellular poly(A)-binding protein (PABP) for the binding of poly(A) tail in vitro. Therefore, the aim of the study is to determine whether the poly(A)-binding feature of coronavirus N protein modulate coronavirus translation and thereby to evaluate how coronavirus regulates translation and replication during infection. Methods: UV-cross linking followed by immunoprecipitation (IP) was used to determine whether N protein is also able to bind to poly(A) tail in vivo. IP was also employed to identify the proteins interacting with N protein and PABP. For the effect of N protein on coronavirus translation, rabbit reticulocyte lysate and HEK-293 cells were used for in vitro and in vivo assay, respectively. Results: In the current study we found that N protein is also able to bind to poly(A) tail in vivo. Since coronavirus N protein is able to bind to poly(A) tail and is correlated with replication, it is hypothesized that N protein can interact with replication-related viral protein instead of translation-related factors. By immunoprecipitation (IP) it was found that N protein can interact with coronaviral non-structural protein 1 (nsp1), but not with translation factor eIF4G, indicating that the binding of N protein to poly(A) tail may be involved in replication and thus inhibit coronavirus translation in infected cells. Using rabbit reticulocyte lysate it was shown that with the increased concentration of N protein coronavirus translation efficiency was decreased probably via binding of N protein to poly(A) tail. Further in vivo study also demonstrated that the translation of coronavirus is inhibited with the increased amount of N protein in cells. Conclusion: Since PABP and BCoV N protein are correlated with translation and replication, respectively, it is concluded that the interplay between the coronaviral poly(A) tail and PABP (for translation) or coronavirus N protein (for replication) may regulate the use of the genome between translation and replication and thus prevent the two events occurring on the same coronavirus genome during infection.

Keywords: coronavirus, Nucleocapsid protein, Translation

17th International Congress of Virology 357

RNA Protein Interaction

17th International Congress of Virology 358

Abstract Submission

RNA protein interactions

PO784

CVB3 infection upregulates host protein PSF to positively regulate viral IRES activity

P. Dave 1, S. Das*, B. George*

1Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Objectives: The 5’ UTR of Coxsackievirus B3 (CVB3) contains internal ribosome entry site (IRES), which allows cap- independent translation of the viral RNA. The 5’-terminal cloverleaf structure regulates viral replication, translation and stability. Although, cloverleaf RNA is extensively characterized both structurally and functionally, limited information is available regarding host protein profile interacting with the cloverleaf RNA. We would like to investigate the RNA-protein interaction at the Cloverleaf and IRES element of CVB3 RNA

Methods: Using RNA affinity chromatography and UV cross linking assays, we have identified several host proteins interacting with CVB3 cloverleaf RNA. Further, we characterize the role of host protein PSF in CVB3 life cycle using various molecular biology techniques.

Results: We find that host protein PSF (PTB associated splicing factor) interacts with the cloverleaf RNA as well as the IRES element. PSF was found to be an IRES trans acting factor (ITAF) for efficient translation of CVB3 RNA. Cytoplasmic abundance of PSF increased during CVB3 infection and this is regulated by phosphorylation status at two different amino acid position. Interestingly, PSF protein was up-regulated in CVB3 infection. CVB3 2A protease expression induced PSF protein levels. We report for the first time the presence of an IRES element in the 5’UTR of PSF mRNA, which is activated during viral infection. Interestingly, we find PSF IRES is also positively regulated by PTB, which is known to regulate CVB3 IRES.

Conclusion: We find that host protein PSF is an important host factor for CVB3 life cycle and it helps in viral RNA translation. Results suggest new mechanisms of regulations of ITAFs during viral infection, where an ITAF undergoes IRES mediated translation, sustaining its protein levels in condition of translation shut-off.

Keywords: None

17th International Congress of Virology 359

Abstract Submission

RNA protein interactions

PO785

DDX3 protein regulates viral translation through its helicase activity

H.-S. Chen*, R. Y. Wang 1

1Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan

Objectives: Japanese encephalitis virus (JEV) belongs to the family flaviviridae and is transmitted between animals and human host by mosquitoes. DDX3, an RNA helicase gene, is believed to play some important roles in the replication of several viruses including JEV. However, little information is shown the mechanism of DDX3 involved in the viral infection cycle. In this study, we explore the potential mechanism of DDX3 regulating JE viral replication and translation in vitro.

Methods: Knockdown of DDX3 protein in the BHK21 cells was achieved by transfection of shDDX3-lentivirus. To target DDX3, we employed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 bound to DDX3 and perturbed its activity. Polysome fractionation assay was carried out to analyze the translational efficiency. Co-Immunoprecipitation assay was carried out to analyze DDX3 and translational complexes interaction. RT-PCR and plaques assay was carried out to analyze the viral RNA accumulation and viral titer.

Results: Knockdown of DDX3 in the BHK21 cells resulted in the reduction of JE viral nonstructural proteins, NS3 and NS5, in the BHK-21 cells. The viral RNAs (both of positive and negative sense) were reduced in the shDDX3-transfected cells. RK-33 treatment of BHK21 cells significantly decreased the viral translational efficiency evaluated by polysome fraction assay. Conversely, the low DDX3-expressing cells exhibited few changes in the viral translational efficiency implying that DDX3 perturbed the viral translation through binding to its 3’-untranslated region. Moreover, our data suggest that DDX3 participates in translation initiation of targeted viral RNA as well as viral protein production via its helicase activity. We showed that DDX3 interacting with host translational complexes to enhance the viral translation. Lastly, we observed the significant reduction of viral titer in the RK-33 treatment BHK21 cells indicating the essential role of DDX3 involved in JEV infection cycle.

Conclusion: DDX3 is critical for viral translation step during JEV infection through its helicase activity.

Keywords: DDX3, JEV, translation

17th International Congress of Virology 360

Late breaking abstract

RNA protein interactions

PO786

Host protein HuR interacts with Coxsackievirus B3 cloverleaf RNA and displaces PCBP2 to restricts viral replication

B. George 1,*, P. Dave 1, S. Das 1

1Microbiology and cell biology, INDIAN INSTITUTE OF SCIENCE, BANGALORE, Bengaluru, India

Objectives: Coxsackievirus B3 (CVB3) is the major pathogen of human viral myocarditis. The genome of CVB3 is single- stranded, positive-sense RNA of 7.4 kb. Viral replication takes place in the cytoplasm of host cells. The genome consists of 5’UTR, a long ORF followed by 3’ UTR. The 5′ terminal cloverleaf structure within the 5’UTR is a cis-acting element that interacts with the viral polymerase and host protein PCBP2. This interaction is known to be essential for viral RNA replication. Recently, we have shown the interaction of PTB associated splicing factor (PSF) with the cloverleaf RNA (Dave et al., 2017, NAR). The objective of the present study is to study the interplay among host proteins at the CVB3 cloverleaf RNA and to study the consequence in CVB3 life cycle.

Methods: We used RNA affinity chromatography to identify the host proteins binding to CVB3 cloverleaf RNA. Partial silencing and overexpression of cloverleaf interacting protein (HuR) were done to study its role in CVB3 life cycle. UV crosslinking and immunoprecipitation assay was performed to study the interplay between cloverleaf interacting proteins.

Results: Using RNA affinity chromatography, we have demonstrated that the host nuclear protein HuR also interacts with the CVB3 cloverleaf structure. We have observed that overexpression of HuR inhibits CVB3 RNA replication, whereas partial silencing relieves the repression. It appears that HuR might not regulate the IRES-mediated translation of CVB3 RNA. Interestingly, total HuR mRNA, as well as protein levels, were found to be induced during CVB3 infection. We also found that HuR relocates from nucleus to cytoplasm upon CVB3 infection. Further, we have shown by UV-crosslinking and immunoprecipitation experiments that HuR displaces PCBP2 at the cloverleaf RNA. Cross talk among host proteins such as HuR, PSF, PCBP2 and the viral polymerase at the cloverleaf RNA during CVB3 replication will be discussed

Conlusion: We hypothesise that the ternary complex between PCBP-2, 3CD and cloverleaf RNA formed at the 5’end of the viral genome might be disrupted due to the displacement of PCBP2 at the cloverleaf RNA resulting in the inhibition of viral RNA replication. Inhibition of CVB3 replication by host protein HuR might be the cellular response to restrict levels of CVB3 replication.

Keywords: Cloverleaf RNA, Coxsackievirus B3, Human antigen R

17th International Congress of Virology 361

Abstract Submission

RNA protein interactions

PO787

INSIGHTS INTO THE ATP POWERED RNA TRANSLOCATION OF DENGUE VIRUS NON-STRUCTURAL PROTEIN 3

C. Swarbrick 1,*, C. Basavannacharya 1, K. Wing Ki Chan 1 2, S. A. Chan 1, D. Singh 1, W. W. Phoo 3, J. Lescar 3, D. Luo 3, S. Vasudevan 1 2

1Emerging Infectious Diseases, Duke-NUS, 2Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 3Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Objectives: Dengue virus is a positive single strand RNA virus of the genus Flavivirus within the Flaviviridae family which are an increasingly global concern with almost 70 human disease causing viruses including Zika virus. There are no directly acting antivirals against Dengue virus and the replication complex (RC), the site of viral replication is poorly understood despite our knowledge of 3D structures of the key viral enzymes, namely the NS3 protease/helicase and NS5 methyl transferase/RNA dependent RNA polymerase.

Methods: Our research aims to explore the interaction between NS3, RNA and ATP utilising a combined biophysical and biochemical approach. Prior structures of NS3Hel were solved in the presence of cSLA12, a complementary sequence to a conserved 5’UTR 12 base sequence in DENV1-4.

Results: We have solved the structure of the NS3 helicase domain in complex with a conserved nucleotide sequence from the 5’UTR of Dengue virus. From this structure number of site-directed mutations were generated and investigated for their effects on ATPase and helicase activities revealing a mechanism of sequence specific recognition and the importance of this sequence within viral replication. Furthermore reverse genetics studies with DENV2 infectious clone indicate that sequence specific RNA interactions are essential for virus replication.

Conclusion: Our findings have important implications on understanding mechanistic roles of these residues on the functional activity of ATPase and helicase activity of dengue virus NS3 when they are interacting directly with the RNA bound in the RNA binding tunnel.

Keywords: dengue virus, non-structural proteins, RNA protein interactions

17th International Congress of Virology 362

Abstract Submission

RNA protein interactions

PO788

MODULATION OF A MULTI-FUNCTIONAL HOST CELL PROTEIN DURING PICORNAVIRUS INFECTIONS

A. Holmes*, B. Semler 1

1Microbiology and Molecular Genetics, University of California, Irvine, Irvine, United States

Objectives: The Picornaviridae family of viruses is responsible for many highly prevalent human diseases worldwide. These include but are not limited to: the common cold, hepatitis, and myocarditis, which are caused by human rhinoviruses, hepatitis A virus, and coxsackieviruses (CVB), respectively. Given their limited coding capacity, picornaviruses must repurpose host factors to carry out their replication cycles. One such host protein is the DNA repair enzyme, tyrosyl-DNA phosphodiesterase 2 (TDP2). Picornaviruses use TDP2 to cleave the bond between the viral RNA and a protein called VPg (Viral Protein genome-linked), the primer for RNA synthesis. TDP2 cleavage of the VPg-RNA linkage is regulated during picornavirus infections, resulting in various forms of the RNA that are either linked to VPg or unlinked. Notably, viral RNA destined for RNA packaging (a late step in the infection) must be VPg-linked, while VPg is absent from polysome-associated viral mRNAs. However, given that there is no apparent change in TDP2 enzymatic activity during the viral replication cycle, the mechanism by which TDP2 regulation occurs is unknown. We sought to determine how TDP2 is regulated during picornavirus infections. We hypothesize that TDP2 is physically blocked from accessing VPg-linked RNA to allow viral RNA encapsidation to take place.

Methods: We used confocal fluorescence microscopy to monitor changes in TDP2 subcellular localization throughout viral infection. We also assessed TDP2 co-localization with replication complex-associated proteins and double-stranded RNA, which was used as a read-out for TDP2 proximity to nascent, VPg-linked RNA. Additionally, we confirmed the results of the immunofluorescence assays by Z-stack analysis.

Results: We found that TDP2 is sequestered to the cell periphery at late times during infection of HeLa cells by poliovirus, the prototypical picornavirus, and CVB3. Additionally, in line with our hypothesis, TDP2 is adjacent to, but does not co- localize with markers of viral replication complexes. Surprisingly, a different pattern emerges during poliovirus infection of other cell types, namely human neuroblastoma cells. TDP2 co-localizes with replication complex-associated proteins and double-stranded RNA late during the infection of this cell type.

Conclusion: These results suggest the following: 1) The mechanism of TDP2 sequestration is cell type-specific and likely involves both cellular and viral proteins; 2) Enzymatically active TDP2 may remain associated with replication organelles at late times of picornavirus infection, despite the fact that the VPg-RNA linkage must be preserved to allow for RNA packaging. Altogether, the results present a complex picture of TDP2 regulation during picornavirus infection that warrants further investigation.

Keywords: picornavirus, TDP2, VPg

17th International Congress of Virology 363

Abstract Submission

RNA protein interactions

PO789

THE INVOLVEMENT OF STAUFEN PROTEIN IN THE TRANSLATION OF ENTEROVIRUS 71

R. Y. Wang 1,*, P. OU 1

1Department of Biomedical Sciences, Chang Gung University, Kweishan, Taiwan

Objectives: Enterovirus 71 (EV71) is belonging to a single-strand and positive-sense RNA virus. The emerging infection of EV71 causes an important issue of the public health in the world, especially in the Asia. Staufen protein, a double-strand RNA binding protein, was first discovered on the studies of developmental stages in the Drosophila melanogaster. The Staufen1 protein was reported to increase the virus protein production, viral RNA replication and virions release in different RNA viruses, such as HIV, HCV and Influenza virus, respectively. e staufen-knockout cells using the ribosome fractionation assay and the reduced viral RNA stability was also detected by means of RNA stability assay indicating a major functional role of staufen protein involved in facilitating viral translation during EV71 infection.

Methods: The Staufen-knockout cell line was created by CRISPR for the functional studies. The RNA pull-down assay was carried out to investigate the interaction between viral RNA and staufen protein.

Results: We demonstrated the staufen protein can specific bind to the untranslated region of EV71 viral RNA (5’UTR). The RNA-binding domain 2-3 (RBD2-3) of staufen protein is responsible for this binding ability. Next we created a staufen- knockout cell line using the CRISPR approach to further characterize the functional roles of staufen protein interacting with viral RNA. Both of the viral RNA copy number and viral protein expression were downregulated in the staufen-knockout cells compared to the wild type naive cells. Moreover, dysregulation of viral RNA translation was observed in the staufen- knockout cells using the ribosome fractionation assay and the reduced viral RNA stability was also detected by means of RNA stability assay indicating a major functional role of staufen protein involved in facilitating viral translation during EV71 infection.

Conclusion: We detected the delay of EV71 propagation in the staufen-knockout cells was due to the impairment of viral translation. Overall, we dissected the functional relevance of staufen protein in EV71 infection cycle and provided the mechanism role of staufen protein participating in viral RNA translation racting with viral RNA. Both of the viral RNA copy number and viral protein expression were downregulated in the staufen-knockout cells compared to the wild type naive cells. Moreover, dysregulation of viral RNA translation was observed in the staufen-knockout cells using the ribosome fractionation assay and the reduced viral RNA stability was also detected by means of RNA stability assay indicating a major functional role of staufen protein involved in facilitating viral translation during EV71 infection.

Keywords: Staufen protein, Enterovirus71, Translation

17th International Congress of Virology 364

Abstract Submission

RNA protein interactions

PO790

UGGT1 ENHANCES VIRAL PATHOGENICITY BY PROMOTING VIRAL RNA SYNTHESIS AND REPLICATION COMPLEX FORMATION

P.-N. Huang*, S.-R. Shih 1

1Research Center for Emerging Viral Infections , Chang Gung University, Guishan Dist., Taiwan

Objectives: Positive-strand RNA viruses are adept at hijacking host cell machinery to promote viral propagation, such as the formation of replication complexes (RCs) containing viral and host proteins on intracellular membranes to facilitate virion assembly and avoid detection by host defense mechanisms. However, the processes by which RCs are assembled and the host proteins involved have not been fully elucidated as yet.

Methods: To better understand how the EVA71 3D polymerase associates with host proteins and other components of the viral replication machinery at RCs, we sought to identify novel host factors that associate with the 3D polymerase in RCs. Accordingly, an anti-3D monoclonal antibody was used to perform immunoprecipitation assays, in order to purify proteins associating with the 3D polymerase in infected cells. We immunoprecipitated mock-infected and EVA71-infected cell lysates with this anti-3D monoclonal antibody, and subsequently identified seven major protein bands that appeared in the EVA71- infected lysates but not in the mock-infected lysates. We excised the protein bands that specifically associated with the 3D polymerase, and subjected them to MALDI-TOF MS analysis.

Results: Here, we show that the endoplasmic reticulum protein UGGT1, a key regulator of the unfolded protein response (UPR) host defense mechanism, interacts with the 3D polymerase of enterovirus A71 (EVA71) to enhance RC formation, elevate viral RNA synthesis, and promote viral replication. Knockout of Uggt1 reduced viral pathogenicity in animal studies.

Conclusion: These findings highlight the role to which viruses can hijack key host proteins to promote viral replication, and may serve as the basis for the development of novel anti-viral strategies.

Keywords: enterovirus A71, RNA polymerase, viral RNA synthesis

17th International Congress of Virology 365

Viral Diagnostics

17th International Congress of Virology 366

Abstract Submission

Viral diagnostics PO791

Comparison of the Aptima HIV-1 and HCV Quant Dx assays with the HIV and HCV Abbott real time assays S. May 1, E. Adamska 1, J. Tang* 1Clinical Microbiology/Virology, University Hospitals of Leicester, Leicester, United Kingdom

Objectives: The Aptima HIV-1 Quant Dx and HCV Quant Dx assays utilise the transcription-mediated amplification (TMA) method for the detection and quantification of HIV-1 RNA and HCV RNA. The aim of this study was to compare the performance of the Aptima TMA against the Abbott RealTime HIV-1 and HCV viral load assays.

Methods: The correlation, accuracy and reproducibility of the quantitative results between the Aptima and Abbott HIV-1 and HCV viral load assays were compared. For the HIV-1 assay testing, 89 clinical samples previously tested with the Abbott assay were selected, including 69 known HIV-1 positive samples. Testing was also performed on the 3rd WHO (World Health Organization) HIV-1 international standard and the 2nd WHO international reference panel for HIV-1. For the HCV assay testing, 82 clinical samples previously tested with the Abbott assay were selected, including 59 known HCV positive samples. The 5th WHO international standard for HCV NAT testing and the NIBSC (National Institute for Biological Standards and Controls, Herts, UK) 4th HCV RNA panel were also used to test the performance of the Aptima HCV Quant Dx assay.

Results: Comparison of the two HIV-1 assays found that they were correlated (R2 =0.87, Lin’s concordance coefficient =0.92), with a mean difference of measurement (according to the Bland-Altman method) of 0.35 log10 =2.24 copies/ml. The Aptima assay detected the HIV-1 WHO international standard from 4.27 down to 1.26 log10 copies/ml with good linearity (R2 =0.96). The Aptima HIV-1 assay was able to detect all subtypes from the 2nd HIV-1 WHO international reference panel. Results from the comparison of the two HCV assays also found that these two assays were highly correlated (R2 =0.96, Lin’s concordance coefficient =0.95), with a mean difference (according to the Bland-Altmann method) of -0.07 log10 =9.93 th copies/ml. The Aptima assay detected the 5 HCV WHO international standard from 4 down to 1 log10 IU/ml with good linearity (R2 =0.95). All genotypes included in the NIBSC 4th HCV RNA RNA panel were detected using the Aptima assay. Good reproducibility was seen in both the HIV-1 and HCV Aptima assays from testing clinical samples in triplicate.

Conclusion: The Aptima HIV-1 and HCV Quant Dx assays demonstrated comparable performance to the Abbott assays for the detection and quantification of HIV-1 and HCV viruses. In addition, the Aptima platform offers significantly more hands-off (walk-away) time than the Abbott m2000 for both of these HIV and HCV viral load assays. Both of these assays also take less time to complete on the Aptima (approximately 3 hrs) than on the Abbott m2000 (approximately 6 hrs), thus reducing the turnaround time for result reporting.

Keywords: Aptima Abbott viral load comparison, HCV, HIV

17th International Congress of Virology 367

Abstract Submission

Viral diagnostics PO792

Development and Evaluation of a SYBR Green Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue, Zika and Chikungunya Viruses. H. Chen*

Objectives: Dengue virus (DENV) Zika virus (ZIKV) and Chikungunya virus (CHIKV) have emerged as the most important arthropod-borne viruses responsible for large and geographical epidemics. Accurate diagnosis of these three viruses remains challenging due to their similar clinical manifestation, common transmission vectors, geographical distribution and seasonal correlation. We aimed to develop and evaluated a rapid, cost-effective molecular assay to simultaneously detect, quantify and differentiate DENV-1, 2, 3, 4 ZIKV and CHIKV using SYBR Green I-based one-step multiplex real-time RT- PCR.

Methods: In the present study, we developed and evaluated a rapid, cost-effective molecular assay to simultaneously detect, quantify and differentiate DENV-1, 2, 3, 4 and CHIKV using SYBR Green I-based one-step multiplex real-time RT- PCR. DENV is serotyped based on melting temperature analysis of PCR amplicon that is specifically corresponding to only each of DENV-1, 2, 3, 4, ZIKV and CHIKV.

Results: The detection limit of the assay was determined to be 50 RNA copies/reaction for DENV-1, 10 RNA copies/reaction for DENV-2, 20 RNA copies/reaction for DENV-3, 5 RNA copies/reaction for DEN-4, 20 RNA copies/reaction for ZIKV and 10 RNA copies/reaction for CHIKV. Furthermore, our assay is not cross-reacting with the panel of RNA viruses validated in this study. In addition, the present assay was evaluated using clinical serum samples and the sensitivity for DENV-1, 2, 3, 4 and CHIKV was found to be 78.58%, 92.86%, 92.86% 92.86%, and 96.7%, respectively, and the specificity of the assay is 100%.

Conclusion: Our assay has the potential as a point-of-care clinical molecular diagnostic platform for DENV, ZIKV and CHIKV in acute-phase patient serum samples.

Keywords: Dengue serotyping, real time PCR, zika and chikungunya viruses

17th International Congress of Virology 368

Abstract Submission

Viral diagnostics PO793

DIFFICULTIES IN DISTINGUISHING ENTEROVIRUS FROM RHINOVIRUS IN COMMERCIAL RESPIRATORY MULTIPLEX ASSAYS H. Kirk-Granger 1, C. Holmes 2, J. Tang 2,* 1Public Health Laboratory Birmingham, Public Health England, Birmingham, 2Clinical Microbiology/Virology, University Hospitals of Leicester, Leicester, United Kingdom

Objectives: There are now many commercial multiplex respiratory PCR assays available, but most cannot distinguish between human enteroviruses (EVs) and human rhinoviruses (RVs). This distinction is clinically important as rhinoviruses rarely disseminate beyond the respiratory tract to cause systemic disease, whereas enteroviruses do so frequently. In a typical neonatal/infant sepsis workup, respiratory samples are often taken. If a test cannot distinguish between EV and RV there is a risk of either discharging the baby home too early (without a period of observation for any systemic complications), or of admitting the baby for longer than necessary. Whilst in the process of transitioning from an in-house to a commercial respiratory multiplex PCR assay, a limited comparison was performed of the ability of three assays to distinguish between EV and RV infections.

Methods: For any EV/RV positive on our routine screening assay (AusDiagnostics, 16-well-Cat 20612, AusDiagnostics Pty Ltd., Beaconsfield, NSW, Australia), we attempted to identify whether the samples contained just EV or RV or both. Two assays were used for this testing on aliquots from the same sample: the FTD21 kit (Fast-track diagnostics Ltd, Siema, Malta) performed in our laboratory, and a sendaway to a commercial diagnostic laboratory MP (Micropathology Ltd., Coventry, UK). For EV, PCR results from the UK national reference laboratory (Enteric Virus Unit, Public Health England, Colindale. London,UK) were used as the ‘gold’ standard, where all EV positive samples were routinely confirmed by VP1 sequencing for EV serotyping. There was no ‘gold’ standard equivalent for RV testing. Each respiratory sample (nasopharyngeal aspirate, or nasal/throat swab in virus transport medium) was split for testing in each of the assays, as required.

Results: A total of 29 samples that were EV/RV POS on the AusDiagnostics assay were sent for further testing. For EV POS samples, surprisingly, there was 0% concordance between the FTD21 and MP assays on which samples were EV only POS. When compared against the PHE gold standard, FTD21 called 3/12 (25%) correctly as EV only POS, but 9/12 (75%) were deemed false POS (where PHE EV was NEG). The two assays agreed 100% on the EV only NEG samples, implying that these were RV only POS. For MP, there was 0/11 (0%) concordance with PHE for EV only POS samples. For EV only NEG, 5/11 (45.5%) samples were concordant on both assays. Only 2/11 (18.2%) samples that were MP EV/RV POS also showed as PHE EV only POS. The other 4/11 (36.4%) MP EV/RV POS samples were PHE EV only NEG, suggesting that these were RV only POS.

Conclusion: Without sequencing, none of these commercial assays/services for EV/RV testing, were able to reliably distinguish between EV and EV. Such EV/RV POS results need to be interpreted with caution, clinically.

Keywords: Enterovirus, Respiratory virus, rhinovirus

17th International Congress of Virology 369

Abstract Submission

Viral diagnostics PO794

NOVEL GLYCO-NANOTECHNOLOGY INCREASES SENSITIVITY OF DIAGNOSIS DURING THE 2015 DENGUE OUTBREAK IN TAIWAN T.-S. Ho 1 2,*, H. Lee 1, Y. Suda 3, C.-C. Liu 1 2 1Department of Pediatrics, National Cheng Kung University Hospital, 2Center of Infectious Disease and Signaling Research, National cheng kung university, Tainan, Taiwan, 3Department of Chemistry and Biotechnology, Kagoshima University, Kagoshima, Japan

Objectives: Dengue is the most prevalent mosquito-borne viral disease in people. About more than 390 million cases of dengue fever are estimated to occur each year globally. Early recognition of dengue is challenging because initial symptoms are often non-specific. In 2015, a cumulative total of 43,784 dengue cases, including 212 deaths have been confirmed in Taiwan. Previously we found that sugar-chain immobilized gold nano-particles (SGNP) can increased the efficacy of dengue RNA extraction from clinical samples. This study aimed to evaluate the sensitivity and specificity of the SGNP-based kit on molecular diagnosis of dengue.

Methods: Patients admitted to the National Cheng Kung University Hospital with virologically or serologically confirmed dengue infections were included in the current study. The epidemiological characteristics, clinical manifestations and laboratory data of these patients were analyzed from chart records. Clinical specimens were stored in -80 degree Celsius until use and applied to SGNP-based and commercial kits.

Results: Totally 109 patients and 20 healthy controls were tested. The overall detection rates for commercial extraction control and the SGNP kit was 76% (57/75) and 91.7% (100/109) respectively. The SGNP method could detect the DENV in 83% (15/18) of ‘negative’ samples defined by commercial method with a favorable clinical outcome. However, the inconsistence between two methods is minimal.

Conclusion: The results showed that the new SGNP-based method increased the sensitivity of dengue virus detection with smaller sample volume and shorter processing time compared with commercial kit. Further clinical validation in different disease stages will foster a more accurate diagnostic tool for dengue infections.

Keywords: Dengue virus, glyco-nanotechnology, in vitro diagnostics

17th International Congress of Virology 370

Late breaking abstract

Viral diagnostics PO795

Sensitive detection of Zika virus antigen in patients’ whole blood as an alternative diagnostic approach F. M. Lum 1,*, C. Lin 2, O. Susova 3, T. H. Teo 1, S. W. Fong 1 4, T. M. Mak 2, L. Lee 5, C. Y. Chong 6, D. Lye 5, R. Lin 2, A. Merits 7, Y. S. Leo 5 8 9, L. F. P. Ng 1 10 1Singapore Immunology Network, Agency for Science, Technology and Research, 2National Public Health Laboratory, Ministry of Health, Singapore, Singapore, 3Blokhin Russian Cancer Research Center, Ministry of Health of the Russian Federation, Moscow, Russian Federation, 4Department of Biological Science , National University of Singapore, 5Institute of Infectious Diseases and Epidemiology, Tan Tock Seng Hospital, 6KK Women’s and Children’s Hospital, Singapore, Singapore, 7Institute of Technology , University of Tartu, Tartu, Estonia, 8Lee Kong Chian School of Medicine, Nanyang Technological University, 9Saw Swee Hock School of Public Health , National University of Singapore, Singapore, Singapore, 10Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom

Objectives: Epidemics caused by the re-emergence of Zika virus (ZIKV) warrant the need to develop new diagnostic measures to complement currently used detection methods. Our earlier work has demonstrated that immune cells of the myeloid lineage, in particularly the monocytic-like cells, are susceptible targets of ZIKV. Therefore in this study, we explored the detection of ZIKV antigen in patients blood monocytes as a plausible diagnostic approach.

Methods: Whole blood samples were obtained at the acute and early convalescent phase from ZIKV-infected patients during the Singapore outbreak in Aug-Sep 2016. Presence of ZIKV antigen was determined by flow cytometry staining for intracellular ZIKV NS3 with a ZIKV-specific polyclonal antibody. The presence of ZIKV antigen was determined in the CD45+CD14+ monocytes.

Results: Data showed that ZIKV NS3 antigen could be detected and the levels of detection were further categorized into 3 groups – high (>40% positive), moderate (between 10-40% positive) and low (<10% positive). While majority of patients showed a decrease in the amount of ZIKV antigen detected at later time points, some patients displayed higher levels as the disease progressed.

Conlusion: Our data highlights an alternative approach in using flow cytometry for the sensitive detection of ZIKV antigen in whole blood. Importantly, the detection of ZIKV NS3 antigen would also indicate an on-going active viral infection. This study also further confirms the role of CD14+ monocytes as an important cellular target for ZIKV infection during the viremic phase.

Keywords: diagnosis, Monocytes , Zika virus

17th International Congress of Virology 371

Abstract Submission

Viral diagnostics PO796

VALIDATION OF NUCLEIC ACID EXTRACTION METHODS ON A QIASYMPHONY FOR THE PCR TESTING OF MULTIPLE POST-MORTEM TISSUE TYPES E. Adamska 1, C. Holmes 1, J. Tang 1,* 1Clinical Microbiology/Virology, University Hospitals of Leicester, Leicester, United Kingdom

Objectives: The ability to extract nucleic acids from many different tissue types is important for post-mortem investigations. Common tissue types include: liver, lung, trachea, larynx, brain, spleen, placenta, heart and bowel. Different PCR assays are used on the different tissue types, depending on the deceased patient’s history. The most common targets are: cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), enterovirus, adenoviruses, parvovirus B19 (PV B19), herpes simplex virus types 1/2 (HSV-1/2), influenza A/B, respiratory syncytial virus A/B (RSV A/B), parainfluenzavirus types 1-4 (PIV 1-4), human metapnuemovirus (hMPV), coronaviruses, rhinoviruses and bocavirus. The aim of this project was to validate a universal tissue nucleic extraction method compatible with our current laboratory procedures to allow streamlined PCR testing by assays already used in-house.

Methods: Over 300 tissue samples were received during 2013-2016. These archived tissues (frozen at -80 oC) had already been tested at a commercial laboratory elsewhere. From the laboratory archive of PM tissue samples (frozen at -80°C), 7 samples that were known to be positive for at least one of the viruses listed above were chosen as being representative of the different tissue types: 2 lung and 1 each of spleen, liver, trachea, larynx and bowel. A further 16 negative tissue samples were chosen at random. Tissue samples thawed at room temperature. A 50 mg piece of tissue was cut and placed in a 2 mL tube with 440 µL of ATL buffer, to which 40 µL of Proteinase K was added, incubated at 56oC (shaking at 900 rpm for at least an hour) then stored overnight at 4 oC. The lysate was split into two 2x240 µL tube aliquots then extracted using a QIASymphony. One tube was extracted using the High Tissue Content (HTC) protocol (without an internal control), and the second using the Complex 200 (C200) protocol (with an MS2 internal control). The HTC and C200 elution volumes were 100 µL and 110 µL, respectively. The DNA concentration was measured using the Nanodrop 1000. All eluates were tested by real-time PCR for human RNase P (RNP) gene, HSV/VZV and respiratory viruses using standard in-house PCR protocols, including a specific PCR targeting the MS2 internal control for the C200-extracted samples. All PCRs were performed on Rotor-Gene Q thermal cycler.

Results: All samples were lysed after one hour, though some of the more friable samples (spleen and brain) were already lysed after thawing. This may have been due to them being frozen beforehand – fresh tissue samples may require more time for complete lysis. The DNA concentrations for these 7 samples, extracted using both HTC and C200 protocols, were similar despite the difference in elution volumes. All samples amplified during RNP PCR showed comparable results (comparing Ct values) when extracted with both protocols (paired t-test p-value = 0.384).

Conclusion: Qiagen recommend nucleic acid extraction from tissues be performed using their dedicated tissue extraction protocols. This study has demonstrated that the C200 extraction protocol worked equally well and extracted nucleic acids as efficiently as the HTC protocol from multiple tissue types. As the C200 protocol is compatible with the routine extraction protocols already in use in our laboratory, it was chosen for further validation. The 110 µL elution volume using this protocol allowed for multiple PCR targets to be tested. The MS2 and RNP PCRs were successfully used as extraction and run controls, respectively.

Keywords: Extraction, Multiple tissue types, PCR

17th International Congress of Virology 372

Virus Discovery

17th International Congress of Virology 373

Abstract Submission

Virus discovery PO828

A MAVRIC APPROACH TO VIRUS DISCOVERY IN AUSTRALIAN ARTHROPODS C. O'Brien*, S. Hall-Mendelin 1, J. Hobson-Peters 2, B. McLean 2, D. Warrilow 3, H. Bielefeldt-Ohmann 2, A. Lew-Tabor 4, G. Deliyannis 5, A. Allen 5, R. Hall 2 1Public Health Virology, Forensic and Scientific services, Queensland Health, 2School of Chemistry and Molecular Biosciences, The University of Queensland, 3School of Chemistry and Molecular Biosciences, Queensland Health, 4Centre for Animal Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, 5Zoetis Australia Research and Manufacturing, Sydney, Australia

Objectives: Haematophagous arthropods are responsible for transmitting a number of significant human and animal pathogens, including Japanese encephalitis virus in mosquitoes and, Tick-borne encephalitis virus and Crimean-Congo Haemorrhagic fever virus in ticks. More recently these arthopods have been implicated in the emergence of severe human pathogens such as Zika virus and severe fever with thrombocytopaenia virus. Despite this, most routine arbovirus surveillance in Australia focuses on the detection of common arboviral pathogens and there is little to no surveillance in ticks.

Methods: Using a novel system which detects double-stranded RNA, a replicative intermediate produced during most viral infections, we have developed a high-throughput system which allows for sequence-independent detection of at least three arbovirus families by fixed-cell ELISA.

Results: Using this system we have identified a number of new viruses from Australian mosquitoes including a divergent negevirus and birnavirus. We are now optimising this system in conjunction with next generation sequencing to explore the virome of the Australian paralysis tick Ixodes holocyclus. To date, sequencing has revealed the presence of an iflavirus with similarity to the honey bee pathogen deformed wing virus and at least two bunyavirus sequences with similairy to known tick-borne pathogens.

Conclusion: This work has expanded our knowledge of the virome of the Australian tick and mosquito populations and provides precedent for further arbovirus screening in Australian ticks.

Keywords: arbovirus vectors, virus discovery

17th International Congress of Virology 374

Viral Diseases

17th International Congress of Virology 375

Abstract Submission

Viral disease

PO797

DEVELOPMENT OF A MOUSE MODEL FOR NEUROVIRULENCE OF ENTEROVIRUS D68

K. Chumakov*, D. Kouiavskaia 1, O. Mirochnitchenko 1

1Office of Vaccines Research and Review, FDA Center of Biologics Evaluation and Research, Silver Spring, United States

Objectives: Enteroviruses are among the most prevalent human pathogens. While causing predominantly mild or inapparent infections, they are implicated in a number of serious diseases that include poliomyelitis, meningoencephalitis, and other neurological conditions, severe respiratory infections, herpangina, hand-foot-and-mouth disease, myocarditis, type 1 diabetes, and others. Factors that determine their pathogenicity remain largely unknown. Enterovirus D68 (EV-D68) is associated with sporadic respiratory disease, occasionally causing outbreaks. The EV-D68 infections confirmed during the relatively large outbreak in the western part of the United States in 2014 were occasionally accompanied by acute flaccid myelitis (AFM). While it is not unusual for enteroviruses to case neurological disease, AFM was not previously reported for EV-D68. What factors affected the pathogenicity of 2014 strains and did they acquire neurotropism? Does this represent emergence of a potentially dangerous human pathogen? To answer these questions, we are developing an animal model to study neuropathogenicity of EV-D68.

Methods: Adult mice are generally not susceptible to enterovirus infections, whereas neonatal mice and knockout mice without Interferon alpha beta receptor (IFNAR KO) can be infected with different enteroviruses. We have infected newborn IFNAR mice with different strains of EV-D68 and observed for clinical signs.

Results: We have found that neonatal IFNAR KO mice develop signs of illness, including paralysis, after peripheral inoculation with 2014 strains of EV-D68, but not with reference strain Fermon isolated from a pneumonia case in 1962.

Conclusion: Neonatal IFNAR mice were found to be susceptible to infection with some EV-D68 strains and to develop limb paralysis. This model could be used for comparison of pathogenicity of EV-D68 strains, studies of its pathogenesis, as well as for testing antiviral drugs and vaccines.

Keywords: Acute flaccid myelitis, animal model, IFNAR mice

17th International Congress of Virology 376

Abstract Submission

Viral disease

PO798

UNDERSTANDING THE NEUROVIRULENCE OF ENTEROVIRUS 71

H. Yeo 1,*, O. Sessions 2, B. Yan 3, J. Chu 1, V. Chow 1, S. Alonso 1

1Department of Microbiology and Immunology, National University of Singapore, 2Program in Emerging Infectious Diseases, DUKE-NUS, 3Department of Laboratory Medicine, National University Hospital, Singapore, Singapore

Objectives: To identify the viral determinants involved in EV71 neurovirulence

Methods: Based on the differences in amino acid sequences between 3 virus clinical isolates which displayed different clinical outcomes in the 2-week old AG129 mouse model of EV71 infection, virus mutants were generated by site-directed mutagenesis from an infectious clone. The viral titers in different cell lines were quantified via plaque assay after infection with all the mutants and compared to the WT strain. Immunofluorescence assay and quantitative real-time PCR (qRT-PCR) was also carried out. Finally, two-week-old AG129 mice were infected with selected mutant. Clinical scores were determined and the viral titers in organs were quantified.

Results: Two-week-old AG129 mice infected with EV71 clinical isolate S41 resulted in neurological symptoms and progressive limb paralysis associated with viral accumulation and neuronal damage in the central nervous system of infected mice, while infection with myotropic MS and C2 strains did not cause any clinical symptoms or disease. Upon alignment of the polyprotein sequences of the three clinical strains based on their virulence in AG129 mice, several mutations of interest were found in VP2, 3A, 3C and 3D regions. Site-directed mutagenesis was carried out based on these mutations on the infectious clone of S41. Growth kinetics of the mutants in various cell lines showed that the VP2149ItoK mutant (position 149 of the VP2 protein - isoleucine to lysine) had minimal replication in NSC-34 cells, a mouse motor neuron cell line. Reverse transfection with purified viral RNA genome and qRT-PCR revealed that the mutant was able to replicate in NSC- 34 but was unable to establish a secondary infection, thus suggesting that the VP2149ItoK mutant was inhibited at the entry step. In vivo, mice infected with the mutant strain remained healthy throughout and did not display limb paralysis. Furthermore, whereas virus titers in the limb muscles were comparable to those measured in mice infected with the WT virus, significantly lower virus titers were found in the spine and brain from mice infected with the mutant strain.

Conclusion: A single mutation at the VP2 region at position 149 from isoleucine to lysine was found to be critical for EV71 in establishing neurovirulence in AG129 mice and in motor neuron NSC-34 cells. The inhibition appears to be at the entry step of the virus infection cycle.

Keywords: enterovirus A71, Mouse Model, neurovirulence

17th International Congress of Virology 377

Viral Evolution & Population Genetics

17th International Congress of Virology 378

Abstract Submission

Viral evolution & population genetics

PO799

A MOLECULAR ANALYSIS OF PORCINE CIRCOVIRUS TYPE 2 ISOLATES CIRCULATING IN KOREAN DOMESTIC SWINE FARMS

S.-H. Cha*

Objectives: Porcine Circovirus type 2 (PCV2) has been recognized as one of the primary etiological agents that cause PCVAD (PCV2 associated disease) including PMWS (Postweaning Multisystemic Wasting Syndrome) and PRDC (Porcine Respiratory Disease Complex) in swine population worldwide. PCV2 is a non-enveloped single-stranded circular DNA virus of family Circoviridae. The genome of PCV2 is approximately 1.76 kb in length and contains at least 3 open reading frames (ORFs). ORF2, encoding capsid protein, is known to be associated with immunogenicity and pathogenicity of PCV2, and often used to examine genetic diversity and monitor molecular evolution of the virus. Recently-circulating genotypes of PCV2 include PCV2a, PCV2b PCV2c and PCV2d based on ORF2 sequences. In this study, field PCV2 collected in 2015 were sequenced and analyzed for genetic variation based on ORF2 nucleotide sequence.

Methods: 70 Lung and serum samples were collected from local pig farms nationwide in 2015. Total DNA was extracted from serum and lung using DNeasy mini Kit (QIAGEN) according to the manufacturer's protocol. Subsequently, polymerase chain reaction (PCR) was carried out using Takara TaqTM and primer sets derived from sequences of the ORF2 and entire PCV2 genome. The amplified PCR products were purified by commercial kit and sequenced (Macrogen, Korea). The results were compared with other 60 PCV2 ORF2 sequences available in GenBank. Multiple sequence alignments and construction of phylogenetic trees were performed using CLC Main Workbench 7.0.3 and Mega 6 program. Bootstrap values were calculated on 1000 replicates of the alignments to assess the confidence limits of the branching.

Results: A total of 15 sequences of PCV2 ORF2 and 9 entire genomes were obtained. PCV2 field strains were classified in 3 genotypes; PCV2a, PCV2b, and PCV2d. Phylogenetic analysis found 7% (1/15) to be PCV2a, 27% (4/15) to be PCV2b, and 67% (10/15) to be PCV2d. The percent identity of the nucleotide sequences among genotypes varies from 99.29% to 100% in PCV2b and from 99.72% to 100% in PCV2d. The appearance of PCV2d (mPCV2b) genotype in local pig farms accords with previous studies reporting genotype shift to PCV2d in Korea.

Conclusion: Since emergence of PCV2 mutant possibly due to vaccine failure has been recently reported, phylogenetic studies of field PCV2 circulating different geographical areas in Korea are necessary for updating genetic information for PCVAD control and current vaccine selection or renewal.

Keywords: Molecular analysis, Poricne circovirus, swine farms

17th International Congress of Virology 379

Abstract Submission

Viral evolution & population genetics

PO800

CO-CIRCULATION OF DIVERGENT HANTAVIRUS LINEAGES IN SOREX SHREW SPECIES IN MONGOLIA AND JAPAN

S. Arai 1,*, B. Boldgiv 2, B. Boldbaatar 3, K. Taylor 4, K. Nakata 5, K. Tanaka-Taya 1, H. J. Kang 6, S. Morikawa 1, R. Yanagihara 6, K. Oishi 1

1National Institute of Infectious Diseases, Shinjuku, Japan, 2National University of Mongolia, 3Institute of Veterinary Medicine, Mongolian State University of Agriculture, Ulaanbaatar, Mongolia, 4Hokkaido University, Sapporo, 5Hokkaido Forestry Research Institute, Bibai, Japan, 6University of Hawaii, Honolulu, United States

Objectives: Recently, genetically distinct hantaviruses have been detected in multiple species of shrews (order Eulipotyphla, family Soricidae) in Europe, Asia, Africa and North America. Seewis virus (SWSV), originally found in the Eurasian common shrew (Sorex araneus) in Switzerland, is now known to be widespread across the geographic range of its reservoir host. In Siberia, Russia, SWSV has also been reported in the tundra shrew (S. tundrensis) and Siberian large- toothed shrew (S. daphaenodon), suggesting host sharing. The objective of this study was to determine if SWSV and other hantaviruses are hosted by Sorex shrews in Mongolia and Japan.

Methods: Total RNA, extracted from lung tissues of 21 Laxmann’s shrews (S. caecutiens), six Eurasian least shrews (S. minutissimus), one flat-skulled shrew (S. roboratus) and 40 tundra shrews, captured near Hövsgöl Lake in Mongolia, during 2010 and 2011, and 11 Laxmann’s shrews, 48 slender shrews (S. gracillimus) and 174 long-clawed shrews (S. unguiculatus), captured in several areas of Hokkaido, during 2008 and 2016, were analyzed for hantavirus RNA by nested RT-PCR.

Results: Hantavirus RNAs were detected in five tundra shrews, two Laxmann’s shrews and one Eurasian least shrew in Mongolia and in 17 long-clawed shrews in Japan. Pair-wise alignment and comparison of partial S-, M- and L-segment sequences indicated SWSV in four tundra shrews in Mongolia and a genetically distinct hantavirus, designated Sarufutsu virus (SRFV), in two long-clawed shrews in Japan. Two Laxmann’s shrews, one Eurasian least shrew, one tundra shrew and 15 long-clawed shrews, all captured during the same periods and locations, harbored a highly divergent hantavirus, named Altai virus (ALTV), which was genetically distinct from SWSV and other Sorex-borne hantaviruses in Eurasia, including Yakeshi virus in the taiga shrew (S. isodon), Qian Hu Shan virus in the greater striped-backed shrew (S. cylindricauda), Asikkala virus in the Eurasian pygmy shrew (S. minutus), Kenkeme virus in the flat-skulled shrew and Artybash virus in the Laxmann’s shrew.

Conclusion: ALTV, first found in a Eurasian common shrew in western Siberia, appears to be hosted by multiple Sorex shrew species in Mongolia and Japan. The co-circulation of ALTV and SWSV and of ALTV and SRFV among syntopic soricine shrew species in Mongolia and Japan suggests a complex evolutionary history.

Keywords: Co-circulation, Hantavirus, Sorex shrew

17th International Congress of Virology 380

Abstract Submission

Viral evolution & population genetics

PO801

COMPLETE GENOMIC SEQUENCING OF RUBELLA VIRUS USING NEXT GENERATION SEQUENCING

M.-H. Chen 1,*, E. Abernathy 1, T. F. F. Ng 2, K. W. Radford 1, J. P. Icenogle 1

1Vaccine Preventable Viral Diseases Branch , 2Polio and Picornavirus Laboratory Branch , Centers for Disease Control and Prevention, Atlanta, United States

Objectives: Next Generation Sequencing (NGS) has been shown to generate genomic sequences quickly and economically. Analysis of the complete genomic sequences of rubella virus (about 9762 nucleotides) has the potential to improve the ability to track transmission patterns and identify the source of imported viruses. The purposes of the study include establishing methods for whole genomic sequencing of rubella virus by NGS and characterizing the viruses from the viral genomes.

Methods: Two workflows were used to conduct genomic sequencing of rubella virus using Illumina MiSeq NGS technologies: the metagenomic approach (pathogen-independent) allows unbiased sequencing of viral pathogens and therefore allows the detection of new variants and resolves co-infections while the amplicon-based method (pathogen- dependent) gives high coverage and thus provides sequences with confidence and consistency. Ninety-six rubella samples, including ninety isolates from both archival collections and recent importations and six clinical specimens, were subjected to random amplification without rubella specific primers. Amplicons that covered the whole genomes of sixty-nine rubella samples, including 11 clinical specimens, were generated. The libraries by both methods were sequenced using Illumina MiSeq platform.

Results: Nearly complete genomic sequences were obtained by both pathogen-independent and pathogen-dependent methods, although low coverage was observed for the 20 terminal nucleotides at each end of the genomes. The sequences of 90 rubella isolates derived from libraries made by pathogen-independent method, not surprisingly, had lower coverage across the entire genome compared to the sequences obtained using the pathogen-dependent workflow. Nevertheless, the contigs from most sequences covered at least 60% of the genome with at least 10-fold coverage. When comparing the sequences obtained using the pathogen-independent method to sequences derived by the Sanger method, very few mismatches were identified, confirming the fidelity of the NGS method.

Conclusion: Developing two NGS workflows for whole genomic sequencing enabled us to rapidly double the number of genomic sequences of rubella virus available in the GenBank. We found that some viruses had unique deletions in the intergenic region of the genome. Metagenomics analysis of the Next Generation sequence data detected a possible co- infection of rubella and herpes simplex 1 virus in one individual. In addition, the complete genomic sequence of a virus isolated from a fatal rubella encephalitis case in an immunized individual with primary immune deficiency had more than 130 variant nucleotides and 44 amino acid substitutions, among which 16 were unique substitutions. These improved methods will be useful to help understand evolution of rubella virus and to monitor genetic changes in the current circulating strains.

Keywords: next generation sequencing, rubella virus, whole genome

17th International Congress of Virology 381

Abstract Submission

Viral evolution & population genetics

PO803

EVIDENCE FOR CO-CIRCULATION OF GENETICALLY DISTINCT STRAINS AND OCCURRENCE OF REASSORTMENT EVENT OF HUMAN ROTAVIRUS B IN BANGLADESH

N. Kobayashi 1,*, A. Meijisoe 1, S. Nahar 2, S. Paul 2, M. Hossain 2, Y. Wang 3, N. Urushibara 1, M. Kawaguchiya 1, A. Sumi 1

1Hygiene, Sapporo Medical University, Sapporo, Japan, 2Microbiology, Mymensingh Medical College, Mymensingh, Bangladesh, 3Virology, Wuhan CDC, Wuhan, China

Objectives: Rotavirus B (RVB) is a rare pathogen of acute gastroenteritis among children and adults, and has been detected mostly in several Asian countries (China, India, Bangladesh, Nepal, Myanmar). Human RVB strains detected to date has been genetically highly conserved, and only a single virus strain is considered to be circulating in an area. However, RVB strains with different RNA patterns were detected in Bangladesh. The objective of this study is to elucidate genetic diversity of these human RVB strains by whole genomic analysis.

Methods: Fourteen RVB strains were detected by PAGE (polyacrylamide gel electrophoresis) from Jan. to Mar. 2014 in faecal specimens of patients with acute gastroenteritis in Mymensingh, located in the central-northern Bangladesh. In the PAGE, two distinct RNA patterns (E1 and E2) were observed. Nucleotide sequences were determined for VP7 gene (all the strains) and VP4, VP6, VP7, NSP1-5 genes (two strains each of E1 and E2) by RT-PCR and direct sequencing. Sequence analysis was performed with LALIGN server and MEGA5 software.

Results: For all the gene segments, sequence identities among strains with the same RNA pattern were higher (99%– 100%) than those between strains with different RNA patterns (94–98%). Although all the gene segments of RVB strains were grouped into Indian–Bangladeshi lineage, VP1–3, VP6, VP7, NSP1, NSP2 and NSP5 genes of strains with E1 and E2 types were assigned to distinct sublineages S1 and S2, respectively. E1-strains clustered with Bangladeshi RVB strains reported previously (e.g., Bang117), while E2-strains with those from India (e.g., NIV-1048101), Myanmar, and Nepal. In contrast, VP4, NSP3 and NSP4 genes of both E1 and E2 RVB strains were classified into sublineage S2. These findings indicated that two genetically distinct RVB strains were simultaneously circulating in Mymensingh, Bangladesh. RVB strains with E1 electropherotype were suggested to be reassortants acquiring three gene segments (VP4, NSP3 and NSP4 genes) from the foreign RVB in the genetic background of indigenous Bangladeshi RVB represented by the strain Bang117.

Conclusion: The present study revealed that the two distinct RVB strains were circulating in the study site, and provided evidence for reassortment event between indigenous RVB strain in the north-central Bangladesh and another RVB predominating in wide area of South Asia.

Keywords: Rotavirus B, molecular epidemiology, reassortment, human

17th International Congress of Virology 382

Abstract Submission

Viral evolution & population genetics

PO805

Evolutionary Phylodynamics of Korean Parechoviruses Reveals a HPeV1 and HPeV3 Recombination Event

V. T. Tran 1,*, H. Dang Thanh 1, W. Kim 1,*

1Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Korea, Republic Of

Objectives: Human parechovirus (HPeV) is known to be a causative agent of respiratory and gastrointestinal illnesses, acute flaccid paralysis encephalitis, meningitis and neonatal sepsis.

Methods: Stool sample preparation: Stool samples were collected from children under 5 years of old with acute gastroenteritis at Chung-Ang University Hospital from January 2014 to October 2015.

RNA extraction and HPeV detection

Nucleotide sequencing and phylogenetic analysis

Recombination Analysis

Results: Of 391 fecal specimens collected from January 2014 to October 2015 from children with acute gastroenteritis in Seoul, South Korea, 221 (56.5%) were found to be positive for HPeV. To investigate evolutionary phylodynamics of Korean HPeVs, full genome sequence of 4 strains was determined and compared to the other HPeV isolates in the GenBank. Phylogenetic analysis indicates that CAU15-2-11, CAU15-2-14, CAU15-2-48, CAU15-2-70 strains might be a product of more than one genomic recombination event which occurred among HPeV1 and HPeV3 strains. The genes encoding the structural proteins VP0, VP3, VP1 and 2A showed a high degree of similarity with the corresponding genes of the HPeV3 strains isolated in Taiwan in 2011 (TW-03067-2011) and in Canada in 2002 (Can82853-01). By contrast, the genes encoding structural proteins 2B, 3A, 3B, 3C and 3D revealed a high degree of similarity with the corresponding genes of the HPeV1 strain isolated in Taiwan in 2010 (TW-71594-2010).

Conclusion: Our data provided important insights into recombination events, which may prove valuable for predicting the emergence of circulating HPeVs strains in acute gastroenteritis patients.

Keywords: None

17th International Congress of Virology 383

Abstract Submission

Viral evolution & population genetics

PO806

Hepatitis B virus genotype C population bottlenecks in East Asia during the 2003 SARS-CoV outbreak suggests complex competition dynamics of two co-circulating pathogens affecting host mobility

H.-F. Liu 1,*, S. Y. C. Lin 2, B. D. Rife 3, M. Salemi 3

1Department of Medical Research, Mackay Memorial Hospital, New Taipei City, 2Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, 3Department of Pathology, University of Florida, Gainesville, United States

Objectives: Hepatitis B virus (HBV) is highly prevalent in South and East Asia and its chronic infection is an important risk factor of liver cirrhosis and hepatocellular carcinoma. Ten genotypes, A to J, have been described so far across different geographical areas, with HBV genotype C (HBV/C) and D most frequently associated with disease progression. In East Asia, HBV genotype C is highly prevalent in South Korea, Hong Kong, and Japan. In China, genotype C is predominant in the northern regions.

Methods: All HBV genotype C full genome sequences with known sampling time and country of origin were mined from GenBank database and aligned. Phylogenies were reconstructed by neighbor-joining, maximum likelihood, and Bayesian inference methods. The time-scaled phylogeny and evolutionary rate were analyzed using Bayesian Markov Chain Monte Carlo algorism, and demographic shaping with nonparametric estimates of effective population size over time of HBV genotype C was performed by Skygrid method. The phylogeographic analysis was performed by BEAST software.

Results: The most recent common ancestor of HBV/C was traced back to the early 1900s in China, where it eventually diverged into two major lineages that gave rise to distinct epidemic waves spreading to other East Asian countries as well as the United States. Demographic inference of viral effective population size over time indicated similar dynamics for both lineages, characterized by exponential growth since the early 1980s, followed by a significant bottleneck in 2003 and another increase after 2004.

Conclusion: By using state of the art phylogeography techniques, we show that distinct epidemic lineages emerged from China during the 1930s-1960s and spread exponentially across different Eastern Asian countries in the following decades prior to a significant population bottleneck in 2003, which possibly correlated with restriction in human migration during the SARS-CoV outbreak. Indeed, fluctuation in HBV/C population size might reflect the effect of SARS-CoV on human mobility. The complex competition dynamic between two co-circulating pathogens revealed by the present study highlights the need, in the age of globalization, for the development of more realistic spatial ecology models of multiple infectious diseases.

Keywords: HBV genotype C, phylodynamics, SARS, virus population bottleneck

17th International Congress of Virology 384

Abstract Submission

Viral evolution & population genetics

PO807

MULTIPLE SOURCES OF GENETIC DIVERSITY OF INFLUENZA A VIRUSES DURING THE HAJJ

J. C. A. Cobbin 1,*, M. Alfelali 2 3, O. Barasheed 2 4, J. Taylor 5, D. Dwyer 6 7, J. Kok 6 7, R. Booy 8 9 10, E. Holmes 8, H. Rashid 6 7 8

1Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Life and Environmental Sciences and Sydney Medical School, University of Sydney, Camperdown, 2National Centre for Immunisation Research and Surveillance (NCIRS), The Children’s Hospital at Westmead, and the Discipline of Paediatrics and Child Health, Sydney Medical School, University of Sydney, Sydney, Australia, 3Department of Family and Community Medicine, Faculty of Medicine in Rabigh, King Abdulaziz University, Jeddah, 4Research Center, King Abdullah Medical City (KAMC), Makkah, Saudi Arabia, 5Institute for Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital , University of Sydney, 6National Centre for Immunisation Research and Surveillance (NCIRS), The Children’s Hospital at Westmead, 7Discipline of Paediatrics and Child Health, Sydney Medical School, 8Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Life and Environmental Sciences and Sydney Medical School, 9National Centre for Immunisation Research and Surveillance (NCIRS), University of Sydney, 10Institute for Clinical Pathology and Medical Research, NSW Health Pathology, Westmead Hospital, The Children’s Hospital at Westmead, Sydney, Australia

Objectives: Large population sizes and densities at mass gatherings such as the Hajj (Makkah, Saudi Arabia) can contribute greatly to outbreaks of respiratory virus infection, posing significant health risks to attendants, host communities and ultimately the global population if they help facilitate viral emergence. However, little is known about the genetic diversity, evolution and patterns of viral transmission during mass gatherings, particularly how much diversity is generated by in situ transmission compared to that imported from other locations. We aim to characterise the extent, origin and drivers of the diversity of influenza A virus at a genomic level within the setting of the Hajj, including whether sub-consensus mutations can be used to track individual transmission events.

Methods: Nasopharyngeal or throat swabs were collected during the 2013, 2014 and 2015 Hajj from pilgrims presenting with an influenza-like illness (ILI). Deep sequencing of influenza A positive samples was undertaken to describe the genomic-scale evolution of influenza A viruses.

Results: Phylogenetic analysis revealed that the diversity of influenza virus at the Hajj was shaped by multiple introduction events, comprising multiple co-circulating lineages in each year, including those that have circulated in the Middle East and those whose origins lie on different continents. At the scale of individual hosts, the majority of minor variants resulted from de novo mutation with only limited evidence of minor variant transmission or minor variants circulating at sub-consensus level despite the identification of multiple transmission clusters.

Conclusion: Together, these data highlight the complexity of influenza infection at the Hajj, reflecting a mix of global genetic diversity drawn from multiple sources combined with local transmission. The diversity of viruses at the Hajj also highlights the potential for lineage co-circulation during mass gatherings, in turn fuelling segment reassortment and the emergence of novel variants. These findings reemphasize the need for vigilant surveillance of respiratory pathogens at mass gatherings as a public health priority.

Keywords: Evolution , Influenza virus, Transmission

17th International Congress of Virology 385

Abstract Submission

Viral evolution & population genetics

PO808

PHYLOGEOGRAPHY OF SORICINE SHREW-BORNE HANTAVIRUSES IN NORTH AMERICA

H. J. Kang 1, S. Arai 2, S. H. Gu 1, J. A. Cook 3, L. J. Dizney 4, L. A. Ruedas 5, R. Yanagihara 1,*

1University of Hawaii at Manoa, Honolulu, United States, 2National Institute of Infectious Diseases, Tokyo, Japan, 3University of New Mexico, Albuquerque, 4University of Portland, 5Portland State University, Portland, United States

Objectives: Multiple species of Sorex shrews (order Eulipotyphla, family Soricidae) in Eurasia harbor genetically distinct hantaviruses, including Artybash virus in Sorex caecutiens, Asikkala virus in S. minutus, Kenkeme virus in S. roboratus, Qian Hu Shan virus in S. cylindricauda, Yakeshi virus in S. isodon, and Seewis virus in S. araneus, S. tundrensis and S. daphaenodon. The objective of this study was to determine the phylogeography of hantaviruses in soricine shrews in North America.

Methods: Total RNAs, extracted from natural history specimens comprising frozen lung or liver tissues of 337 soricine shrews (109 Blarina brevicauda, 10 Blarina carolinensis, 1 Notiosorex crawfordi, 1 Sorex bairdi, 3 Sorex bendirii, 40 Sorex cinereus, 1 Sorex haydeni, 4 Sorex minutissimus, 69 Sorex monticolus, 1 Sorex nanus, 12 Sorex palustris, 30 Sorex trowbridgii, 12 Sorex tundrensis and 44 Sorex vagrans), captured in widely separated geographic regions of the United States and Canada, between 1980 and 2006, were reverse transcribed and analyzed for hantavirus RNA by nested RT- PCR.

Results: Hantavirus RNAs were detected in tissues of 74 shrews (41 B. brevicauda, 1 B. carolinensis, 1 S. bairdi, 2 S. cinereus, 1 S. haydeni, 14 S. monticolus, 1 S. palustris, 5 S. trowbridgii, 8 S. vagrans). Phylogenetic analyses, using maximum-likelihood and Bayesian methods, based on partial or full-length S-, M- and/or L-segment sequences indicated four distinct hantaviruses: Camp Ripley virus in B. brevicauda, Iamonia virus in B. carolinensis, Ash River virus in S. cinereus, and Jemez Springs virus in S. bairdi, S. haydeni, S. monticolus, S. palustris, S. trowbridgii and S. vagrans. Camp Ripley virus-infected B. brevicauda were found in 12 states (Arkansas, Iowa, Kansas, Michigan, Minnesota, New Hampshire, Ohio, Pennsylvania, Virginia and Wisconsin), and Jemez Springs virus-infected Sorex shrews were captured in Alaska, California, Colorado, New Mexico, Oregon and Washington in the United States and in British Columbia and Yukon Territory in Canada.

Conclusion: The long-held conventional view that rodents are the principal hosts of hantaviruses is not tenable. Close collaborations with museum curators and field mammalogists have accelerated the acquisition of new knowledge about the spatio-temporal distribution of shrew-borne hantaviruses. Moreover, mounting evidence supports the concepts of extensive host sharing and host switching. That is, the same hantavirus species may be harbored by more than one reservoir host species.

Keywords: hantavirus, shrew

17th International Congress of Virology 386

Abstract Submission

Viral evolution & population genetics

PO809

Transcriptome analysis to identify expressed sequences derived from endogenous viral elements

S. Nakagawa 1 2,*, M. Ueda 2

1Department of Molecular Life Science, Tokai University School of Medicine, Isehara, 2Micro/Nano Technology Center, Tokai University, Hiratsuka, Japan

Objectives: In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs) including endogenous retroviruses (ERVs), which are thought to be derived from viral infections in germ cells. Although most EVEs have been inactivated by insertions, deletions, substitutions, and/or epigenetic modifications, a few open reading frames (ORFs) of EVEs are still active and express viral proteins in the hosts. Indeed, several genes derived from EVEs have been found to be functional for host species, such as syncytins for placenta development in various mammals. To identify these elements comprehensively, we developed a database for EVE ORFs in 20 mammalian genomes.

Methods: We used RetroTector and RepeatMasker with RMblast and RepBase to identify EVEs in genomes. For each identified candidate region, we scanned all possible codon reading frames, three in each direction (i.e. six frames). If the longest reading frame in the region does not contain any stop codons encoding more than 80 amino acids (aa), the amino acid sequence was searched by using HMMER with viral motif profiles. Hidden Markov models (HMMs) of the viral motif profiles used in this process were downloaded from the Pfam and the Gypsy databases. Each ORF having at least one HMM profile hit was stored in the database for the corresponding genome. Next, to retrieve EVEs that are missed by the two computational programs, we performed similarity searches using BLAT against each genome using amino acid sequences of endogenous and exogenous retroviruses (including sequences found in our study). We then summarized EVE ORF sequences with viral motifs and encoding more than 80 amino acids by removing overlapping sequences while accounting for reading frames.

Results: A total of 736,771 non-overlapping EVE ORFs for 20 mammalian genomes were found and stored in the gEVE database (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs, all of which can be downloaded. Further, using the gEVE data, we successfully identified various genes including syncytins derived from ERVs obtained from RNA-seq data stored in the NCBI SRA database. This result suggests that NGS data analyses combined with our annotation data enable us to discover hidden functional EVE sequences in genomes.

Conclusion: The gEVE database for EVE ORFs in 20 mammalian genomes is publically available that will contribute the further understanding of EVE sequences in the host genomes.

Keywords: comparative genomics, endogenous retrovirus, Molecular evolution

17th International Congress of Virology 387

Abstract Submission

Viral evolution & population genetics

PO880

Emergence of Dengue virus type 3 genotype I associated with upsurge of DENV3 cases in Klang Valley, Malaysia

K.-K. Tan 1 2,*, N. Zainal 1 2, J. Abd-Jamil 1, N. S. Azizan 1, C. N. Yaacob 1, N. A. A. Che Mat Seri 1, N. I. Samsudin 1, N. H. Mahfodz 1, S. AbuBakar 1 2

1Tropical Infectious Diseases Research and Education Center (TIDREC), University of Malaya, 50603 Kuala Lumpur, Malaysia, 2Department of Medical Microbiology, University of Malaya, Kuala Lumpur, Malaysia

Objectives: Malaysia is a hyperendemic country for dengue, where all four dengue viruses (DENV) co-circulates since the 1960s. The virus demonstrated a cyclical transmission cycle that involved DENV type 1 (DENV1), DENV type 2 (DENV2), and DENV type 3 (DENV3), but not DENV type 4 (DENV4). Surveillance data obtained from WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya demonstrated that since 2008 DENV3 exhibited low transmission rate (<15%) in Klang Valley, Malaysia. A sudden upsurge of DENV3 isolation, however, was observed in 2016. Here, we present a phylogenetic study of the DENV3 strains recovered from 2008 to 2016 to infer the changes of Malaysian DENV3 genetic diversity over time and the relationship between Malaysian strains with other isolates recovered globally.

Methods: A random subset of DENV3 virus samples recovered from 2008 to 2016 was obtained from that WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya. The virus was propagated through one round passage in cell culture. Viral RNA was extracted from cell culture supernatant. Five overlapped fragments spanning the complete genome of DENV3 were amplified and subsequently sequenced using Ion Torrent PGM platform. Sequencing raw reads were assembled using GSNAP algorithms as implemented in Sequencher 5.2.4. Phylogenetic relationships of the Malaysian DENV3 isolates with other isolates recovered globally was determined and presented.

Results: Phylogenetic analyses based on the complete coding region of DENV3 recovered from Klang Valley, Malaysia along with other sequences of representative strains retrieved from the GenBank demonstrated that the Malaysian DENV3 isolates recovered between 2008 to 2016 clustered temporally into two major genotypes. The Malaysian isolates collected before 2016 clustered into the DENV3 genotype III (DENV3/III), while the Malaysian 2016 isolates clustered within DENV3 genotype I (DENV3/I). The 2016 Malaysian isolates formed a Malaysian monophyletic group clustered closely with Indonesian strains recovered in 2008, suggesting a possible Indonesian origin of these Malaysian DENV3/I strains.

Conclusion: Our findings demonstrated the emergence of DENV3/I in 2016 where completely replaced the pre-existing circulating DENV3 strains, DENV3/III. This coincided with the upsurge of DENV3 isolation in Klang Valley, Malaysia. Continuous study to monitor changes in the virus population structures is important to provide insights into the virus transmission dynamics locally.

Keywords: Arbovirus, Dengue virus, Malaysia

17th International Congress of Virology 388

Abstract Submission Viral evolution & population genetics

PO868

AN IN SILICO INVESTIGATION OF ENTEROVIRUS TYPE 71 MOSAIC GENOME STRUCTURE G.-W. Chen 1, Y.-N. Gong 1,* 1Computer Science & Information Engineering, Chang Gung University, Taoyuan, Taiwan, Province of China

Objectives: Enterovirus type 71 (EV71) is a member of the Enterovirus genus that belongs to the family Picornaviridae. EV71 is a major viral agent associated with a wide range of clinical outcomes in human, from hand- foot-mouth disease to severe neural complications. Due to the genetic homology shared by many enterovirus genotypes/serotypes, these viruses are known to frequently exchange their genetic segments through genomic recombination. Moreover, the recombinants may further exchange their genomes to produce mosaic genomes. SimPlot is a tool commonly used to reveal genetic recombination of enteroviruses. It uses a sliding window to scan through the aligned query genome with respect to a number of template genomes to identity a breakpoint featuring significant pairwise percent identity discontinuity from one template to another. To successfully identify the tentative recombination pattern of a potentially highly mosaic genome, the choice of appropriate template genomes becomes critical. Methods: With thousands of enterovirus genomes available in the database, a systematic way to include needed template(s) in order to reveal the mosaic genome structure is needed. In this study, we took a sample EV71 genome from a local patient and used BLASTN to know that it resembles to a database EV71 genome known as a triple- recombinant (with 2 cross-overs) consisting of EV71 genotype C (part 1, from 5’ end up to VP1/2A juncture, ~3,760- nt), EV71 genotype B (part 2, ~1,656-nt), and CA16 (part 3 towards the 3’ end, ~1,971-nt). This mosaic structure is confirmed by SimPlot with the inclusion of template genomes of all three viruses. To further elucidate tentative mosaic component(s) of this particular EV71 genome, we performed BLASTN of part 2 and part 3 segments respectively, and found genomes that are genetically distinct from the two already identified mosaic components EV71 genotype B and CA16. Results: We found a new mosaic component from CA4 which resembles to our EV71 genome better than the originally found EV71 genotype B in the 2B/2C region. With the inclusion of this CA4 genome as the templates in SimPlot, we conclude that our EV71 genome a quadruple-recombinant with 3 cross-overs rather than a triple- recombinant shown earlier. Conclusion: Mining the enterovirus genome database helps in finding appropriate template genomes to be included in SimPlot to explore possible mosaic genome structure for a given enterovirus genome. This approach can be iteratively practiced to reveal more mosaic components, if they ever exist in the database.

Keywords: enterovirus A71, recombination, sequence database

17th International Congress of Virology 389

Virus Entry, Fusion, Assembly & Budding

17th International Congress of Virology 390

Abstract Submission

Virus entry, fusion, assembly & budding PO831

CELLULAR SPHINGOMYELIN PLAYS AN ESSENTIAL ROLE IN INFECTION OF RUBELLA VIRUS N. Otsuki*, M. Sakata 1, K. Saito 2, Y. Mori 1, K. Okamoto 1, K. Hanada 2, M. Takeda 1 1Department of Virology 3, National Institute of Infectious Diseases, Musashimurayama, 2Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjyuku-Tokyo, Japan

Objectives: Rubella is a mild disease characterized by low-grade fever and a morbilliform rash. Importantly, it causes multiple congenital defects in neonates born from mothers who suffered from rubella during the pregnancy. Rubella virus (RuV) is an enveloped RNA virus that enters cells via receptor mediated-endocytosis and low pH-dependent membrane fusion. A previous study suggested that cellular phospholipids support RuV infection. This study focused on the relationship between RuV infection and cellular sphigomyelin (SM).

Methods: (1) RuV shows hemmagglutination (HA) activity on goose erythrocytes. Goose erythrocytes were pretreated with sphingomyelinase (SMase) and HA activity of RuV was analyzed. (2) Vero cells were pretreated with SMase and infected with RuV to show the effect of SMase on RuV infection. (3) Serine palmitoyl transferase (SPT) is the key enzyme which functions at the first step of the sphingolipid biosynthetic pathway and myriocin inhibits the SPT activity. Thus, myriocin decreases levels of cellular SM and its intermediates. To investigate the relationship between RuV infection and SM biosynthesis, Vero cells were pre-incubated with myriocin and infected with RuV. (4) To investigate the roles of SM at the early step of RuV infection, a single-infection type RuV-virus-like-particle (RuV-VLP) was used. RuV-VLP expresses the luciferase as a reporter. Vero cells were incubated with RuV-VLP at 4℃ for 120min, and then incubated at 37℃ for 0, 20, 60, and 120min. After that, cells were incubated at 35℃ in presence of SMase for 3 days, and the luciferase activity in cells was measured. (5) To investigate whether depletion of cellular SM affects the replication of RuV genome, RuV-RNA replicon cells were used. The replicon cells constitutively express an artificial RuV genome encoding only RuV non-structural protein gene, C protein gene, and luciferase gene. The replicon cells were incubated with SMase or myriocin for 2days, and then the luciferase activity in cells was measured. (6) Finally, to investigate the physical interaction of RuV particles with SM, liposomes floatation assay was performed using liposomes containing SM, cholesterol and/or phosphatidylcholine.

Results: The HA titer has been greatly reduced, when goose erythrocytes were pretreated with SMase. These data indicated that SM on the erythrocyte membrane plays an important role in the RuV HA activity. The infectivity of RuV in Vero cells was greatly decreased by SMase or myriocin in a dose-dependent manner. These data indicated that cellular SM is necessary for RuV infection. Assays using RuV-VLP demonstrated that SM was necessary at the early step of RuV infection, because RuV-VLP infection measured by luciferase was strongly suppressed only when SMase-treatment was started at 0min and 20min after RuV-VLP infection. On the other hand, luciferase activity was no longer affected, when SMase-treatment was started at 120min after RuV-VLP infection. The luciferase activities of replicon cells were not affected at all, even when these cells were incubated with SMase or myriocin. These data indicated that SM was not necessary for the RuV genome replication in cells. The liposomes floatation assay revealed that RuV binds to liposomes, when the liposomes contain SM.

Conclusion: Cellular SM plays an essential role in RuV HA reaction and infection. Furthermore, the SM plays an important role in the early step of RuV infection, but was not necessary for the genome replication of RuV in cells. Finally, the floatation assay suggested that SM is a key molecule for the RuV attachment to cells.

Keywords: rubella virus, sphingomyelin, Virus entry

17th International Congress of Virology 391

Abstract Submission

Virus entry, fusion, assembly & budding PO832

ENHANCEMENT OF HUMAN ENTEROVIRUS 71 INFECTION BY CHLORPROMAZINE REVEALS AN UNKNOWN ENTRY PATHWAY OF ENTEROVIRUSES J. Yan 1,*, M. Yuan 1, J. Xun 1, C. Chen 1, Y. Zhang 1, S. Zhang 1 1Scientific Research Center, Shanghai Public Health Clinical Center, Shanghai, China

Objectives: Human Enterovirus A71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine or dynasore, which inhibit clathrin and dynamin dependent endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete.

Methods: Various cell lines susceptible to EV71 infection were tested. The EV71 infection and cell entry was monitored by flow cytometry and immunofluorescence. Chlorpromazine, dynasore and siRNA were utilized to explore the roles of various endocytic components. The effect of chlorpromazine on viral entry or post-entry activities was dissected using EV71 replicon and pseudoviral experiments.

Results: Chlorpromazine suppressed EV71 infection in various cell lines tested. However, it enhanced viral infection in A549 cells. Further studies showed that the maximum EV71 infectivity in A549 cells was time-consuming, and chlorpromazine did not shorten the process. Surprisingly, dynasore, the dynamin inhibitor, also enhanced EV71 infection in A549 and Hela cells, while it inhibited infections in other cells tested. Mechanistically, chlorpromazine did not increase EV71 binding to A549 cells and failed to enhance viral translation and replication, but enhanced viral entry by using EV71 replicon- RNA and pseudo-viral infection. Immunofluorescence microscopy further confirmed these findings. Using siRNA experiment, we found that the enhancement of EV71 infection by chlorpromazine did not require the component of clathrin mediated endocytosis and dynamin. Finally, the infection enhancing effect of chlorpromazine was also corroborated in Coxackivirus A16 infections.

Conclusion: Although chlorpromazine and dynasore could inhibit EV71 cell entry as previously reported in most cell lines tested, these inhibitors may enhance viral infection on particular cell types. This enhancement was due to increased viral entry and not other steps in viral life cycles. Current study indicated a novel dynamin-independent entry pathway utilized by Enteroviruses that cause Hand-Foot-and-Mouth Disease.

Keywords: clathrin, dynamin, enterovirus A71

17th International Congress of Virology 392

Late breaking abstract

Virus entry, fusion, assembly & budding PO834

Molecular interactions between Vaccinia virus envelope proteins A27 and A26 K. Chen 1,*, K. Carillo 1, P. Sterbova 1, G. Coronel 1, J.-C. Hsiao 1, W. Chang 2, D.-L. Tzou 1 1Institute of Chemistry, Academia Sinica, 2Institute of Microbiology, Academia Sinica, Taipei City, Taiwan

Objectives: In the current study, we were in an attempt to uncover the interactions between A26 and A27 at the molecular level and the residues responsible for pH sensing at N-terminus of A26 are to be reported.

Methods: Proteins A27 and A26 samples were expressed using BL21 (DE3) E. coli in LB medium. For NMR analysis, the protein samples were expressed with N15 isotope labeling using M9 medium. Circular Dichroism, Heteronuclear Single Quantum Coherence (HSQC) NMR experiment, and Surface Plasmon Resonance (SPR) experiment were done to determine the interaction between vaccinia virus envelope proteins A27 and A26.

Results: Vaccinia virus (VV) that infects various cell lines and animals is the prototype of the Orthopoxvirus genus of the Poxviridae family. VV envelope protein A27 is known for virus cell attachment that binds to heparan sulfate on cell surface. Aside from this, A27 serves as anchor for VV A26 protein, acting as a critical role in pH sensing that might regulate virus cell entry pathway. Previous studies showed that A27 is able to bind to A26 via its C-terminal domain. And the A27-A26 complex is tethered to vaccinia virus membrane via interaction between A27. The protein-protein interaction between A27 and A26 was shown to be either partially or completely interrupted by N-terminal or C-terminal mutations. Noteworthy there are six cysteine residues present in A26 protein sequence. Among them, Cys43 and Cys342 form intra-molecular disulfide bond, whereas Cys441 and Cys442 are involved in the formation of inter-molecular disulfide bonds with Cys71 and Cys72 residues of A27, respectively.

Conlusion: In addition to the disulfide bonds, it has shown that there are other interactions that stabilize the A27-A26 protein complex.

Keywords: A26 protein, A27 protein, vaccinia virus

17th International Congress of Virology 393

Abstract Submission

Virus entry, fusion, assembly & budding PO835

PROHIBITIN: A NOVEL VIRAL RECEPTOR AND HOST REPLICATION FACTOR FOR NEUROVIRULENT EV71 I. Too 1,*, E. L. Tan 2, J. J. H. Chu 1, S. Alonso 1 1Department of Microbiology and Immunology, National University of Singapore, 2Center for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore

Objectives: Enterovirus 71 (EV71) is considered as the most important neurotropic virus after the eradication of poliovirus. With no clinically approved vaccines or antiviral drugs against EV71, elucidating the mode of action involved in EV71 neuropathogenesis is essential.

Methods: Our lab has recently established a novel in vitro infection model to understand the neurovirulence of EV71 using the murine motor neuron cell line, NSC-34. Using this novel model, a 2-dimensional gel electrophoresis proteomic approach was employed to profile the differential expression of host proteins during viral infection.

Results: By mapping the interactions between EV71 and host cellular proteins, prohibitin (PHB) was identified as a potential host factor involved in EV71 infection cycle. Down-regulation of PHB expression by siRNA gene knockdown confirmed the role of PHB in EV71 infection of NSC-34 cells. Furthermore, using specific antibodies, we showed that blocking PHB expressed at the cell surface impaired viral entry in NSC-34 cells. In addition, we generated strong experimental evidence supporting that intracellular PHB expressed on the mitochondrial membrane interacts with non-structural proteins (3CD) of EV71, thus suggesting a role of this host protein during the viral genome replication.

Conclusion: Together, these findings support that PHB plays an important role during EV71 infection cycle in NSC-34 cells, at both the virus entry and replication steps. Importantly, similar observations were made with human neuroblastoma cells but not with human muscle cells, thus suggesting that the role of PHB during EV71 infection is limited and specific to neuronal cells, and contributes to the neurovirulence of EV71.

Keywords: enterovirus A71, neurovirulence

17th International Congress of Virology 394

Virus Surveillance, Epidemiology & Public Health

17th International Congress of Virology 395

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO837

180-Nucleotide Duplication in the G Gene of Human metapneumovirus A2b Subgroup Strains Circulating in Yokohama City, Japan, since 2014

M. Saikusa 1,*, C. Kawakami 1, N. Nao 2, M. Takeda 2, S. Usuku 1, T. Sasao 1, K. Nishimoto 1, T. Toyozawa 3

1Yokohama City Institute of Public Health, Yokohama, 2Department of Virology 3, National Institute of Infectious Diseases, Musashimurayama, 3Yokohama City Public Health Center, Yokohama, Japan

Objectives: The aim of this study was to strengthen our understanding of the epidemic behavior of human metapneumovirus (HMPV) by providing virological data on the distribution pattern and genetic evolution of HMPV.

Methods: In Yokohama City between January 2013 and June 2016, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) were collected from patients suffering upper or lower acute respiratory infections (ARIs) in 16 sentinel hospitals and clinics participating in the National Epidemiological Surveillance of Infectious Diseases (NESID), instituted by the Infectious Diseases Control Law in Japan, and in eight other medical institutions. The de-identified clinical specimens were subjected to multiplex RT–PCR, which identifies 15 major respiratory viruses. The clinical specimens that were positive for HMPV were analyzed further. The G gene cDNAs of the HMPV strains were amplified by RT–PCR and sequenced by direct sequencing. Phylogenetic analyses were performed with the maximum likelihood method in the MEGA software (ver. 7.0.20). Potential acceptor sites for N- and O-linked sugars were predicted with the NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc 3.1 programs (http://www.cbs.dtu.dk/services/NetOGlyc/), respectively. The overall rate of evolutionary change (nucleotide substitutions per site per year) in the HMPV G gene was estimated with the BEAST 2 program (http://beast2.org/), which uses a Bayesian Markov Chain Monte Carlo (MCMC) approach (BEAST 2: A Software Platform for Bayesian Evolutionary Analysis, http://dx.doi.org/10.1371/journal.pcbi.1003537). The year in which 180-nucleotide duplication (180nt-dup) occurred in the HMPV G gene was estimated based on the mean rate of nucleotide substitutions for the HMPV G gene.

Results: HMPV was detected in 91 specimens, accounting for 7.0% of the total specimens, and the nucleotide sequences of the G genes of 84 HMPV strains were determined. Among the 84 strains, six, 43, 10, and 25 strains were classified into subgroups A2a, A2b, B1, and B2, respectively. Approximately half the HMPV A2b subgroup strains detected since 2014 had a 180nt-dup in the G gene and clustered on a phylogenic tree with four classical 180nt-dup-lacking HMPV A2b strains prevalent between 2014 and 2015. The 180nt-dup causes a 60-amino-acid duplication (60aa-dup) in the G protein, creating 23–25 additional potential acceptor sites for O-linked sugars. The evolutionary rate of the HMPV G gene was estimated to be 4.3 × 10−3/site/year (95% highest probability density: 3.2–5.4 × 10−3/site/year). Based on these data, 180nt-dup was predicted to have occurred in the ancestral strain between 2011 and 2013.

Conclusion: Our data demonstrate a 180nt-dup in the G gene of HMPV and suggest that 180nt-dup occurred between 2011 and 2013. The HMPV A2b strains containing 180nt-dup (A2b180nt-dup HMPV strains) became major epidemic strains within 3 years. The detailed mechanism by which the A2b180nt-dup HMPV strains gained an advantage that allowed their efficient spread in the community and the effects of 60aa-dup on HMPV virulence must be clarified.

Keywords: G gene, Human metapneumovirus, Molecular epidemiology

17th International Congress of Virology 396

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO838

Assessment of household level transmission of Zika virus infection in Singapore

R. L. Lim 1,*, M. I. Chen 1

1Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore

Objectives: A Zika virus (ZIKV) outbreak occurred Singapore in August 2016. We conducted a prospective cohort study to assess the extent of household level transmission of ZIKV, assess if there were possibly any undiagnosed infections, as well as identify the epidemiological features associated with ZIKV infections in Singapore.

Methods: A total of 21 participants from 7 households were recruited into this study in October 2016, including 7 index patients (IP) and 1 non-index patient confirmed with ZIKV infection at Tan Tock Seng Hospital Communicable Disease Centre (TTSH CDC) as well as 13 members of their households. All participants answered a brief questionnaire regarding recent illness, travel and risk factors as well as provided blood and mid-stream urine samples at recruitment and at 3 - 6 months post-recruitment. Urine samples were tested for ZIKV RNA via real-time polymerase chain reaction (RT-PCR). Blood samples were processed to isolate serum, and serum was analyzed by serological testing.

Results: All ZIKV-confirmed patients were symptomatic and displayed a variety of symptoms including fever, rash, conjunctivitis, joint pain or swelling. The first five households were from known ZIKV-outbreak areas. In the first household with 6 members, the IP and his elder brother were diagnosed with ZIKV infection and with onset of fever and rash beginning two days apart. In the second household with 3 members, the IP reported onset of fever and rash 11 days after the onset of rash in her elder sister. The IP’s mother and elder sister were both not diagnosed with ZIKV infection. In the third, fourth, and fifth households, only the IP reported fever and rash lasting for 1-6 days while all other household members were asymptomatic and had no recent overseas travel history. The sixth household was living in a non-ZIKV-outbreak with no recent dengue outbreaks. Only the IP, but not her husband, reported onset of fever and rash lasting a day; the IP was working approximately 1-2 kilometres away from the largest Zika cluster in Singapore. In the seventh household, which lived in a non-ZIKV-outbreak area (but with recent dengue outbreaks), only the IP, but not his wife and mother, reported rash but with no fever lasting 7-8 days; the IP stayed in Brazil for a month for the 2016 summer Olympics. All urine samples tested negative for ZIKV at the time of recruitment. Of the 7 urine samples collected from the IP, 6 were collected beyond day 19 after symptom onset while 1 was collected on day 7 after symptom onset. Serology results are still pending.

Conclusion: Screening for household infections using RT-PCR to detect ZIKV RNA in urine samples had very poor yield in our study. While some of this may be due to the long gap between onset of symptoms in the IP and sample collection in the household members, we note that, of 14 non-IP household members, only 1 was diagnosed with ZIKV. However, there was one other individual with symptoms suggestive of ZIKV infection which was not diagnosed. It remains unclear if there was any household level transmission of ZIKV, and our serology results, which are still pending, may provide more definitive answers as to whether the IP’s household members were exposed to ZIKV.

Keywords: Epidemiology, Outbreak, Zika

17th International Congress of Virology 397

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO839

CHARACTERIZATION OF MOLECULAR EPIDEMIOLOGY AND GENETIC DIVERSITY OF IMJIN VIRUS (MJNV) IN CROCIDURA LASIURA, REPUBLIC OF KOREA

S.-H. Lee 1,*, W.-K. Kim 1, J. S. No 1, J.-A. Kim 1, J. I. Kim 1, S. H. Gu 2, H.-C. Kim 3, T. A. Klein 4, M. Park 1, J.-W. Song 1

1Microbiology, Korea university, Seoul, 25th R&D Institute, Agency for Defense Development, Daejeon, Korea, Republic Of, 35th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247, APO AP 96205-5247, 4MEDDAC-Korea, 65th Medical Brigade, Unit 15281, APO AP 96205, United States

Objectives: Hantaviruses (family Bunyaviridae) are enveloped negative-sense RNA viruses including large (L), medium (M), and small (S) segments. In humans, hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in America. In East Asia, annually 100,000 cases of HFRS have been reported in the Republic of Korea (ROK) and China. HFRS is mainly caused by Rodent-borne hantaviruses, e.g. Hantaan virus, Seoul virus, and Puumala virus. Recently, shrew-borne viruses were very likely able to infect humans in Africa, but the pathogenicity remained unclear. Since the question raises as to whether the shrew-borne virus can infect a human and induce a disease, the definition of molecular epidemiology and phylogeographic analyses of Imjin virus (MJNV), a shrew- borne hantavirus in ROK, are required for the surveillance of MJNV infection.

We first discovered and isolated MJNV from the Ussuri white toothed shrews (Crocidura lasiura) in highly HFRS endemic areas, ROK. In this study, dynamic circulation and viral loads of MJNV in various tissues of shrews captured from the endemic areas, Gyeonggi and Gangwon provinces, were demonstrated by serological and molecular analyses. The coding region of MJNV tripartite genomes was obtained and phylogenetic analyses revealed the geographical diversity and genetic exchanges, recombination and reassortment of MJNV.

Methods: C. lasiura was collected in Gangwon and Gyeonggi provinces during 2011 to 2014. Indirect immunofluorescent- antibody assay (IFA) was examined for the presence of MJNV-specific IgG. Total RNA was extracted from tissues, followed by performing nested RT-PCR for genomic sequencing of MJNV. Viral loads of MJNV RNA in the various tissues were measured by quantitative RT-PCR. Phylogenetic trees were generated using Maximum-likelihood (ML) methods. Recombination Detection Program 4 (RDP4) applied to the analysis of MJNV genomic organization based on open reading frames of the L, M, and S segments.

Results: Of a total of 96 C. lasiura, four (4.2%) shrews were seropositive for anti-MJNV IgG and MJNV RNA was detected from nine (9.4%), respectively. The preponderance of MJNV-infected shrews was male (17.1%) and adult (13.3%) consistent with the gender- and weight-specific prevalence of hantaviruses in other species. The viral load of MJNV RNA in various tissues, including lungs, livers, kidneys, and spleens, was varied and higher amounts in sero- and PCR-positive (IFA+PCR+) shrews than IFA-PCR+ shrews. Using coding sequences of MJNV tripartite genomes, phylogenetic analyses demonstrated geographic clustering for the L and S segments while the M segment was the most divergent. Finally, the genomic characterization of MJNV suggested that MJNV 12-2 (Pyeongchang) was compatible with recombination of MJNV L segment. The genome of MJNV 04-55 and 13-1 (Yeoncheon) contained heterogeneous M segment and homologous L and S segments, indicative of reassortants.

Conclusion: In conclusion, the prevalence of MJNV described the preferential infection in male and adult shrews, ROK. Profiling of MJNV RNA in various tissues of the shrews may reflect the dynamic infectious status and circulation of MJNV in natural reservoirs. Phylogeographic and genomic characterization of MJNV indicated the highly divergent viral population including the natural occurrence of recombination and reassortment. Thus, these findings provide better understandings for the molecular epidemiology, dynamic circulation and genetic diversity of shrew-borne hantaviruses. 17th International Congress of Virology 398

Keywords: Hantavirus, Molecular epidemiology, Phylogenetic analysis, Dynamic circulation, Genetic exchange- recombination and reassortment

17th International Congress of Virology 399

Late breaking abstract

Virus surveillance, epidemiology & Public Health

PO840

Clinical Study and Detection of MCPyV in Hospitalized Children with Respiratory Tract Infection

L. Zheng*, T. Wei

Objectives: To investigate the prevalence and clinical features of MCPyV in hospitalized children with respiratory tract infection (RTI) in Beijing.

Methods: 200 nasopharyngeal aspirates (NPA) were collected from 200 hospitalized children aged <14 years old with RTI in Beijing Friendship Hospital China from January 2014 to April 2014. LTAg gene of MCPyV was detected with an established TaqMan real-time PCR and confirmed by sequencing. Primers and probe used for this assay were 5- TCTGGGTATGGGTCCTTCTC-3 (forward primer), 5-CATGGTGTTCGGGAGGTATATC-3 (reverse primer), and FAM- ACTGGGAGTCTGAAGCCTGGGA-TAMRA. The resulting amplicon was 79bp. All the MCPyV-positive specimens were screened for other common respiratory viruses, including RSV, HMPV, HBoV, IFV(A, B, C), PIV 1-4, HCoV-229E, HCoV- OC43, HCoV-NL63，HCoV-HKU1, WUPyV and KIPyV.

Results: The detection limit of the real-time PCR assay was 1 copy and detection range was from 1010-100 copies/μl. Coefficient of determination (R2 = 0.997) shows a good linear correlation. The TaqMan-based real-time PCR assay for MCPyV detection did not amplify any other mixed virus, indicating excellent specificity. Of the 200 children with ARI included in the study, and the gender ratio was 1.44:1 (male 112, female 88). The median age was 24 month and the age range was 3 days to 14 years. The prevalence of MCPyV was 3% (6/200), the infected children ranged in age from 6 month to 5 years and children £ 3 years of age accounted for 83.3% (5/6) of cases. 6 MCPyV-positive patients were diagnosed with bronchopneumonia and acute bronchitis. The most common symptoms were cough, fever and asthma. All 6 MCPyV- positive patients were coinfected with other respiratory viruses, of which influenza virus A (IFVA) and respiratory syncytial virus (RSV) were most common. Additionally, their viral loads were lower than 10 copies/ml NPA specimen.

Conlusion: The prevalence of MCPyV was 3% in NPA specimens from hospitalized children with RTI, measured by real- time PCR. Regarding the 100% coinfection rate and the low viral load, MCPyV is not considered to be the cause of respiratory diseases.

Keywords: Merkel cell polyomavirus, Respiratory infection, TaqMan real-time PCR

17th International Congress of Virology 400

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO841

Comparative Seroepidemiology of Coxsackievirus A6, A16 and Enterovirus 71 Infections in Singapore Adults

R. L. Lim 1,*, A. R. Cook 1, N. Z. Hamis 2, P. P. K. Lau 2, S. Chen 2, M. C. Phoon 2, W. M. Koh 1, M. I. Chen 1, V. T. Chow 2

1Saw Swee Hock School of Public Health, 2Department of Microbiology and Immunology, National University of Singapore, Singapore, Singapore

Objectives: Coxsackievirus A6 (CA6), coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are the predominant causative agents causing periodic outbreaks of hand, foot and mouth disease (HFMD) in Singapore. We conducted a cross- sectional study to estimate and compare the seroprevalence of CA6, CA16 and EV71 infections in an adult cohort in Singapore. We also investigated the specificity of neutralizing antibodies against CA6, CA16 and EV71 in sera obtained from BALB/c mice immunized with the respective inactivated viruses.

Methods: We tested 115 serum samples from healthy adults aged 21 to 78 years collected from Tan Tock Seng Hospital during the 2009 H1N1 pandemic in Singapore. The neutralizing antibody levels against CA6, CA16 and EV71 were analyzed by microneutralization test. The seroprevalence rate and geometric mean titer (GMT) of neutralizing antibodies against each of the enteroviruses were calculated. Young, female adult BALB/c mice were immunized intraperitoneally with monovalent, heat-inactivated CA6, CA16 or EV71, together with either PBS control, or an adjuvant (aluminium hydroxide or imiquimod, a toll-like receptor 7 agonist). Microneutralization tests were performed on the sera collected from immunized mice at various time-points.

Results: Overall, high seroprevalence rates were observed in the adult population in Singapore, with CA6 and CA16 seroprevalence being higher than that of EV71. While CA16 and EV71 seroprevalence rates were similar in both genders, CA6 seroprevalence was significantly higher in females than males. Infections of the three enteroviruses exhibited similar seroprevalence rates among different ethnic groups in Singapore. Overall, the GMT of the three enteroviruses displayed a declining trend as age increased. The GMTs for CA6, CA16 and EV71 were 44.2, 52.8 and 22.5 respectively among young parents aged 30 years, compared with 34.5, 24.1 and 17.9 in middle-aged adults aged 50 years. While CA16 and EV71 GMTs were not affected by the presence of children in the household, CA6 GMT was significantly higher for individuals with household members aged less than 5. A significant proportion of individuals possessed neutralizing antibodies against either both CA6 and EV71, or CA16 and EV71. This may imply either multiple infections of CA6, CA16 and EV71, or cross- reactivity of neutralizing antibodies. While this could not be ascertained from the medical history of the study participants, we addressed this question by mouse immunization models. Microneutralization tests on the sera collected from immunized mice revealed neutralizing antibodies against the specific enterovirus, but did not exhibit any non-specific cross-reactivity.

Conclusion: In general, CA6 and CA16 infections are highly prevalent in the adult population in Singapore, and with seroprevalence rates even higher than that of EV71. It is likely that the high seroprevalence of CA6, CA16 and EV71 infections represent multiple enterovirus infections rather than cross-reactivity of enterovirus antibodies. In view of the potentially severe clinical complications of EV71 infection, EV71 candidate vaccines are currently in phase IV clinical trials to evaluate protective efficacy against this important causative agent of HFMD.

Keywords: Coxsackievirus A16 (CA16), Coxsackievirus A6 (CA6), Enterovirus 71 (EV71)

17th International Congress of Virology 401

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO842

CORRELATION BETWEEN SFTS VIRUS ANTIBODY POSITIVE RATE IN WILD DEER AND NUMBER OF SFTS PATIENTS

S. Morikawa*, M. Kimura 1, T. Yoshikawa 2, Y. Kaku 1, E.-S. Park 1, K. Imaoka, M. Saijo 2, K. Maeda 3

1Department of Veterinary Science, 2Department of Virology 1, National Institute of Infectious Diseases, Shinjuku, 3Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan

Objectives: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease, with fatality rate ranging from 2 to 30%, first identified in China in 2009, then found in Japan and South Korea in 2013. SFTS virus (SFTSV), a member of the Phlebovirus genus in the family Bunyaviridae, is transmitted by tick. In Japan, a total of 217 laboratory- confirmed SFTS cases with fatality rate of 22% were reported since 2013. All the patients were reported in the western region of Japan. In Japan, wild Japanese deer and other animals have been shown to have SFTSV antibodies. To understand the significance of SFTSV antibody positive rate in wild deer, serological surveillance of SFTSV in wild deer was conducted in the SFTS endemic and non endemic region.

Methods: Blood samples of captured wild Japanese deer during the hunting season between 2015 and 2017 were kindly supplied from hunters associations and municipalities. Antibodies to SFTSV were detected by SFTSV-specific ELISA and immunofluorescent antibody test. Statistical analysis was performed if there is positive correlation between antibody positive rate in wild deer and number of SFTS patients in the 20 prefectures in Japan.

Results: SFTSV antibody positive deer were found in 17 out of 20 prefectures in Japan and SFTS patients were reported in 10 out of 20 prefectures between 2015 and 2016. Pearson’s positive correlation with correlation coefficient of some 0.7 was observed between SFTSV antibody positive rate in wild deer and number of SFTS patients in the prefectures so far tested.

Conclusion: SFTSV is widely distributed in Japan, however, antibody prevalence in wild deer has been shown to be significantly high in the endemic region. SFTSV is maintained in an animal-tick interface, and wild deer are considered to play a crucial role in the interface. In the present study, we have shown that antibody prevalence in wild deer is correlated with number of SFTS patients, thus it is important to conduct serosurveillance in animals to evaluate risk of SFTS in the region.

Keywords: Seroepidemiology in animal, severe fever with thrombocytopenia syndrome, SFTS virus

17th International Congress of Virology 402

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO843

CYCLOVIRUS DETECTION IN CHILEAN CHILDREN AND ADULTS WITH AND WITHOUT RESPIRATORY DISEASE BY REAL-TIME PCR

V. Luchsinger 1,*, E. Torres 1, Y. Prades 1, C. Moreno 1, D. Benadof 2, R. Pizarro 3, M. Ruiz 4, L. Avendaño 1

1Virologia, ICBM, Facultad de Medicina, Universidad de Chile, 2Laboratorio, Hospital R. Rio, 3Medicina, Hospital L Cordova, 4Medicina, Hospital Clinico U Chile, Santiago, Chile

Objectives: Cyclovirus (CyCV) is a member of the recently proposed genus of the family Circoviridae detected in insects, birds and mammals; includes naked viruses with a small circular ssDNA. It was first detected in humans in 2010; it has been reported in 3-15% of sick children and 0.7%>33.3% in asymptomatic. The only published study of respiratory specimens (Phan et al. 2014) displayed CyCV detection in 3.3% of 120 nasopharyngeal aspirates of Chilean children with acute respiratory infections (ARI). Infection frequency in adults is unknown. The aim of this study was to detect CyCV in adults and children with and without respiratory disease.

Methods: CyCV was studied in 372 respiratory specimens: 105 from hospitalized adults with community-acquired pneumonia (CAP) between 2012 and 2014; 101 from hospitalized infants with ARI during 2016; 104 from asymptomatic adults between September and December 2016, and 62 from children without respiratory disease rolled up between November 2016 and January 2017 in Santiago, Chile. Total nucleic acids from samples were extracted by MasterPure kit (Epicenter®) and a fragment of rep protein gene of CyCV was amplified by real time polymerase chain reaction, using primers and probes designed by Beacon Designer 8.0® and the Kapa PROBE® kit in a RotorGene6000®. Quantitative variables and qualitative were compared with Mann Whitney test and Fisher´s test, respectively.

Results: CyCV was detected in 57/105 (54.3%) adults with CAP and 18/104 (17.3%) asymptomatic adults (p<0.001). In 15/101 (14.9%) children with ARI and in 22/62 (35.5%) infants without respiratory disease was amplified CyCv (p<0.01). In the 4 studied groups male ratio was similar between individuals with or without CyCV (p>0.05). Median age of CAP adults with CyCV was 67 years (range 20-89 years) and without CyCV was 69 years (range 21-87 years) (P=0.7). Median age of ARI children with CyCV was 3 months (range 0.7-10 months) and without CyCV was 5 months (range 0.4-11 months) (P=0.6). Twenty five amplicons of 150bp from adults were purified and sequenced. The identity of all of them was over 95% compared to the four previously identified CyCV Chilean strains.

Conclusion: This is the first report of CyCV in adults with and without respiratory disease. Infection frequency was significantly higher in adults with CAP than asymptomatic adults and children with and without ARI and all percentages were higher than previously published. Cyclovirus could be a new respiratory pathogen in adults.

Keywords: children respiratory infection , Cyclovirus, respiratory disease

17th International Congress of Virology 403

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO844

Dengue seroprevalence of healthy adults in Singapore shows a sustained low level of dengue Force of Infection

L. K. Tan, S. L. Low*, J. Ong, S. Lam, H. H. Tan, L. W. Ang, W. Y. Wong, R. Chua, L. C. Ng, A. R. Cook

Objectives: Singapore is a dengue endemic city state and in the past two decades has experienced periodic dengue epidemics. Due to asymptomatic, subclinical and misdiagnosed dengue infections, routine national dengue notifications do not capture all dengue infections and thus do not reflect the true intensity of disease transmission. To better assess the true infections and disease control efforts in Singapore, age-stratified serosurveys were conducted on the local populace in 2009 and after the largest dengue outbreak in 2013. Fusing these data with those from previous sero-surveys, the long-term temporal changes in the force of infection (FOI), i.e. the rate at which susceptible individuals were infected, was estimated using Bayesian computational methods.

Methods: Dengue serosurveys were conducted on healthy non-remunerated voluntary blood donors (n=3,627; age 16-60 years in 2009 and n= 3,813; age 16-60+ years in 2013). Their residual sera were screened for dengue IgG using the Panbio dengue IgG Indirect ELISA.

Age-specific dengue seroprevalence data independently collected in 2004 and from a pediatric cohort in 2009 were synthesized with our data to estimate the historical dengue FOI. The total number of samples used for analysis was 12,576. The modelled annual FOI enabled us to estimate the basic reproduction number (R0, defined as the number of secondary cases which one case would produce in a completely susceptible population), infections averted and proportion of notified cases among true infections.

Results: The age weighted dengue IgG prevalence in 2013 was 49.8% (95% CI: 48.4 to 51.1%) and the prevalence increased with age. In the age group 16 to 60 years, there was a significant decline (Fisher’s exact test, P < 0.05) in the weighted IgG prevalence from 2009 (50.8%, 95% CI: 49.4 to 52.3%) to 2013 (44.8%, 95% CI: 43.3 to 46.3%). The year-on- year estimates of dengue FOI from 1930 to 2013 was above 50 per 1000 per year in 1960s, gradually declined over the next two decades, , after which it remained stable from mid-1990s at around 10 per 1000 per year. The reproduction number (R0) also declined since the 1960s. The change in age structure of the population was found to be insufficient to explain the reduction in transmission, and some of the decline may be attributed to the sustained national vector control program. The estimated proportion of notified cases among true infections improved from 1:12 in 2005–2009 to 1:7 in 2010–2013. The number of non-infected residents in 2013 across age were projected to be 2.8 million, 2.1 million, 0.96 million and 0.16 million if FOI was held fixed since 1960, 1970, 1980 and 1990 respectively. No additional infections were averted among residents based on the FOI estimate in 2000 and a wide credible interval was observed.

Conclusion: Our results show that a substantial proportion of the population remains susceptible to dengue infections. The improved notified case to infection rates signifies that the national notification system that relies on diagnosed cases has improved over time. The data also suggest that the magnitudes of epidemics cannot be fairly compared across calendar years and current disease control program remains applicable.

Keywords: Dengue fever, force of infection, seroprevalence

17th International Congress of Virology 404

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO845

DETECTION OF AN EMERGING ETIOLOGICAL AGENT OF VIRAL DIARRHOEA WITH THREE SEGMENTED GENOMIC RNA PROFILE IN KOLKATA, INDIA

E. Anthony*

Objectives: In course of an ongoing surveillance program in collaboration between NICED, Kolkata, India and Okayama University, Japan, faecal specimens (n=1924) received during 2014-2016 from Infectious Disease and Beliaghata General Hospital and Dr Bidhan Chandra Roy Postgraduate Institute of Pediatric Sciences were screened to understand the disease burden and etiology of pathogens associated with diarrhoea.

Methods: During the screening for etiological agents (rotavirus and other non-rotaviral agents) we detected an emerging enteric pathogen showing 3 segmented genomic RNA profile by 2% agarose gel electrophoresis and ethidium bromide staining, in 184 diarrhoea cases. The first genomic segment of the emerging virus was seen to migrate above the 1st segment of rotavirus i.e. <3302 bp; the 2nd segment was below the 6th segment of rotavirus i.e. >1356 bp and the 3rd segment was closer to the 10th segment of rotavirus approx. >1074 bp in the agarose gel.

Results: 91 cases were positive for emerging agent with three segmented genomic RNA profile (Fig. 1) as the only detectable etiological agent, in different age groups - viz. >1yr-3 cases, >2 yrs. -1 case, >3 yrs. -3 cases, >5 yrs. -12 cases; >12 yrs. -5 cases, >18yrs. -6 cases. The age group of adult cases was 18-40 yrs. - 30 cases, >60 yrs. -19 cases, 60-90 yrs. -12 cases. Besides acute watery diarrhoea, other clinical symptoms associated with these positives were (a) vomiting in 28.57% (26 cases); (b) vomiting with abdominal pain in 34% (31 cases). All three symptoms viz. fever, abdominal pain and vomiting was recorded in 37.36% (34 cases). 93 positives with emerging etiological agent were of mixed infection.

Conclusion: Thus, this emerging virus has appeared as another etiological agent besides Rotavirus, Astrovirus, Norovirus, Sapovirus, Picobirnavirus and Adenovirus hitherto reported from Kolkata, India.

17th International Congress of Virology 405

Image:

Keywords: Emerging RNA Virus, Three segmented RNA genome profile, Viral gastroenteritis

17th International Congress of Virology 406

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO846

DETECTION OF HANTAAN VIRUS RNA GENOME FROM ANTI-HANTAAN VIRUS IGG SERONEGATIVE APODEMUS AGRARIUS IN HIGHLY ENDEMIC AREAS IN KOREA, DURING 2013-2016.

J. S. No 1,*, W.-K. Kim 1, J.-A. Kim 1, S.-H. Lee 1, J. Jung 2, D. Lee 3, D. H. Song 3, H.-C. Kim 4, T. A. Klein 5, J.-W. Song 1

1Department of Microbiology, Korea University, Seoul, 2The Armed Forces Medical Center, Seongnam, 35th R&D Institute, Agency for Defense Development, Deajeon, Korea, Republic Of, 45th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247, APO AP 96205-5247, 5MEDDAC-Korea, 65th Medical Brigade, Unit 15281, APO AP 96205, United States

Objectives: Hantaan virus (HTNV), of the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in human. Recently, the viral RNA of Hokkaido virus (HOKV), a genus of Hantavirus, was detected in anti-PUUV seronegative grey red-backed voles (Myodes refocanus). The outbreak of HPS occurred in the USA showed high percentage of hantavirus RNA positivity from seronegative rodents. Although the majority of epidemiologic studies has found that rodents are seropositive for hantavirus-specific immunoglobulin, the discovery of hantavirus RNA in seronegative hosts has led to an investigation of the presence of HTNV RNA in rodents captured in the endemic areas. In this study, we demonstrated the presence of HTNV RNA from anti-HTNV IgG seronegative rodents in the highly HFRS-endemic areas in the Republic of Korea (ROK).

Methods: Apodemus agrarius, natural reservoir of HTNV, was captured in Gyeonggi and Gangwon provinces from 2013 to 2016. Indirect immunofluorescent antibody (IFA) test was performed in 458 A. agrarius and total RNA was extracted from lung tissues and analyzed by HTNV M segment-specific RT-PCR. The phylogenetic tree was generated by Maximum likelihood method using MEGA 5. RT-qPCR was performed targeting the HTNV S segment in the lung, liver, kidney, and spleen of rodents.

Results: A total of 69 (15.1%) of 458 A. agrarius were positive for anti-HTNV IgG antibodies and 61(88.4%) of them harbored the HTNV M segment. To investigate the presence of HTNV RNA in seronegative rodents, we performed RT-PCR for the HTNV M segment. Hantaviral RNA was detected in Seven (1.8%) of the anti-HTNV IgG seronegative rodents captured in Gyeonggi province. Phylogenetic analysis demonstrates that HTNV strains from anti-HTNV IgG seronegative rodents form geographically specific clusters. To identify the difference amounts of HTNV RNA, RT-qPCR was performed in various tissues of anti-HTNV IgG seronegative and HTNV RNA positive (IFA-PCR+) rodents. Cycle threshold (Ct) value was in the range of 27–36 in IFA-PCR+ rodents. Anti-HTNV IgG seropositive and HTNV RNA positive (IFA+PCR+) rodents showed lower Ct values (large amounts of HTNV RNA) than the IFA-PCR+ rodents in all of tissues examined in this study.

Conclusion: Detection of the HTNV RNA genome in the seronegative rodents may be a means of identifying the circulation and early infectious stage of HTNV. In addition, HTNV RNA was detected only in seronegative rodents from Gyeonggi province. These data may reflect that the HTNV is actively circulated among the rodent population in the Gyeonggi province. In conclusion, our data suggest the dynamic circulation of HTNV in highly endemic areas, providing important insights into the epidemiology and virus-host interaction of hantaviruses in nature.

Keywords: Epidemiology, Hantavirus, Virus-host interaction

17th International Congress of Virology 407

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO847

Diversity of Japanese encephalitis virus after replacement of genotype in Japan.

K. Matsui*, S. Tajima, F. Kato, M. Daibata, T. Takasaki

Objectives: Japanese encephalitis virus (JEV) is an important mosquito-borne virus infection disease in east southern Asia. There are five genotypes of JEV, of which genotype 3 (G3) has been circulating until the early 1990s and has been increasingly replaced by genotype 1 (G1) since the 1990s in Japan. Several JEV strains were isolated between 2000 and 2016 but they were identified based only nucleotide sequence and were not analyzed biological characterization. We have isolated G1 virus from swine serum in 2006, 2012, 2013 and 2014 with 1, 1, 2 and 1 strains, respectively. Although these sequences differed at the nucleotide level their amino acid sequences were very similar.

Methods: To investigate phenotypic differences, we characterized 5 strains, including vaccine strain Beijing-1, and compared multiplication kinetics in mammalian Vero cells plus C6/36 and NIID-CTR mosquito cell lines. In addition, sensitivity of interferon was measured using A549 cells, and neutralization with a JEV specific monoclonal antibody (mAb 503b) by plaque reduction neutralization test (PRNT).

Results: There were no apparent differences in multiplication kinetics by the five JEV strains in the three cell types. Similarly, there was no difference in sensitivity of INF-γwere no difference of these isolates. However, the Beijing-1 strain tighter binding to mAb 503b than the five isolates, which could not be distinguished in binding to mAb 503b.

Conclusion: Overall, our results indicate that the isolates differed in nucleotide sequence but there was no difference of multiplication in cell culture, binding to mAb and sensitivity of INF-γ.

Keywords: Japanese encephakitis virus, genotype 1, biological characterization

17th International Congress of Virology 408

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO848

Epidemiological Stasis of Pandemic Norovirus GII.4 may be Mirrored in the Genotypic Transition Identified in the Environmental Occurrence Survey

Y. S. Jeong 1,*, E. S. Koo 1

1Biology, Kyung Hee University, Seoul, Korea, Republic Of

Objectives: The presence of human norovirus in the aquatic environment can cause outbreaks related to recreational activities and the consumption of norovirus-contaminated clams. In this study, we investigated the relative dominance of norovirus genogroups I (GI) and II (GII) based on open reading frame 1 (ORF1) or ORF1-ORF2 sequences detected in the coastal aquatic environment neighboring clam fisheries in South Korea. We also analyzed newly emerged GII sequences considered as causative epidemic candidates in the study area.

Methods: In the phase I study from March 2014 to February 2015, a total of 504 water samples were collected periodically from four coastal areas (A–D areas; total sites = 63), of which 44 sites were in estuaries (clam fisheries) and 19 were in inflow streams. In the phase II study from March 2015 to May 2016, we collected 182 water samples from 26 sites of the inflow upstreams in three coastal areas (B, C, and D areas) to monitor the norovirus distribution prior to massive dilution by marine water. One hundred liters of environmental water per a sample was passed through Nano-Ceram filters according to standard procedures. Subsequent elution and concentration processes followed USEPA method 1615. PCR-amplified products of the target gene were purified and cloned. Human norovirus sequences were identified and aligned using ClustalX. Phylogenetic relationships of the aligned sequences were determined using MEGA6.

Results: Sequencing analysis of the ORF2 region C in the phase I study revealed that 20.6% of the water samples were contaminated with GI (13.3%) or GII (16.6%). A total of 229 human norovirus sequences were identified in the water samples, and phylogenetic analysis showed that the sequences clustered into eight GI genotypes (GI.1, 2, 3, 4, 5, 6, 7, and 9) and nine GII genotypes (GII.2, 3, 4, 5, 6, 11, 13, 17, and 21). During this study period, we observed that various genotypes of GII were often codominant rather than showing single dominance of GII.4. Interestingly, dominant genotypes appeared to be replaced rapidly and somewhat periodically. Based on ORF2 region C sequence analysis in the phase II study, there was a much higher prevalence of GII.17 and GII.21 than of GII.4 until September 2015. Additional sequence analyses of the ORF1-ORF2 junction and ORF2 from the water samples revealed that the GII.17 sequences in this study were closely related to the novel strains GII.P17-GII.17, the main causative variants of the 2014–2015 human norovirus outbreak in China and Japan. Furthermore, the GII.21 sequences were identified as GII.P21-GII.21 variants and contained new amino acid variations in the blockade epitopes of the P2 domain. Since December 2015, however, the detection frequency of GII.4 Sydney 2012 capsid sequence has increased and overtaken those of both GII.17 and GII.21 at the beginning of 2016. The ORF1 genotype of this re-emerging GII.4 variant was identified as GII.P16, which has not been combined to pandemic GII.4 series thus far.

Conclusion: This study highlights four issues: 1) a strong correlation between norovirus occurrence in inflow streams and coastal areas nursing clam fisheries; 2) the codominances involving various genotypes of both GI and GII appeared to reflect the epidemiological stasis of the GII.4 pandemic variants during the study period; 3) the novel GII.P17-GII.17 variants appeared to co-circulate in Far-East Asia; 4) re-emergence of the GII.4 Sidney 2012 recombined with a different RNA- dependent RNA polymerase genotype (GII.P16) in environmental settings may predispose the area to a future pandemic.

Keywords: coastal aquatic environment, epidemiological stasis of GII.4, human norovirus

17th International Congress of Virology 409

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO849

Extensive genomic recombination among the circulating viruses of Enterovirus A species

K.-M. Lee 1,*, S.-R. Shih 1 2

1Department of Medical Biotechnology and Laboratory Science, College of Medicine, 2Research Center for Emerging Viral Infections, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan

Objectives: Enterovirus A71 (EV-A71), which is a species of the enterovirus genus belonging to the family Picornaviridae, is mainly associated with hand, foot, and mouth disease, but sometimes results in severe neurological complications and even death in young children. In Taiwan, several subgenotypes of EV-A71 identified during epidemics, including B4, C2, and C4, have been shown to be recombinant. Viral recombination can greatly affect the virulent properties (e.g., antigenicity and cell tropism) of the existing virus and lead to the prevalence of emerging viruses. However, the causal relationship between recombination and epidemics has yet to be clarified.

Methods: In this study, we performed a full-genome analysis of 61 clinical isolates of EV-A71 collected from 2005 to 2014 in Northern Taiwan. We found the periodic circulation of different subgenotypes, and the prevalent strain in the 2005, 2008, 2010, and 2012 epidemics are C4, B5, C4, and B5, respectively. In contrast to the ladder-like tree-structure (a characteristic of the continuous evolution) of the subgeotype B5 in the phylogram, the C4 type showed the scattered distribution, suggesting the co-existence of various C4 strains. We combined our results with other full-genome sequences of EV-A71 and several coxsackieviruses retrieved from GenBank to comprehensively analyze the recombination among the co- circulating viruses of Enterovirus A species (EV-A) in Taiwan

Results: Our results showed that the subgenotype B3, which is also recombinogenic with the coxsackievirus A16 (CV- A16)-like P3 region, participated in the intra-typic recombination of EV-A71 and led to the emerging of the subgenotype C4 and C2-like strain. In addition, such kind of EV-A71 (P2)/CV-A16 (P3) signature could also be found in several co-circulating coxackieviruses including CV-A2, CV-A4, CV-A6, and CV-A12, probably through multiple recombination events involving the inter-typic recombination of EV-A71/C4 and CV-A2, and intra-typic recombination among coxsackieviruses.

Conclusion: The recombination analyses clearly show that most, if not all, recombination breakpoints in EV-A have non- random distributions and are most likely to occur between structural gene (P1) and cis-replication element (cre) of P2 region, indicating that the specific combination of P1 and nonstructural region (P2 and P3) might subject to natural selection. In addition, the widespread existence of EV-A71/CV-A16 signature in a variety of circulating viruses of EV-A also suggests that this specific genomic assembly might be benefic for the viruses. However, whether the signature could increase the fitness or virulence of the recombinant viruses remain to be addressed.

Keywords: Coxsackievirus, enterovirus A71, recombination

17th International Congress of Virology 410

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO850

GENETIC REASSORTMENT AND DIVERSITY OF HANTAAN VIRUS IN HIGHLY HFRS-ENDEMIC AREAS, REPUBLIC OF KOREA.

J.-A. Kim 1,*, W.-K. Kim 1, J. S. No 1, S.-H. Lee 1, D. Lee 2, D. H. Song 2, M.-S. Park 1, H.-C. Kim 3, T. A. Klein 4, J.-W. Song 1

1Microbiology, Korea University, Seoul, 25th R&D Institute, Agency for Defense Development, Daejeon, Korea, Republic Of, 35th Medical Detachment, 65th Medical Brigade, Unit 15247, APO AP 96205-5247, 4MEDDAC-Korea, 65th Medical Brigade, Unit 15281, APO AP 96205, United States

Objectives: Hantaan virus (HTNV), a negative sense RNA virus of the Family Bunyaviridae, is the causative agent of a rodent-borne disease known as Hemorrhagic Fever with Renal Syndrome (HFRS). Gangwon and Gyeonggi provinces are highly endemic areas of HFRS in the Republic of Korea (ROK). However, previous studies conducted epidemiological and phylogeographic analyses of Hantaan virus (HTNV) in Gyeonggi province. The present study first demonstrated the geographic distribution and phylogenetic diversity of HTNV in Gangwon province.

Methods: Rodents (Apodemus agrarius) were captured in the Gangwon and Gyeonggi provinces of the ROK, from 2003 to 2014. A total of 5,929 A. agrarius were examined for anti-HTNV IgG from sera and a partial sequence of M segment (1,982-2,327nt) from lung tissues of the seropositive rodents using immunofluorescence antibody (IFA) test and HTNV- specific RT-PCR, respectively. Whole genome sequences of HTNV were recovered using conventional RT-PCR and Rapid Amplification cDNA Ends PCR. Phylogenetic and Recombination/Reassortment analyses were performed using the whole sequences of HTNV tripartite genomes.

Results: Of 5,929 A. agrarius, 774 (13.1%) of A. agrarius were seropositive for anti-HTNV IgG. HTNV-specific RT-PCR showed that 70.3% (544/774) A. agrarius were positive for the partial sequence of HTNV M segment. To investigate the molecular diversity of HTNV in the endemic areas, the whole genome sequences of 34 HTNV strains were completely obtained, consisting of Cheorwon, Hwacheon, and Yanggu in Gangwon province and Paju, Pocheon, and Yeoncheon in Gyeonggi province. Phylogenetic analysis showed the geographic distribution and diversity of HTNV in the ROK. Using Recombination Detection Program 4 (RDP4), the recombination/reassortment analysis suggested a possible reassortment of HTNV L segment in nature.

Conclusion: This study determined the geographic distribution and phylogenetic diversity of HTNV in Gangwon and Gyeonggi provinces. The phylogeographic analysis of HTNV showed the genetic diversity and a likely natural HTNV reassortant in Gangwon and Gyeonggi provinces. These observations may provide a better understanding of molecular evolution and epidemiology of hantaviruses.

Keywords: Hantaan virus, Phylogeographic diversity, Reassortment

17th International Congress of Virology 411

Late breaking abstract

Virus surveillance, epidemiology & Public Health

PO851

IUMS 2017 Singapore - Gladys YEO - Low risk of Zoonotic Transmission from Civet Cats in Singapore

G. Yeo*

Objectives: The environmental surveillance of infectious diseases could offer early detection of an emergence of zoonotic diseases. The establishment of an effective surveillance system is essential for the understanding of the ecology of a disease, pathogen epidemiology and ultimately for the development of disease control programmes. The emergence of SARS in 2003 epitomized the threat of dangerous pathogens to global biosecurity; and the association between SARS and civet cats highlighted the need for surveillance and risk assessment of animal-borne diseases in our local population of Paradoxurus hermaphroditus, or palm civet cats.

Methods: Here, we initiated a study to estimate the size of our local population of civet cats, namely the common palm civets. We investigated infection for a range of diseases in these civet cats living in Singapore between 2012 and 2015.

Results: Using molecular methods of detection, none of the the civet cats (n = 278) in the study was found to be infected with coronavirus (CoV), lyssavirus, Hantavirus, Rickettsia spp. and Leptospira spp. Furthermore, none of the sera specimens contained any antibodies that were specific to SARS-CoV, rabies virus, Dengue virus and Leptospira spp. Faecal swabs (n = 36) collected were also negative for Leptospira spp. To our knowledge, this is the first report on the investigation of pathogens and antibody prevalence in common palm civets in Singapore.

Conlusion: Nevertheless, wild animals constitute a potential zoonotic risk to other pathogens which should not be overlooked. For that, a framework for preparedness, management and mitigation of any zoonotic risk from civet cats would need to be established in the event of any future emergence of zoonotic diseases.

Keywords: Civet Cats, Surveillance, Zoonotic Transmission

17th International Congress of Virology 412

Late breaking abstract

Virus surveillance, epidemiology & Public Health

PO852

IUMS 2017 Singapore - LIM Xiao Fang - Surveillance of Zoonotic Pathogens in Singapore Bats

L. Xiao Fang*

Objectives: Bats are important potential reservoirs and vectors for the transmission of emerging infectious diseases (EID). Many highly pathogenic viruses such as SARS coronavirus and rabies-related lyssavirus have crossed the species barrier to infect humans and other animals. Till date, only a few studies are dedicated to understand the bat’s ecology and their role in zoonotic pathogens transmission in Singapore.

Methods: We have conducted an island wide survey on bats’ roosting sites and determined the species, demographic and population size for 15 locations. It was found that the Lesser Dog-faced bat is the most commonly found species in Singapore. Guano samples collected from three of the major roosting sites were screened for pathogens such as CoV, rabies-related lyssavirus, Japanese encephalitis and Leptospira spp. by PCR. Besides these samples, random guano samples submitted by our collaborators were also subjected to similar screening.

Results: Through the surveillance program, we have detected Bat CoV (Group 2, clade d; Betacoronavirus) from 3 pools of samples collected from Lesser dog-faced bats in Singapore. This virus was found to be phylogenetically closely related to the HKU9 (2007) and Diliman-1525G2 (2008). HKU9 originated from Guandong province of Southern China where the SARS epidemic surfaced in 2002 and has less than 70% identities to all known coronavirus.

Conlusion: Through the study, we have identified the major bat roosting sites and are conducting periodic survey of the guano samples as part of continual surveillance for risk assessment of bats borne diseases.

Keywords: Bats, Surveillance, Zoonotic Pathogens

17th International Congress of Virology 413

Late breaking abstract

Virus surveillance, epidemiology & Public Health

PO853

IUMS 2017 Singapore - Marcella KONG - Risk of Japanese Encephalitis Virus Transmission in Singapore

M. Kong*

Objectives: Japanese Encephalitis (JE) is one leading cause of viral neurologic disease in Asia, caused by the Flavivirus, Japanese Encephalitis Virus (JEV). The transmission cycle of JEV is kept within water birds, swines and humans as reservoirs, amplifying hosts and accidental dead-end hosts respectively. Culex mosquitoes, particularly Culex tritaeniorhychus, is a primary vector for transmission. The JE endemic situation in Singapore was improved after the abolishment of pig farms in 1992, with only a few sporadic cases in recent decades. However, JEV antibodies were detected in animals at offshore islands and the peripheral parts of Singapore in early 2000, which may be an indicative of JE transmission among wildlifes. A collaborative study between National Parks Board and National Environment Agency of Singapore was thus initiated to determine the risk of JEV transmission on the main city island.

Methods: Areas with the presence of both animal hosts and vectors involved in JEV transmission were identified through active site surveillance. Mosquito diversity at these locations was determined and the potential JE vectors were subjected to blood meal preference analysis and the PCR screening for the presence of JEV. The study was coupled with passive surveillance of animal sera collected from migratory birds, donor birds, wild boars and sentinel chickens.

Results: Till date, we have detected mosquito pools with JEV genotype 1 and 2. Consistent with the previous studies, JEV antibodies were also detected in different animal hosts. These results implied the continuous JEV transmission with the presence of suitable hosts and vectors in Singapore.

Conlusion: It is thus important to have a periodic, if not continual surveillance program to access and evaluate the impending risk of JE in Singapore.

Keywords: Mosquito, Japanese Encephalitis Virus, Surveillance

17th International Congress of Virology 414

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO855

PREVALENCE OF RESPIRATORY PATHOGENS IN ADULTS WITH ACUTE FEBRILE ILLNESS IN SINGAPORE

M. Mah 1,*, M. Linster 1, M. Moorthy 1, Z. Zhu 1, J. Lim 1, L. Ch'ng 1, Y. Su 1, C. Tam 1, R. Coker 2, J. Low 3, E. E. Ooi 1, G. Smith 1

1Program in Emerging Infectious Diseases, Duke-NUS Medical School, 2Saw Swee Hock School of Public Health, National University of Singapore, 3Duke-NUS Medical School, Singapore, Singapore

Objectives: The Early DENgue infection and outcome study (EDEN) enrolled patients presenting with acute febrile illness at primary care centres in Singapore. We performed a retrospective screen and analysis of the obtained nasopharyngeal samples for nineteen viral targets using Luminex technology (NxTag RPP). The identification of the aetiological agents from patients presenting with uncharacterized acute fever will enhance our understanding of disease illness and spread.

Methods: A total of 2950 respiratory nasopharyngeal samples were collected from adults that presented at five polyclinics in Singapore with an acute onset of fever (≥38°C) within less than 72 hours between December 2007 to February 2013. These samples were previously tested negative for dengue virus. Viral RNA was extracted from samples stored in universal transport medium, followed by PCR identification of 22 pathogens using the Luminex NxTAG Respiratory Pathogen Panel multiplex approach.

Results: The percentage presence of a viral respiratory pathogen overall was 41.2% (1214/2950). The viral targets from the panel were detected with the following frequencies: influenza A virus, 19.1% (564/2950) [seasonal H1N1, 0.3% (9/2950), pandemic H1N1, 11.0% (325/2950), seasonal H3N2, 7.8% (230/2950)]; influenza B virus, 6.8% (201/2950); parainfluenza viruses 1-4, 2.1% (62/2950); seasonal human coronaviruses, HKU1, OC43, NL63 and 229E, 2.7% (81/2950); human respiratory syncytial virus/human metapneumovirus, 1.9% (59/2950); rhinovirus/enterovirus, 8.0% (237/2950); adenovirus, 1.0% (29/2950); human bocavirus, 0.1% (4/2950).

Conclusion: To our knowledge, this has been a first study investigating the prevalence of a comprehensive panel of respiratory viruses in the general adult population in Singapore. The identification of respiratory viruses provides new insight into undifferentiated acute febrile illnesses of the population in Singapore and will help to target vaccine development and pathogen diagnostics.

Keywords: febrile illness, Public health, Respiratory tract infections

17th International Congress of Virology 415

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO856

QUALITY RISK MANAGEMENT (QRM)-BASED APPROACH TO ASSURE THE VIRAL SAFETY OF POLYDEOXYRIBONUCLEOTIDE DERIVED FROM SALMON SPERM OR TESTICLE

I. S. Kim*, J. E. Bae 1, J. M. Han 1

1Biological Sciences and Biotechnology, Hannam University, Daejeon, Korea, Republic Of

Objectives: Sodium polydeoxyribonucleotide (PDRN) has been used as the medicinal and cosmetic product for treating decubitus, wound, or scar, and skin graft and skin care. Most of PDRN is obtained from salmon sperm or testicle by an extraction process. Therefore the use of PDRN necessarily entails some degree of virus contamination risk. For ensuring the safety of PDRN it is necessary to manage the virus risk according to ICH guideline, Quality Risk Management Q9.

Methods: Quality risk management is a systematic process for the assessment, control, communication and review of risks to the quality of the drug (medicinal) product across the product lifecycle. For QRM-based approach to assure the viral safety of PDRN, two systematic measures such as virus testing for raw materials and viral clearance validation were adopted.

Results: For the testing of adventitious viruses, the most common assay is in vitro assay for the presence of viral contamination in which the test material is inoculated into susceptible cell lines such as the African green monkey kidney cell line Vero and the human diploid fibroblast cell line MRC-5, and the readout is a visible cytopathic effect. The manufacturing process contains heat treatment procedure at 90°C for 3.5 hours and 84% ethanol treatment for 15 hours. These procedures have the potential to inactivate viruses. In order to evaluate the efficacy of heat treatment and ethanol treatment for inactivation of possible contaminating viruses, a variety of experimental model viruses including Infectious hematopoietic necrosis virus, Viral hemorrhagic septicemia virus, Hepatitis A virus, and Porcine parvovirus were chosen. In vitro virus testing showed that the raw materials do not contain viruses susceptible to Vero or MRC-5 cell line. Virus clearance validation study revealed that all the viruses tested were completely inactivated during heat treatment and ethanol treatment.

Conclusion: These results indicate that the manufacturing process for PDRN from salmon sperm or testicle has sufficient virus-reducing capacity to achieve a high margin of virus safety.

Keywords: Polydeoxyribonucleotide , Quality Risk Management , Viral safety

17th International Congress of Virology 416

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO857

RESOURCE IMPACT OF MANAGING SUSPECTED MERS PATIENTS IN A UK TEACHING HOSPITAL

J. Veater 1, N. Wong 1, I. Stephenson 2, S. Atabani 3, J. Tang 1,*

1Clinical Microbiology/Virology, 2Infectious Diseases Unit, University Hospitals of Leicester NHS Trust, Leicester, 3Public Health Laboratory Birmingham, Public Health England, Birmingham, United Kingdom

Objectives: To analyse the cost of additional infection prevention measures for patients admitted with suspected Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection over the standard precautions used for other seasonal respiratory viruses; including a cost analysis of our hospital’s policy of maintaining enhanced precautions until MERS-CoV infection had been excluded, even despite an alternative laboratory-confirmed diagnosis in some cases; and to compare the difference in cost and turnaround time between sending a sample out to a reference laboratory to test for MERS-CoV versus performing the testing in-house.

Methods: The medical notes of 5 patients admitted with febrile respiratory illnesses in October 2015 after returning from MERS-CoV endemic areas were reviewed. The duration of admission, time to positive in-house respiratory viral PCR result and time to negative MERS-CoV reference lab result were calculated for each patient. The additional costs for each patient were then calculated based on the cost per unit for each addition piece of personal protective equipment (PPE), the estimated extra staff time required for additional patient visits, the additional staff training required to work in enhanced containment conditions, the extra cleaning measures required, and additional laboratory costs for processing potential MERS-CoV samples versus those for testing for the usual seasonal respiratory viruses. The clinical features, identified pathogens, treatment and outcome for each patient were also summarised.

Results: All 5 patients tested positive for influenza A/H1N1, A/H3N2 or B viruses and negative for MERS-CoV. Notably, all patients presented ahead of the UK seasonal influenza epidemic activity suggesting they acquired their infection abroad, such that pre-travel immunisation may have prevented their infections. Throat swabs from all patients tested positive by in- house PCR assays for influenza A/H1N1, A/H3N2 or B viruses within 31 h (median: 23 h) from collection. MERS-CoV PCR results were available to clinicians between 25 h and 57 h from collection (median: 50 h) and patients remained in medium- secure negative-pressure isolation for a mean of 39 h (median: 45 h). All patients recovered. The cost for additional PPE equipment was relatively low, and most of the additional laboratory costs were borne by the reference laboratory. However the cost of additional staff time required for each visit was more significant. In total, the additional costs from all of these contributions were calculated at £119 per patient per day. Some additional costs were not quantifiable, such as the cost of individual negative pressure room ventilation or disposal of enhanced PPE waste as they were indistinguishable from that of the whole ward.

Conclusion: In-house PCR testing could potentially reduce the costs of managing patients admitted with suspected MERS- CoV. However the benefit of an earlier diagnosis and a reduction in the use of enhanced PPE measures on the clinical ward side would then need to be weighed against the potential extra costs on the diagnostic laboratory side, including the extra costs of sustaining the availability of the MERS-CoV test kits throughout the year for relatively few sporadic cases of suspected MERS cases being admitted who then require testing for MERS-CoV.

Keywords: Infection control cost effectiveness, Influenza, MERS

17th International Congress of Virology 417

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO858

SEROEPIDEMIOLOGICAL AND MOLECULAR EPIDEMIOLOGICAL STUDY OF HEPATITIS B VIRUS AMONG LOCAL RESIDENTS: A COMMUNITY-BASED STUDY IN 2012 AND 2016, WUHAN, CHINA

X. Zhou 1,*, B. Chen 2, B. Pang 1, N. Kobayashi 3, Y. Wang 1

1Division of Microbiology, 2Division of Infectious Disease, Wuhan Centers for Disease Prevention and Control, Wuhan, China, 3Sapporo Medical University School of Medicine, Sapporo, Japan

Objectives: China has historically been a highly endemic area for hepatitis B infection. To learn the hepatitis B virus infection status, and analyze the dynamic changes of immunity levels, HBV serological status of local residents was investigated through a community-based study in Wuhan, 2012 and 2016. At the same time, the molecular epidemiological study of hepatitis B virus, including both viral loads assay and the phylogenetic analysis, was performed from all HBsAg positive samples.

Methods: Local residents have been recruited by stratified cluster random sampling, and each participant’s information and serum were collected. Serological markers including HBsAg, anti-HBs, anti-HBc, HBeAg and HBeAb were assayed by enzyme linked immunosorbent assay (ELISA). DNA was extracted from all HBsAg positive samples. HBV-DNA load was determined by quantitative real-time PCR assay, and S gene sequence was determined from nested-PCR product. Alignment and phylogenetic analysis were performed using MEGA 7.0 software. Data was analyzed by Epi Info version 7.2.0.1.

Results: A total of 4 901 questionnaires and 4 877 blood samples were collected. Among these, 5.54% (270/4 877), 38.94% (1 899/4 877) and 66.64% (3 250/4 877) participants were positive for HBsAg, anti-HBc and anti-HBs, respectively. As an indicator of vaccine-mediated immunity, isolated anti-HBs was present in 64.93% participants (1 868/4 877). Of all HBsAg- positive participants, 12 (4.44%) were positive for both HBsAg and HBeAg. HBsAg prevalence was higher in adults over the age of 23 (8.02–10.60%) compared with that in children under the age of 12 (2.07–3.41%; p<0.01) and other age groups (3.23–6.40%; p<0.01), increasing with age (p<0.01). HBsAg prevalence in males (5.06%) was almost the same as that in females (5.95%) (P>0.05). There was no significant difference in the prevalence between urban and rural areas (2.52% and 3.56%, respectively, P>0.05). Viral loads were detected in 267 HBsAg-positive participants, and 53 (19.85%) were positive. Serological status of HBsAg (+)/anti-HBc (+)/HBeAg (+) had the highest HBV-DNA load, and their DNA positive rate were significantly higher than that of other serum markers combinations. Viral loads of serological status of HBsAg (+)/anti-HBc (+), HBsAg (+)/HBeAb (+)/anti-HBc (+) and anti-HBs (+)/HBeAb (+)/anti-HBc (+) were decreasing in turn. The HBV-DNA load had significant difference in those serum markers combinations mentioned above (P<0.05). Two HBV genotypes were identified. The majority was genotype B (51/53), followed by C (2/53). The S gene sequences showed high identities (98- 99%) to those of neighboring countries and region such as Japan, Malaysia, South Korea, Australia and Taiwan.

Conclusion: HBsAg prevalence among children has decreased in Wuhan. The highest HBsAg prevalence in adults over age 23 suggests urgent need of expanded vaccination to adults. To understand the infection status exactly, a combined assay for both HBV serum markers and viral loads is recommended. This is the first molecular epidemiological study on HBV of local residents which will facilitate understanding the molecular epidemiology and long-term evolutionary dynamics of HBV.

17th International Congress of Virology 418

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Keywords: Hepatitis B Viral Load, Phylogenetic Analysis, Seroepidemiological Survey

17th International Congress of Virology 419

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO859

Serotyping of Adenovirus Detected from Influenza-Like Illness Surveillance in the Philippines, 2006-2012

C. Calzado 1 2,*, V. L. Arguelles 2, J. L. Foronda 2, A. Tandoc 2, H. Oshitani 3, M. Saito 3, M. Okamoto 3

1Graduate School, University of Santo Tomas, Manila, 2Department of Virology, Research Institute for Tropical Medicine, Muntinlupa, Philippines, 3Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japan

Objectives: Human adenoviruses (HAdv) are DNA viruses belonging to the genus Mastadenoviridae, and can cause various serotype-specific diseases, such as acute respiratory infections, conjunctivitis, and acute gastroenteritis. In the Philippines, 871 HAdv were isolated from influenza-like illness (ILI) cases in the National Influenza Surveillance from 2006- 2012; however, the serotypes are unidentified. We conducted this study to determine the circulating serotypes, since existing data regarding adenoviruses and its serotypes are limited in the country.

Methods: Nasopharyngeal and/or oropharyngeal swabs were collected through national Influenza-Like Illness (ILI) surveillance from 2006 to 2012. Samples were inoculated into HEp2c cells and observed daily for adenovirus-like cytopathic effects. HAdv were confirmed through immunofluorescence microscopy and isolates were subjected to neutralization test using serotype-specific antisera.

Results: From 2006 to 2012 a total of 37, 590 ILI cases were seen in the National Influenza Surveillance. HAdv infection was identified in 871/37,590 (2.3%) of the cases and represented 871/6,221 (14%) of all viral respiratory pathogens detected. We randomly selected 136 HAdv isolates from sentinel sites from 2006-2012 and have detected seven serotypes: 41/136 (30%) HAdv-2; 29/136 (21%) HAdv-3; 22/136 (16%) HAdv-1; 17/136 (13%) HAdv-5; 11/136 (8%) HAdv-7; 3/136 (2%) HAdv-6; 1/136 (0.7%) HAdv-4 and 12/136 (8%) are unsubtypeable.

Conclusion: HAdv are also seen in cases of influenza-like illness. Serotype identification should be included as part of testing algorithms in surveillance systems to detect new serotypes and better understand its epidemiology and pathogenesis. Results obtained from this study will provide additional information to guide the prevention and control strategies of the Department of Health, provide data needed for vaccine component preparation, and open doors to future studies on the prevalence and epidemiology of HADv serotypes in the country.

Keywords: adenovirus, Epidemiology, serotyping

17th International Congress of Virology 420

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO860

The Risk Factors in the Initiating, Spreading and Containing of Major Epidemics of Dengue in Tainan, Taiwan

P.-W. Shih 1,*, T.-C. Chan 2, H.-Y. Ko 3, Z.-R. Li 3, L.-Y. Wang 4, C.-T. Fang 3, C.-C. Ku 5, C.-C. King 3

1Institute of Epidemiology and Preventive Medicine, National Taiwan University (NTU), 2Research Center for Humanities and Social Science, Academia Sinica, 3Institute of Epidemiology and Preventive Medicine, College of Public Health, NTU, 4Institute of Public Health, College of Medicine, National Cheng-Kung University, 5Institute of Immunology, College of Medicine, Taipei, Taiwan

Objectives: Dengue is a seasonal vector-borne viral disease in Tainan (located in southern Taiwan) with different magnitude of outbreaks in various years. Most annual total numbers of indigenous dengue cases in this city were lower than 506, much smaller than those in Kaohsiung. However, large-scale outbreaks did occur in 2007 and 2015 caused by dengue virus serotype 1 (DENV-1) and DENV-2, respectively. The aims of this study were to fully understand the risk factors involved from the beginning through different waves to the ending stages of these two outbreaks for better monitoring of dengue cases and prediction in epidemic trends in future years.

Methods: Because Aedes aegypti mosquitoes have an important role in dengue clusters, we used geographical information system (GIS) to search for clusters of dengue cases by SaTScan software. Li is the smallest administration unit and also an area for prevention/control of mosquitoes has been conducted. Therefore, Li-specific data for different variables were collected. All these Li-specific risk factors were analyzed within each cluster. To fully understand possible different risk factors (population density, age structure, flooding areas etc) might be associated with various epidemic stages (from early to middle and late periods), univariate analyses were conducted. Then, the geographically weighted model was used for multivariate analyses.

Results: Both the 2007 and 2015 outbreaks started from the same Annan and North Districts of Tainan City. The diffusion of dengue cases had similar trends from the East to the West toward the estuary. In 2015, by the time reached to the middle stage of the epidemic, population density became more important. The dengue cases in 2015 were more clinically severe than those in 2007, with the highest incidence and case fatality rates among those with age over 60 years.

Conclusion: In conclusion, these results imply that mosquito surveillance plus health education are both important in those common areas involved in the initiation of dengue outbreak. In addition, residents living in high population density areas should be taught by usage of mosquito repellant and prevention measures. Furthermore, age-stratified syndromic surveillance should be tested in a pilot study through international collaboration, while elderly dengue cases had lower and shorter fever plus atypical symptoms/signs.

Keywords: Dengue , Risk factors, Taiwan

17th International Congress of Virology 421

Abstract Submission

Virus surveillance, epidemiology & Public Health

PO862

Near patient sampling to assist infection control for a tetraplegic, chronic adenovirus-infected paediatric patient.

J. Tang*, A. Patel 1, R. Cannon 1, S. Moran 2, E. Hoyle 3, M. Pareek 4

1Clinical Microbiology/Virology, 2Leicester Children's Hospital, 3Infection Prevention and Control, University Hospitals of Leicester, 4Infection, Immunity, Inflammation, University of Leicester, Leicester, United Kingdom

Objectives: A 6-year old tetraplegic, bed-bound girl with a tracheostomy on long-term ventilation became chronically infected with a seasonal respiratory adenovirus (serotype C1) infection. Regular nasopharyngeal aspirates (NPAs) showed AdV loads varying from 1000 copies/ml to 163 million copies/ml, using a quantitative DNA PCR assay over a 4-month period. Due to this high level of AdV being potentially shed into the environment from the respiratory tract of the patient, all healthcare staff were required to wear personal protective equipment (PPE), consisting of: surgical masks, face visors, gloves and gowns each time they attended this patient for educational and play visits, as well as the daily maintenance of her tracheostomy. The paediatric team were therefore concerned whether this would affect her psycho-social development as only the staffs’ eyes, but no other facial expression were visible to the patient, each time they visited. Near-patient air- sampling was initiated to assess the actual exposure risk to staff of airborne AdV from this patient, to determine whether such a high level of personal protective equipment was really necessary.

Methods: An SKC Button Sampler powered by an AirChek XR5000 pump (both supplied by SKC Ltd., Blandford Forum, UK) was used for all the air sampling. This collected air onto a dissolvable gel filter at a rate of 4 L/min. At the end of the sampling period, the gel filter was carefully extracted from the filter, dissolved in 2 ml of virus transport medium (VTM), extracted (QIAsymphony DSP Virus/Pathogen Kit, Qiagen, Manchester, UK) and run on the Ausdiagnostics (Respiratory Viruses 16-WELL, cat. 20602, AusDiagnostics UK Ltd., Chesham, UK) assay with a claimed lower limit of detection of 1075 AdV copies/ml. Various positions around the patient’s head and room were sampled for varying durations, multiple times, over several weeks, to detect the presence of airborne AdV DNA. The air-sampling sessions were coordinated to coincide with the days on which routine NPAs were taken from the patient for monitoring the AdV levels.

Results: In total 9 air-samples were taken: 2 from the bedside table (~1-1.5 m away from the patient’s head, static sampling, sampled for 4 hrs); 2 during daily suctioning of the tracheostomy site (~0.5-1 m away, worn on collar by physiotherapist, 30- 40 mins); 3 during daily suctioning of the tracheostomy (~0.3-0.5 m away, hand-held over the tracheostomy site, 30-40 mins); 1 during an educational session (~0.5-1m away, worn by the educational therapist, 40 mins); 1 from the over-the- bed table (0.3-0.5 m away, static sampling, 14.5 hrs). No AdV was detected in any of these samples. Further control experiments involving spiking the gel filters prior to extraction/PCR with the patient’s AdV showed no inhibition, and minimal impact of the dilution factor due to the gel filter being dissolved in 2 ml VTM, compared to the neat NPA PCR signal.

Conclusion: As a result of these experiments, the recommendation for PPE for attending this child was relaxed, surgical masks and face visors were no longer required, with no detrimental effects upon the staff to this date. Although infection prevention and control guidelines are necessarily precautionary and generally universal to avoid confusion, near patient air- sampling can be used to refine the PPE requirements in specific patient cases where the need arises.

Keywords: adenovirus, air-sampling, Infection control

17th International Congress of Virology 422

Abstract Submission Virus surveillance, epidemiology & Public Health

PO893

Molecular epidemiology of viral conjunctivitis in the southern region of South Korea, 2012-2016 : Adenovirus, Enterovirus D. W. Park*, Y. S. Lee 1, H. J. Park 2, S. H. Kim, J. W. Park 2, E. S. Kim 2, H.-Y. Kee 2, J. K. Chung 1GWANGJU HEALTH UNIVERSITY, 2Health and Environment Institute of Gwangju, Gwangju, Korea, Republic Of

Objectives: Epidemic keratoconjunctivitis (EKC) and acute hemorrhagic conjunctivitis (AHC) are a common disease caused by human adenovirus(HAdV) and enterovirus, respectively, in South Korea. But, there are very limited data regarding to trend of occurrence to control of viral conjunctivitis in South Korea. The main objective of this study was to determine the trend of types of adenovirus and enterovirus causing viral conjunctivitis in the southern region of South Korea. Methods: We collected conjunctival swabs from 492 suspected patients with viral conjunctivitis from 6 ophthalmic hospitals in Gwangju Metropolitan City in South Korea between 2012 and 2016. Both protocols RT-PCR were performed using Human Enterovirus/Adenovirus Detection kit (POSTBIO Inc. Gyeonggi-do, South Korea) for detection of positive according to the manufacturer’s instructions. After detection of 249 HAdV samples and 19 enterovirus samples among 492 samples, we conducted conventional PCR with 268 samples and sequencing reaction as described previously. Results: Of 492 samples, HAdV and enterovirus were detected in 249 samples (50.6%) and 19 samples (3.9%) respectively by Real- Time PCR. The serotype analysis showed HAdV-8 in 183 samples (73.5%), HAdV-37(5.6%) in 14 samples, and HAdV-3(3.6%), HAdV-4(3.6%) in 9 samples respectively. And coxsackievirus A24 (CA24) in 8 samples (42.1%) and coxsackievirus B1 (CB1) in 4 samples (21.1%) were detected respectively. We found the two cases of coinfection with HAdV and enterovirus. Conclusion: HAdV-8 and CA24 known as main cause of viral conjunctivitis worldwide were found to be the predominant serotype in our study. But, because predominant type is a little different for each year like HAdV-3 and 4 in 2013, HAdV-37 in 2014 and 2016, CB1 in 2014, further studies of serologically HAdV and enterovirus are necessary to control viral conjunctivitis in South Korea.

Keywords: Adenovirus, Enterovirus, South Korea, Viral conjunctivitis

17th International Congress of Virology 423

Virus Vaccines

17th International Congress of Virology 424

Abstract Submission

Virus vaccines

PO811

A NEW REPLICATION-DEFECTIVE, VACCINIA-DERIVED, CHO-MANUFACTURED, VACCINE VECTOR SYSTEM (SCV) CO-EXPRESSING CHIKUNGUNYA AND ZIKA VIRUS STRUCTURAL GENES IS EFFECTIVE IN PRECLINICAL STUDIES.

N. Prow 1,*, P. Eldi 2, T. Cooper 2, L. Liu 2, K. Diener 2 3, P. Howley 4, J. Hayball 2 3, A. Suhrbier 1

1Inflammation Biology, QIMR Berghofer Medical Research Institute, Brisbane, 2School of Pharmacy and Medical Sciences, University of South Australia, 3Robinson Research Institute and Adelaide Medical School, University of Adelaide, Adelaide, 4Sementis Ltd, Melbourne, Australia

Objectives: Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging mosquito-borne viruses that have recently caused large outbreaks of human disease globally. Both viruses are transmitted by Aedes aegypti and Aedes albopictus. Currently there are no licenced vaccines for use in humans for either virus. Herein we seek to design and test in preclinical models a replication-defective vaccinia-derived vaccine vector system (SCV) that co-expresses the structural proteins of both CHIKV and ZIKA viruses.

Methods: A novel vaccinia-derived, replication-defective, vaccine vector platform termed Sementis Copenhagen Vector (SCV) has been developed. SCV vaccines are incapable of generating viral progeny in vivo due to the targeted deletion of D13L, an essential viral assembly gene. SCV is manufactured in CHO cells expressing D13L and the host-range factor, CP77, providing an industry standard production process. A SCV-CHIK/ZIKV vaccine co-expressing the structural protein cassettes of both CHIKV and ZIKV was generated and tested in established mouse models.

Results: A single vaccination with the SCV-CHIK/ZIKV vaccine was able to induce antibody responses in both wild-type and interferon receptor knockout mice within 2 weeks post-vaccination. Challenge studies in mouse models will be undertaken to assess vaccine-mediated protection against viral infections.

Conclusion: The SCV platform has the capacity for multiple large recombinant inserts and the ability for production using industry standard manufacturing processes. The SCV-CHIK/ZIKV vaccine provides an attractive vaccine candidate to provide protection against both CHIKV and ZIKV; arboviruses that often co-circulate and transmitted by the same mosquito species.

Keywords: Chikungunya virus, vaccinia virus, zika virus

17th International Congress of Virology 425

Abstract Submission

Virus vaccines

PO812

ACTIVE AND PASSIVE IMMUNIZATION WITH MODIFIED NS1 WING DOMAIN PROVIDE IMMUNE PROTECTION AGAINST DENGUE VIRUS INFECTION

Y.-C. Lai 1,*, T.-M. Yeh 2

1The Institute of Basic Medical Sciences, 2Departments of Medical Laboratory Sciences and Biotechnology Medical College, National Cheng Kung University, Tainan, Taiwan

Objectives: Dengue virus (DENV) infection used to be in tropical and subtropical regions only but is now a global issue. Although a tetravalent dengue vaccine has been licensed, the efficacy against all four serotypes of DENV and the risk of antibody dependent enhancement induced by vaccination made the implementation of this vaccine in general population still arguable. On the other hand, immunization with nonstructural protein-1 (NS1) has been known to protect mice against dengue infection. However, the molecular mimicry between NS1 and host proteins makes it have the potential risk to induce autoreactive antibodies if whole NS1 is used as vaccine. To reduce this risk, it is necessary to identify the epitopes recognized by NS1 antibodies which can protect from DENV infection and do not cross-react with host proteins.

Methods: Based on the phage display epitope recognized by an anti-NS1 mAb 33D2 which recognized a conserved NS1 wing domain (NS1-WD) region but not host proteins, we synthesized a modified NS1-WD peptide to immunize mice and evaluate the protective effects of mice against four serotypes of DENVs challenge after active or passive immunization against this modified NS1-WD peptide both in vitro and in vivo.

Results: We found that both modified NS1-WD immune sera and mAb 33D2 can neutralize dengue infection and NS1- induced endothelial permeability in vitro. In addition, both active immunization with modified NS1-WD peptide and passive transfer of mAb 33D2 into mice can alleviate DENV-induced disease development and inhibit NS1-induced endothelial permeability in vivo.

Conclusion: Our results demonstrated that modified NS1-WD peptide can induce antibodies against DENV-induced infection and disease, suggesting that this modified NS1-WD peptide is a promising dengue vaccine candidate.

Keywords: dengue, Nonstructural protein 1, Vaccine

17th International Congress of Virology 426

Abstract Submission

Virus vaccines

PO813

Broad immune response elicited by immunization with multi-subtype virus-like particles (VLPs) co-localizing H5, H7, H9 and H10 hemagglutinins.

I. Tretyakova 1,*, R. Hidajat 1, X. Sun 2, J. Belser 2, T. Tumpey 2, P. Pushko 1

1Research and Development, Medigen, Inc., Frederick, MD, 2Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, United States

Objectives: Avian influenza (AI) viruses caused deadly pandemics in the past and currently represent growing threat to public health. Severe human infections with high fatality rates were reported for H5, H7, H9 and H10 subtypes of AI. Currently there is no approved vaccine for H7, H9 or H10 influenza. It’s impossible to predict which AI virus and HA subtype may mutate into highly transmissible variant and potentially start a new pandemic. As a preparedness measure universal or broadly protective vaccines are being developed against potential pandemic AI strains. Here we report on our experimental broad VLP vaccine for H5, H7, H9 and H10 subtypes of AI. We hypothesized that virus-like particles (VLP) containing four different HA antigens can elicit immune response to all four subtypes. We also sought to test cross-protective potential of the immune sera against heterologous influenza antigens.

Methods: Quadri-subtype influenza VLPs were expressed using recombinant baculovirus (rBV) expression system. Four HA genes (H5, H7, H9, and H10), retroviral gag and neuraminidase (NA) expressing cassettes were cloned together in tandem fashion and moved into the baculovirus genome. As a result, all 6 VLP relevant genes were expressed in the same insect cell infected with the rBV, and their proteins assembled into multi-subtype H5.H7.H9.H10 VLP.VLPs were purified from the growth medium of infected Sf9 cells, concentrated and formulated in PBS for characterization, analysis and immunogenicity and challenge experiments in ferrets.

Results: Quadri-subtype VLP vaccine elicited serum antibodies to the homologous H5, H7, H9 and H10 antigens and cross- reacted with multiple clades of H5N1 virus and with the heterologous H10 virus. After heterologous challenge with the H10N1 virus, vaccinated ferrets showed significantly reduced titers of replicating H10N1 virus in the respiratory tract indicating protective effect of vaccination with VLPs.

Conclusion: We conclude that multi-subtype VLP approach can be a feasible approach to prepare broadly protective AI vaccines.

Keywords: immune response, influenza vaccine, virus like particles

17th International Congress of Virology 427

Abstract Submission

Virus vaccines

PO814

Changes in the growth properties of a rubella RA27/3 vaccine virus after long-term persistence in the cutaneous granuloma of an immune deficient patient

L. Perelygina*, A. Adebayo 1, M. Dorsey 2, S. Plotkin, K. Sullivan 3, J. Icenogle 1

1Centers for Disease Control and Prevention, Atlanta, 2University of California, San Francisco, 3University of Pennsylvania, Philadelphia, United States

Objectives: Rubella (RuV) RNA and proteins were found in an association with cutaneous granulomas in children with primary immune deficiencies (PID). Immunohistochemistry and sequence analysis suggested altered tropism of persisting RuV vaccine viruses. The objective of this study was to determine whether infectious RuV can be found in granulomas and to characterize virus growth properties of the virus in different cell types.

Methods: Infectious RuV was isolated from a skin biopsy specimen from a case of PID by co-culture of tissue homogenates with Vero cells followed by two sequential passages of infected cells. RuV RNA was amplified by RT-PCR and the sequences of 5 overlapping genomic fragments was determined by the Sanger method. To compare the growth properties of the patient virus to the RA27/3 vaccine virus, different cell lines (epithelial, endothelial, fibroblasts) were infected and virus yield was determined by end point titration on Vero cells. The percentage of infected cells was determined by immunofluorescent detection of the E1 protein.

Results: Sequencing of RuV RNA directly from the clinical specimen revealed presence of a heterogeneous viral population with 99% nt identity between the consensus sequence of the clinical samples and the RA27/3 sequence (24 aa substitutions). The isolated p2 virus from the PID case was homogeneous in sequence and contained a 2-aa deletion in p150 protein and 36 aa substitutions located predominantly in the structural proteins. Compared to the RA27/3 vaccine virus, the isolate from the PID case replicated substantially less efficiently in all cell types producing titers that were 2-3 logs lower than the titer of RA27/3. The efficiency of virus entry and lateral spread in all cell types was also reduced in the isolate from the PID case, and these differences were more pronounced in endothelial cells.

Conclusion: The RA27/3 vaccine virus can persist in granulomas of PID patients producing genetically drifted infectious virus with altered growth properties and tissue tropism.

Keywords: persistent infection, rubella vaccine, vaccine safety

17th International Congress of Virology 428

Abstract Submission

Virus vaccines

PO815

DESIGN AND EFFICACY EVALUATION OF A LIPID CORE PEPTIDE-BASED VACCINE AGAINST RSV

N. Jaberolansar 1,*, K. Chappell 1, D. Watterson 1, M. Skwarczynski 1, P. Young 1 2 3, I. Toth 1 2 3 4

1School of Chemistry and Molecular Biosciences, 2Institute for Molecular Bioscience, 3Australian Infectious Diseases Research Centre, 4School of Pharmacy, The University of Queensland, Brisbane, Australia

Objectives: Respiratory syncytial virus (RSV) is a significant cause of the lower respiratory tract infections in infants, young children, and the elderly. The high mortality and morbidity associated with RSV diseases emphasizes the urgent need for an effective RSV vaccine. Traditional vaccine development such as pathogen inactivation was complicated by an induced immunopathology. In addition to live attenuated approaches new vaccine designs have focused on eliciting protective neutralizing antibody responses to specific viral epitopes. The antigenic site Ø at the top of the pre-fusion RSV F glycoprotein was chosen for this study for peptide-based vaccine design.

Methods: This study focuses on the synthesis and analysis of lipid core peptide (LCP) vaccine constructs. The self- adjuvanting LCP delivery system consisted of two copies of lipoamino acid (C16) as a self-adjuvanting moiety, antigenic site Ø (B cell epitope) and Pan DR (PADRE) as a T helper epitope. The coil-promoting sequences from the yeast GCN4 protein were added to the native protein to induce native helical conformation of the incorporated epitope. Peptide and LCP constructs were synthesized using Fmoc solid phase peptide synthesis (SPPS) and characterized by electrospray ionization mass spectrometry (ESI-MS) and analytical high-performance liquid chromatography (HPLC). C57BL/6 mice were immunized with vaccine constructs and sera assessed for antibody production and neutralizing potential.

Results: The LCP construct with modified epitope was able to fold into the desired helical conformation as assessed by circular dichroism and was recognized by pre-fusion specific antibodies (D25) recognizing site Ø on RSV F protein. However immunization of mice with the synthetic vaccine, while inducing high levels of peptide-reactive antibodies failed to elicit neutralizing antibodies.

Conclusion: The results illustrated that this B cell epitope-based peptide vaccine may not sufficiently mimic the same peptide sequence as expressed in the complete RSV pre-fusion F protein. Further optimization of the structural presentation of this immunogenic epitope is required.

Keywords: Peptide vaccine, Respiratory syncytial virus

17th International Congress of Virology 429

Abstract Submission

Virus vaccines

PO816

Development of Next-Generation Enterovirus 71 Vaccines

M.-S. Lee*

Objectives: Enterovirus 71 (EV71) and polioviruses both belong to enterovirus genus. Based on phylogenetic analysis of the most variable VP1 genes, EV71 could be classified into three major genogroups (A, B and C) including 11 genotypes (A, B1-B5, and C1-C5). Since 1997, EV71 has caused life-threatening infections with severe neurologic complications in Asian countries, including Malaysia, Taiwan, Singapore, Brunei, Vietnam, Cambodia and China. Therefore, development of EV71 vaccines is a national priority in these countries.

Methods: Currently, five vaccine candidates have been evaluated in clinical trials. Here are the limitations of these 5 candidates. First, genotypes of these 5 candidates include B2 (Singapore), B4 (Taiwan), and three C4 (China) strains, which are different from the predominant genotype B5 in Taiwan and South-Eastern Asia. Moreover, these vaccine viruses could not grow efficiency in cell cultures (~107 PFU/ml). Second, similar to inactivated poliovirus vaccines (IPV), EV71 forms full (high immunogenicity) and empty (low immunogenicity) virus particles in cell cultures (they are called D and C antigens, respectively in IPV research). Therefore, it is critical to develop assay for quantifying D antigens, which are not well-defined in the 5 vaccine candidates. Third, the current 5 vaccine candidates did not have good potency assay to monitor efficiency of different purification procedures. Fourth, these 5 candidates employ intramuscular injection and it would be desirable to develop intradermal delivery with antigen sparing to improve vaccine supply.

Results: First, we have adapted a genotype B5 virus isolated locally in 2008 to generate high-growth viruses (~108 PFU/ml) in Vero cells. Moreover, the high-growth B5 virus could induce cross-reactive neutralizing antibody titers against multiple EV71 genogroups. Second, we have employed ultracentrifuge to separate EV71 full and empty virus particle and generate D antigen-specific mouse monoclonal antibody. The mouse monoclonal antibody has been used to develop assay for quantifying D antigens. Third, we has employed the D antigen assay to compare efficiency of different purification platforms. Fourth, recent studies on influenza vaccines and IPV have proved the antigen sparing effect of intradermal delivery. Therefore, we are comparing intramuscular and intradermal routes for delivering EV71 vaccines in animal studies.

Conclusion: In conclusion, we have developed next-generation enterovirus 71 vaccine candidates with multiple improvements. We are looking for industry partners to initiate clinical trials.

Keywords: Vaccine validity, enterovirus 71, animal study

17th International Congress of Virology 430

Abstract Submission

Virus vaccines

PO817

Evaluating Cross-protection against Zika virus, Japanese encephalitis virus, Tickborne encephalitis virus, West Nile virus and Yellow fever virus after Dengue Vaccination

J. Chen*, M. L. H. Oh 1

1Infectious Diseases, Changi General Hospital, Singapore, Singapore

Objectives: In this study, we evaluate if the antibodies elicited by CYD-TDV may cross-react and potentially provide protection against other flaviviruses such as Zika virus, Japanese encephalitis virus, tickborne encephalitis virus, West Nile virus and yellow fever virus.

Methods: Sera of 64 CYD-TDV vaccinated volunteers and 23 control volunteers were evaluated using EUROIMMUN (Germany) indirect immunofluoresence test kit for the detection of IgG antibodies against Zika virus, Japanese encephalitis virus, tickborne encephalitis virus, West Nile virus and yellow fever virus.

Results: Interestingly, among the CYD-TDV vaccinated volunteers, 12.5% of them were found to have absence of IgG antibodies against all the flaviviruses tested, including dengue viruses in their sera while 81.3% were found to have IgG antibodies against all serotypes of dengue viruses. Among the control volunteers, 21.7% were found to have IgG antibodies against one or more of the flaviviruses, most probably due to prior exposure. Among the CYD-TDV-vaccinated volunteers who have antibodies against all dengue viruses, 78.1% of CYD-TDV vaccinated were detected to have antibodies against one or more of the flaviviruses tested and 43.8% of them have cross-reactive antibodies against all the flaviviruses tested.

Conclusion: The presence of cross-reactive antibodies against Zika virus, Japanese encephalitis virus, tickborne encephalitis virus, West Nile virus and yellow fever virus in the sera of CYD-TDV vaccinated volunteers could potentially offer cross-protection and will be evaluated in the future using plaque reduction neutralization test.

Keywords: Dengue, Flavivirus, vaccine

17th International Congress of Virology 431

Abstract Submission

Virus vaccines

PO818

FMDV Multi-VP1e emulsified with ISA 201 induces protection against lethal FMDV challenge

J.-S. Lee*

Objectives: In this study, we evaluated a novel recombinant protein-based vaccine for foot-and-mouth disease viruses (FMDV).

Methods: To construct multi-epitope-based protein for FMDV vaccine, we selected epitope sites containing G-H loop and C-terminus known as major immunogenic sites in VP1 region from three serotypes FMDVs, O1/Manisa/Turkey/69, A/Pocheon/001/KOR/2010 and Asia1/Shamir /89, which had ever occurred or threatened in Korea and we conjugated each epitope sites. Through the E.coli expression system, the multi-VP1e protein was expressed in soluble form and purified by FPLC and IMAC system. By emulsifying with Montanide ISA 201, adjuvant known as efficacious in FMDV protection, we finally prepared recombinant FMDV vaccine candidate and used to immunize animals.

Results: Multi-VP1e protein was expressed in soluble form and purified by FPLC and IMAC system. Mice were inoculated with multi-VP1e protein emulsified with ISA 201 every two weeks by I.M injection and we demonstrated that multi-VP1e protein vaccine can induce humoral and cellular immunity and completely protect from lethal infection of FMDV Asia1/Shamir/89. Additionally, multi-VP1e protein vaccine induced the production of sufficient neutralizing antibodies against each three serotypes of FMDV in pig and effectively protected pigs from FMDV Asia1/Shamir/89 challenge.

Conclusion: Multi-VP1e protein vaccine which derived from E. coli expression system may be a safe and effective vaccine candidate against diverse FMDV. [This study was supported in part by the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Grant no. 315044031)]

Keywords: FMDV, Multi-VP1e, Vaccine validity

17th International Congress of Virology 432

Abstract Submission

Virus vaccines

PO819

Humoral Immune Responses to Influenza A(H1N1)pdm2009 Vaccination in Schoolchildren after Annually Repeated Vaccination

W. Liu 1,*, Y.-H. Lien 1, C.-C. Ku 2, C.-C. King 1

1Institute of Epidemiology and Preventive Medicine, 2Institute of Immunology, National Taiwan University, Taipei, Taiwan

Objectives: The 2009 pandemic H1N1 virus [H1N1pdm09] spread globally since 2009, resulting in cross-country pandemic. World Health Organization had recommended the H1N1pdm09 as one of the three components involved in seasonal influenza vaccine since 2009. Taiwan's government launched mass-vaccination of this novel virus on grade 1-6 elementary schoolchildren in 2009 and then implemented annual vaccination of trivalent inactivated flu vaccine until now. The aim of this study was to investigate how the repeated vaccination of H1N1pdm09 influenced the antibody (Ab) responses before and after vaccination with 2011-12 seasonal flu vaccine among schoolchildren.

Methods: A seroepidemiological cohort study with 935 grade 1-6 schoolchildren from the two elementary schools in Taipei was conducted from Nov. of 2011 to Mar. of 2012. Blood samples were collected at the three time points: pre- vaccination (T1, Nov. 2011), 1-month post-vaccination (T2, Dec. 2011), and ending of the flu season (e.g. 4-month post- vaccination, T3, Mar. 2012). The hemagglutination inhibition (HI) assay was used to measure the participants’ levels of HI Ab against the vaccine antigen prepared from formalin inactivation of A/California/07/2009 (H1N1pdm09) by the Adimmune Corp., Taiwan. Seroprotective Ab was defined as HI titer at least 1:40, and seroprotection rate was the percentage of subjects with a seroprotective Ab. SAS 9.3 was used to conduct student t-tests, chi-square, ANOVA and logistic regression for both univariate and multivariate analyses. Vaccination history used documented records in 2009-2010 and 2010-2011 flu seasons.

Results: Schoolchildren who had received only the 2010 vaccine showed the highest level of geometric mean titer (GMT) of HI Ab and seroprotection rate (SR) at T1 than those had vaccinated only in 2009, in both 2009 and 2010, and those unvaccinated in 2009/2010 [GMT(95%CI): 71.6(41.7-123), 42.0(25.3-69.6), 34.8(28.5-42.6), 28.9(13.1-63.6), p=0.0509; SR%: 71.9, 66.7, 62.5, 29.0%, p=0.627]. However, the GMT at T2 (1-month post-vaccination in 2011-12) was significantly the highest in those without any previous vaccination of H1N1pdm09, followed by those vaccinated only once either in 2009 or in 2010, and those vaccinated in the past two years respectively [GMT(95%CI): 557.2(252.8-127.9), 394.0(258.1-601.3), 434.1(340.6-555.3), 282.5(242.5-329.0), p=0.0025]. After stratifying by HI serotiters at T1, this pattern was more obvious among those with maintaining seroprotective Ab at T1 [GMT=940.3(514.5-1718.6), 419.1 (229.3-765.9), 488.1(360.0-661.7), 366.6(316.5-424.6), P<0.0001], but was inconsistent with those without seroprotective Ab [GMT=254.1(49.6-1300.6), 320.0(203.5-503.2), 320.0(216.2-473.8), 188.1(141.5-249.9), P>0.05].

Conclusion: In conclusion, annually repeated flu vaccination in both 2009 and 2010 neither indicated higher seroprotection rate of anti-H1N1pdm09 Ab in schoolchildren at T1, nor induced stronger Ab responses at T2. Additionally, among schoolchildren without vaccination history, ever infection by H1N1pdm09 (defined as anti-H1N1pdm09 serotiters ≧1:40 at T1) may benefit the Ab responses at the first-time vaccination of A(H1N1)pdm09 in 2011-12 flu season. Further investigation in both T-cell immunity and B-cell memory is needed to understand the different flu vaccine immunogenicity affected by natural infection or repeated vaccination of the split flu vaccine.

Keywords: Humoral Immunity, Antibody response, 2009 Pandemic Influenza A(H1N1) Virus, Schoolchildren, Annual Mass Vaccination, Vaccination Histories, Seroepidemiology, Taiwan

17th International Congress of Virology 433

Abstract Submission

Virus vaccines

PO820

Interaction of intracellular specific IgA (icIgA) with uncleaved influenza virus haemagglutinin (HA0) in patient respiratory exudate cells can inform vaccine design.

T.-W. Kok 1,*, M. Costabile 2, G. Tannock 3, P. Li

1School of Medicine, University of Adelaide, 2School of Pharmacy & Medical Sciences, University of South Australia, Adelaide, 3Burnet Institute Medical Research, Melbourne, Australia

Objectives: Co-localisation of influenza virus HA0 with intracellular IgA (icIgA) in patient respiratory exudate cells.

Inhibition of viral replication by icIgA antibodies has been observed in in vitro studies using epithelial cell lines. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across the polarized cell. In an experimental model of murine influenza, secretory IgA has been shown to be the major mediator of nasal immunity in mice that recovered from influenza infection. Immunity was abrogated by nasal instillation of anti-IgA, but not anti-IgG or anti-IgM. Only polymeric specific IgA prevented upper respiratory tract infection and pathology.

Methods: Mouse antisera against Influenza A H3N2 haemagglutinin (HA0) cleavage loop peptides were used in confocal fluorescence microscopy to show specific staining of wild type virus (H1N1 and H3N2) in clinical specimens and MDCK cell line. The HA0 cleavage loop peptides used for intranasal immunizations in mice were derived from influenza A/H3N2 virus strains. Anti-human IgA (secretory) specific antibodies were used to show colocalization of virus and icIgA.

Results: The results showed specific immunofluorescence staining of Flu A/X31(HA0 uncleaved)-infected MDCK cells as well as icIgA in patient respiratory exudate cells. Both results confirm specific co-localization and suggest interaction between influenza A virus and icIgA in patient respiratory exudate cells. Importantly, the mouse anti-HA0 cleavage site sera stained wild type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. These are the first observations of co-localized influenza A virus and intracellular IgA in clinical specimens.

Conclusion:

Role of icIgA: This report shows the co-localisation of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture positive. These observations suggest that icIgA directed against the conserved HA0 site has a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent “second round” viral replication and spread to other cells. The precise mechanism by which IgA mediates intracellular viral neutralisation remains to be fully elucidated. In conjunction with the haemagglutination inhibition assay, we believe that a rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co- localized Flu virus and icIgA – would contribute strongly to further mucosal immunity studies. Unfortunately, for ethical reasons, virus specific ELISPOT assays that quantify specific IgA responses are unavailable for human subjects.

Significance: This finding will provide more detailed understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps with a specific focus on mucosal viral infections. This will inform the design of more effective vaccines against influenza, respiratory syncytial virus, rotavirus and other viral infections transmitted via the mucosal route.

Keywords: Intracellular IgA Colocalisation Influenza Haemagglutinin Uncleaved

17th International Congress of Virology 434

Abstract Submission

Virus vaccines

PO821

NON-SPREADING LASSA REPLICON PARTICLES AS A VACCINE CANDIDATE

M. Kainulainen 1,*, J. Spengler 1, S. Nichol 1, C. Albarino 1, C. Spiropoulou 1

1Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, United States

Objectives: Lassa is a viral hemorrhagic fever endemic to parts of West-Africa. With annual case numbers estimated at hundreds of thousands - including thousands of deaths - Lassa is a major public health burden in the affected areas. The causative agent, Lassa virus of the family Arenaviridae, is spread primarily by excreta of the "multimammate rat" (Mastomys natalensis), although person-to-person transmission can also occur. Ribavirin has been used to treat cases of Lassa fever. However, there are no approved vaccines to prevent infections. The objective of this project is to develop a method for producing non-spreading Lassa replicon particles and to evaluate them as a vaccine candidate.

Methods: Our laboratory has previously established a reverse genetics system for Lassa virus. We have now modified this system by replacing the glycoprotein precursor (GPC) gene in the viral S genomic segment with a reporter gene, and expressing GPC from a helper plasmid instead. The system produces ‘viral replicon particles’ (VRPs) that can enter susceptible cells and replicate, but fail to spread due to lack of newly synthesized glycoprotein GPC. For efficient production of the VRPs, we established a Vero E6-based cell line that expresses Lassa GPC from a codon-optimized open reading frame. The vaccine potential of VRPs will be evaluated in guinea pig model of Lassa infection.

Results: The VRPs are rescued reproducibly using GPC helper plasmid. The stable cell line displays glycoprotein processing closely resembling that seen in cells infected with the actual virus. These cells enable a 1000-fold amplification of rescued VRPs in a single passage as well as practical means to scale up their production. To improve detection of our vaccine candidate’s replication by host cell innate immunity sensors, we have studied relative contributions of Lassa NP and Z in inhibiting these responses.

Conclusion: We have established a system for efficient generation of Lassa replicon particles. The particles have been characterized in cell culture systems and will be evaluated as a vaccine candidate in Lassa infection model.

Keywords: Innate immunity, Lassa virus, Viral replicon particle

17th International Congress of Virology 435

Abstract Submission

Virus vaccines

PO822

PRECLINICAL DEVELOPMENT OF AN EBOLA VIRUS VACCINE BASED ON RECOMBINANT SUBUNITS EXPRESSED IN INSECT CELLS

A. Lehrer 1,*, M. Lieberman 1, T. A. Wong 1, T. Geisbert 2 1Department of Tropical Medicine, University of Hawaii, Honolulu, 2Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, United States

Objectives: Ebola Virus Disease (EVD) is the most prominent example of filovirus disease, however, due to the absence of a commercial market it lacked public and private research resources. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines. The 2013-2016 Zaire Ebolavirus (EBOV) outbreak in several West African countries with more than 28,000 reported cases revealed the true epidemic potential of filovirus infections when entering an urban setting in a highly mobile society. As typical in an epidemic with infectious patients traveling within and from the endemic area, the disease was also exported outside the outbreak region as has been shown with introductions into Nigeria, Mali, and the United States (amongst other countries averting in-country transmission from imported cases). This demonstrated the threat posed to the global public health systems if spread of Ebola or a related filovirus cannot be contained at its source.

Despite significant progress with the clinical development of several EBOV vaccine candidates during and after the West African outbreak, no EBOV specific therapeutics or vaccines have yet received regulatory approval. Our objectives are to develop a safe and effective subunit EBOV vaccine for human use and to define the mechanism of protection and identify immune correlates of protection for Ebola and other filovirus vaccines.

Methods: We have produced soluble recombinant filovirus glycoproteins (GP) and matrix proteins (VP24 and VP40) from EBOV using the Drosophila S2 cell expression system. The immunogenicity and protective efficacy of highly purified recombinant subunits and admixtures formulated with or without clinically relevant adjuvants were evaluated in mice, guinea pigs and macaques.

Results: Strong antigen-specific IgG titers as well as virus neutralizing titers were observed after administering two or three doses of adjuvanted formulations. In mice and non-human primates subunit proteins were also shown to elicit cell mediated immune responses. Analysis of secreted cytokines in batch-cultured, antigen-stimulated splenocytes or PBMC’s demonstrated antigen-induced Th1 and Th2 type responses.

Recombinant vaccine candidates were tested successfully for protection in the mouse model of EBOV. Further studies allowed us to demonstrate that both humoral and cell-mediated immunity are elicited and can mediate protection. Additional immunogenicity and efficacy studies in guinea pigs were focused on optimized antigen dosing, antigenic balance and adjuvantation. Multiple formulations consistently produced strong antibody responses and demonstrated 100% protective efficacy in the EBOV guinea pig model.

Results from studies in two species of non-human primates demonstrate that vaccination with formulations of recombinant EBOV subunits and an emulsion-based adjuvant consistently produces high anti-EBOV IgG and virus neutralizing titers. This prevents viremia subsequent to live virus challenge and protects animals from terminal EBOV disease.

Conclusion: These studies suggest that we have defined a viable Ebola virus vaccine candidate based on non-replicating viral subunits. Current efforts in our laboratory are focused on defining correlates of protection to allow clinical development of a monovalent vaccine candidate for protection against EVD and further formulation optimization towards a trivalent, recombinant subunit vaccine with protective efficacy against EBOV, Sudan ebolavirus (SUDV) and Marburgvirus (MARV) infection. 17th International Congress of Virology 436

Keywords: Ebola virus, filovirus, vaccine

17th International Congress of Virology 437

Abstract Submission

Virus vaccines

PO823

PROCESS DEVELOPMENT OF NEW-GENERATION ENTEROVIRUS 71 VACCINES USING HIGH-GROWTH VACCINE VIRUS

H. Yen*, M.-S. Lee, M.-Y. Chia, A. Y.-C. Hu

Objectives: Since 1997, different EV71 genotypes have caused life-threatening epidemics in Asian countries, including Malaysia, Taiwan, Singapore, Brunei, Vietnam, Cambodia and China. Therefore, development of EV71 vaccines is a national priority in these countries. Currently, five vaccine candidates have been evaluated in clinical trials. However, these five vaccine candidates have the following limitations. In this study, we have adapted high-growth genotype B5 vaccine 8 virus, which could grow to ~10 TCID50/ml in microcarrier-based and BelloCell-based Vero cell culture systems and induce cross-reactive neutralizing antibody responses against the three EV71 major genogroups in rabbits. In addition, an enzyme- linked immunosorbent assay (ELISA) quantified protective vaccine antigens was developed.

Methods: In this study, a microcarrier- system and BelloCell- based systems were used to generate high-growth B5 virus. In the details of the microcarrier system, the virus can be produced by spinner flask and reached to 108 TCID50 titers. Furthermore, the BelloCell system was generated 107-108TCID50 titers. Three purified vaccine antigen groups obtained from this study and one preclinical lot (genotype B4) of Medigen EV71 vaccine candidate were prepared for rabbit immunization. Immune responses in Rabbits were determinated by neutralization antibody production assay. In our study, the neutralizing monoclonal antibody, Mab2016-1, and polyclonal rabbit serum with high titer were purified by protein A affinity chromatography and developed by antigen capture ELISA as quantitative assay for evaluating antigen purification processes. The results of Ac-ELISA were compared with our animal immune responses in each group.

Results: Our data showed that inactivated B5 antigens purified by sucrose continuous gradient separated full and empty particles. Higher immune responses in rabbits were induced by sucrose gradient process than that of iquid chromatography. Besides, the monoclonal antibody, Mab2016-1, provided high neutralization responses in rabbits for EV71. Our data indicated that animal experiment results were consist with the value of Ac-ELISA assay.

Conclusion: In conclusion, the new-generation EV71 genotype B5 vaccine candidate is a warrant for further development.

Keywords: Enterovirus 71, High-growth vaccine virus, Process development

17th International Congress of Virology 438

Abstract Submission

Virus vaccines

PO824

Protective immunity to measles and dengue are induced by a recombinant measles vector expressing dengue envelope protein domain III

C.-H. Pan*

Objectives: Dengue has a major impact on global public health, and the current licenced vaccine is restricted to individuals aged above 9 and shown a limitation to dengue-2 infection. There is still a need of a new and better dengue vaccine for children under 9. Current measles vaccine is safe and eficient vaccine for 1-years-old infents, and also has potential as a vector for antigen delivery.

Methods: In this study, we developed a recombinant measles virus (MV) to express envelope protein domain III (ED3) of dengue-1 to 4. We also evaluated the immunogenicity and protective efficacy of a dengue vaccinein a mouse model.

Results: The expression of dengue ED3 was detected in recombinant MVs infected Vero cells and no significant change in replication was observed between recombinant MVs and the parental MV. The specific interferon-gamma and antibody responses against dengue virus and MV are successfully induced. Neutralizing antibodies against MV and dengue viruses were also detected in the MV-vectored dengue vaccine immunized mice. After challenge with dengue-2, a detectable but significant lower viremia and higher titer of anti-dengue-2 neutralizing antibodies were observed in MV-vectored dengue vaccine immunized mice versus the vector control, suggesting that a protective antibody response against dengue-2 was elicited.

Conclusion: In conclusion, our studies have demonstrated the potential of the MV-vectored dengue vaccine to efficiently induce both MV- and dengue-specific T-cell and antibody responses even in individuals who received the MV vaccination or those pre-infected with dengue viruses. Despite the lower replicative efficiency of MV in mice, the immunity elicited by the MV-vectored dengue vaccine still provides partial protection against DENV-2 infection. This study contributes to the understanding of the MV-vectored dengue vaccine and provides clues for the future application of a 2-in-1 MV and dengue vaccine.

Keywords: dengue vaccine, Dengue virus, measles vector

17th International Congress of Virology 439

Abstract Submission

Virus vaccines

PO825

Purification and characterization of pre-pandemic recombinant virus-like particle (VLP) vaccine against avian- origin H5, H7, H9 and H10 influenza

R. Hidajat*, I. Tretyakova 1, M. Exum 1, A. Tibbens 1, T. Tumpey 2, P. Pushko 1

1Research and Development, Medigen, Inc., Frederick, 2Influenza Division, Centers for Disease Control and Prevention, Atlanta, United States

Objectives: Highly pathogenic avian influenza (HPAI) poses the greatest potential to cause a serious pandemic. Avian- origin influenza subtypes including HPAI have been identified in severe human cases with high fatality rate. Experimental vaccines have been shown to be effective in preventing severe influenza. However, preparation of the vaccines using traditional technologies can take time before they become available in the clinic. In addition, working with wild-type viruses is hampered by biosafety and environmental concerns. Here we developed fast and safe recombinant VLPs with four avian- origin subtypes of hemagglutinin (HA) expressed on the surface. The all four avian-origin subtypes H5, H7, H9 and H10 are known to be able to infect humans. This quadri-valent VLP vaccine candidate is designed to elicit immune response and protect the vaccine recipients from the corresponding influenza strain.

Methods: Recombinant baculovirus (rBV) expressing avian-origin HA subtypes H5, H, H9 and H10 as well as neuraminidase (NA), and bovine immunodeficiency virus gag (Bgag) proteins was constructed using baculovirus expression vector system (BEVS). The rBV was used to infect Sf9 insect cells and the infected cells released multi-subtype H5.H7.H9.H10 influenza VLPs into the cell supernatant. VLP concentration and purification including removal of BV and host DNA were done by using tangential flow filtration (TFF), ion exchange (IEX) column chromatography and followed by ultracentrifugation. The VLPs were assayed for hemagglutination and neuraminidase activities, and reaction to antibodies against H5, H7, H9 and H10 subtypes. The VLPs were also evaluated for the quantities of each HA subtype and for the particle morphology using electron microscopy. The VLPs were then used to vaccinate ferrets. The immunized animals were evaluated for elicited specific serum antibodies, followed by challenge with heterologous virus.

Results: rBV infected Sf9 cells released multi-subtype H5.H7.H9.H10 influenza VLPs into the cell supernatant. During chromatography, VLPs migrated in the flow-through fraction, while majority of the rBV vector particles and host cell DNA were bound to the resin. The purified quadri-valent VLPs showed hemagglutination and neuraminidase activities; and reacted with specific antisera in immunoblotting assay. Electron microscopy showed that these Bgag-based VLPs resembled influenza virions with the diameter of 150-200 nm. Upon immunization with the VLPs, all vaccinated ferrets elicited specific antibodies against H5, H7, H9 and H10. After heterologous challenge with the H10N1 virus, all vaccinated ferrets showed significant reduction titers of replicating virus in the respiratory tract indicating protective effect of vaccination.

Conclusion: We developed the method for construction, production, and purification of quadri-subtype VLPs aiming for multi-valent influenza vaccine candidate. This VLP vaccine candidate is showed to maintain properties necessary for an effective pre-pandemic influenza vaccine. Animal studies confirmed that this vaccine candidate effectively protected the animals against pathogenic challenge, and therefore can be considered in emergency vaccination scenarios involving avian- origin influenza.

Keywords: influenza vaccine, protein purification, virus like particle

17th International Congress of Virology 440

Abstract Submission

Virus vaccines

PO826

Synthetic nucleic acid antibody prophylaxis confers protective immunity in vivo against Chikungunya virus

K. Muthumani 1,*, S. Flingai 1, N. Muruganantham 2, G. Sarangan 3, P. Vijayachari 2, A. S. Khan 4, N. Y. Sardesai 5, J. J. Kim 4, D. B. Weiner 1

1Vaccine Center, The Wistar Institute, Philadelphia, United States, 2Regional Medical Research Centers, Indian Council of Medical Research, Port Blair, 3Department of Microbiology, Sri Ramachandra Medical College & Research Institute, Chennai, India, 4R&D, Inovio Pharmaceutics Inc, Plymouth Meeting, 5R&D, Inovio Pharmaceutics Inc, Philadelphia, United States

Objectives: Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for epidemic outbreaks in Asia, Africa, and the Caribbean Islands. The virus has recently adapted to more prevalent mosquito vectors, which raises concerns for continued pathogenic spread. Currently, neither a vaccine nor targeted therapies are licenced for CHIKV infections. Furthermore, individuals at highest risk of developing severe infections, including elderly and immuno-compromised populations, often respond poorly to traditional vaccine approaches. Previously, it has been shown that monoclonal antibody therapy can effectively control CHIKV infection and disease.

Methods: A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti–CHIKV envelope monoclonal antibody (DMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy.

Results: One intramuscular injection of DMAb produced antibodies in vivo more rapidly than active vaccination with an anti- CHIKV DNA vaccine. This DMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of DMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection.

Conclusion: A DNA-based DMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short- and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

Keywords: Chikungunya virus, DNA Vaccine , Immune response

17th International Congress of Virology 441

Abstract Submission

Virus vaccines

PO827

THE IMMUNE REACTION TO INTRODUCTION OF A VACCINE WITH ADJUVANT

O. Lebedinskaya*, Y. Troynich 1, N. Axmatova 2, E. Axmatova 2

1Hystology, Acad. E.A. Wagner Perm State Medical University, Perm, 2I.I. Mechnikov Research Institute for Vaccines and Sera, Moscow, Russian Federation

Objectives: Adjuvant Chitosan is a natural nontoxic biopolymer produced by the deacetylation of chitin, a major component of the shells of crustaceans.

The aim of our research is to study the adjuvant properties of derivatives of Chitosan on the model of flu and evaluation of molecular-cellular mechanisms of their action on the effectors of innate and adaptive immunity.

Methods: There are 2 group of mouse – both male. 1st group get influenza vaccine Vaxigrip with isotonic NaCl hypodermically and 2nd one get vaccine with solution of chitosan also hypodermically. Sections of thymus and spleen were stained with hematoxylin and eosin, methyl green and Pronina on the brush on RNA, Schiff-iodic acid on Sabadash neutral glycosaminoglycans.

Statistical data processing was performed using morphometric method, additional methods of coloring, immunofermental method, statistic analisis.

Results: Thus during the investigations it was demonstrated that in mice vaccinated with influenza vaccine cortical substance of the thymus has been expanding, the boundary between it and the substance of the brain become fuzzy. The medulla shape has been irregular with a very big unit of Gassal cells and a large number of macrophages. Both in the cortex and in the medulla there have been areas of bare stroma. In the spleen of animals of this group of white pulp is lager that the red one. Follicles are increased in diameter and have merged. The red pulp is moderately hyperemic. No expansion of the cortical substance of the thymus has been noted in the hypodermically vaccined group. In the medulla there are clearly visible cells of the stroma, but places of "barestroma" have not been found.

Conclusion: In conclusion, the experiment shown that adjuvant Chitosan effectively raise immunogenicity and protective efficiency of influenza vaccine.

Keywords: adjuvant , Chitosan , influenza vaccine

17th International Congress of Virology 442

Late breaking abstract

Virus vaccines

PO863

Development of a Recombinant Zika Virus Vaccine based on Insect Cell expressed Subunits

T. A. Wong 1, A. To 1, L. Medina 1, M. Lieberman 1, M. Namekar 1, E. Nakano 1, M. Kumar 1, V. Nerurkar 1, A. Lehrer 1,*

1Department of Tropical Medicine, University of Hawaii, Honolulu, United States

Objectives: Zika virus (ZIKV) is a mosquito-borne single-stranded RNA virus belonging to the family flaviviridae. Although known for almost 70 years, ZIKV was suspected to cause only mild disease until recently when it garnered increased interest by the public health community due to a suspected association with an increase in cases of microcephaly in infants born to mothers infected during pregnancy. As of present time, over 60 countries and territories have experienced symptomatic cases of Zika from local mosquito transmission. As ZIKV plagues many parts of the world, the prevention of birth defects and possibly Guillain-Barré syndrome is eminent. Currently, there is no vaccine or specific therapeutic medical countermeasures available and while DNA, mRNA, measles-vectored and inactivated ZIKV vaccines are in Phase I clinical trials, recombinant non-replicating subunit vaccines may offer a safer and more effective platform.

Methods: Using the Drosophila S2expression system we produced the ectodomain of the ZIKV envelope protein (ZIKV-E). This protein was subsequently purified to ~90% purity using immunoaffinity chromatography. We subsequently tested mouse immunogenicity of formulations with and without adjuvants after one, two and three doses (given in 3-week intervals). This was followed by testing the efficacy of the best formulations in mouse and non-human primate viremia models.

Results: The recombinant subunit proved to be highly immunogenic in BALB/c, Swiss Webster and C57BL/6 mouse strains as shown by significant increases of antigen-specific antibody titers after one, two and three doses. While differences in vaccine responses were seen dependent on the genetic background of each mouse strain, the use of clinically relevant adjuvants lowered the required dose level and maximal IgG titers were achieved after one or two immunizations. Plaque reduction neutralization tests demonstrated that high virus neutralization titers were elicited. These are considered a good correlate of protection in the defense against flavivirus infections. Efficacy of our vaccine candidate formulations was demonstrated in BALB/c and cynomolgous macaque models where significant suppression of viremia was observed in vaccinated animals as compared to naïve controls.

Conlusion: These data demonstrate proof-of-concept and establish recombinant subunit immunogens as an effective vaccine candidate against ZIKV infection. Most importantly, as little as one dose of our tested formulations might induce at least partial immunity, allowing the rapid use and deployment in travelers, and, due to the excellent safety profile of recombinant subunits, potentially even in pregnant women living or traveling in areas with a high risk of ZIKV infection.

Keywords: recombinant subunit, Vaccine, Zika virus

17th International Congress of Virology 443

Viruses and Cancer

17th International Congress of Virology 444

Abstract Submission

Viruses and Cancer

PO864

A RECOMBINANT MEASLES VIRUS BLIND TO SLAM SHOWS ANTI-TUMOR ACTIVITY AGAINST TRIPLE NEGATIVE BREAST CANCER CELL LINES

T. Fujiyuki*, Y. Amagai 1, K. Shoji 1, A. Sugai 1, M. Awano 1, H. Sato 1, M. Yoneda 1, C. Kai 1

1Laboratory Animal Research Center, The Institute of Medical Science, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Objectives: One of the most refractory breast cancer types is triple negative breast cancer (TNBC), in which cells are resistant to both hormones and Herceptine treatments and, thus, often cause recurrence and metastasis. Effective treatments are needed to treat TNBC. We previously demonstrated that a recombinant measles virus, named rMV- SLAMblind, showed antitumor activity against breast cancer cells. Here we examined whether rMV-SLAMblind is effective for treating TNBC.

Methods: Expression level of nectin-4, a receptor for rMV-SLAMblind, was examined using 12 TNBC cell lines with flow cytometry. By inoculating the cells with rMV-SLAMblind expressing EGFP, the infectivity and cytotoxicity of the virus were examined. Anti-tumor effect of rMV-SLAMblind in vivo was examined by using mouse xenograft models of TNBC. For intravenous administration, we used rMV-SLAMblind expressing luciferase to monitor the virus replication by using In Vivo Imaging System.

Results: Nectin-4 was expressed on the surface of 75% of the analyzed TNBC cell lines. rMV-SLAMblind did infect the nectin-4-expressing TNBC cell lines, and significantly decreased the viability of half of these in vitro. Intratumoral injection of rMV-SLAMblind suppressed tumor growth in xenografts of TNBC cell lines. To assess its potential as a treatment for metastatic breast cancer, we performed intravenous administration of the virus to a xenograft, and observed the virus replication in the tumor and significant suppression of the tumor growth.

Conclusion: These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for treating triple negative type and/or metastatic breast cancer.

Keywords: measles virus, oncolytic virus, virotherapy in cancer

17th International Congress of Virology 445

Late breaking abstract

Viruses and Cancer

PO865

Caprine Herpesvirus 1 (CpHV-1) a potential candidate for oncolytic virotherapy.

S. Montagnaro 1, V. Iovane 1, M. V. Puzio 1, R. Ciarcia 1, D. Rocco 2, L. De Martino 1, G. Iovane 1, U. Pagnini 1,*

1Department of Veterinary Medicine and Animal Productions , University of Naples "Federico II", 2ASL Napoli 2 Nord, Naples, Italy

Objectives: Oncolytic virotherapy is a therapeutic approach to cancer treatment that utilizes native or genetically modified viruses that selectively replicate in cancer cells while displaying minimal adverse effects in normal healthy cells. To date, a wide variety of viruses have been evaluated for their oncolytic potential, including DNA viruses. Our group previously demonstrated that Caprine herpesvirus type 1 (CpHV 1) is able to induce apoptosis in several normal cell lines. Recently, we have characterized in more detail the intracellular pathway by which CpHV 1 is able to induce apoptosis by analyzing the gene expression response during the apoptotic phase of CpHV 1 infection in a murine neuroblastoma cell line. Thus, the aim of the present research was to investigate the ability of CpHV-1 to replicate, cause cell death and affect cellular viability in a panel of human cancer cells lines.

Methods: Human breast adenocarcinoma (MDA-MB-468), Human cervical adenocarcinoma (HeLa), Human osteosarcoma (U2OS), Human prostatic adenocarcinoma (PC3), Human lung carcinoma (A549), Human malignant melanoma (A375) and Chronic Myelogenous Leukemia (K562) cell lines were used. Madin Darby bovine kidney (MDBK) cell line was used as control. Citotoxicity was assayed by MTT test; Apoptosis was assayed by using the ApoToxGlo Triplex assay; The espression of apoptosis-related genes was assayed by Western Blot; Viral replication was assayed by TCID50 method.

Results: In a first series of experiments we have analyzed the effect of CpHV1 infection on cell viability by means of the MTT assay at different time post infection and several multiplicity of infection. All cell lines, except K562 cells, showed a marked cytopatic effect, demonstrating an oncolytic potential of CpHV-1 in tested human cancer cells. The reduction of cells viability was associated with significant levels of viral production as assayed by TCID50 after 24 h p.i. for MDBK, 293T, MDA-MD468, A549, U2OS, A375 and after 48 h p.i. for PC3, HeLa and K562 cell lines. Then, we investigated virus induced cytotoxicity, viability, and apoptosis within a single assay well, by using the ApoToxGlo Triplex assay. Analysis of virus infected cells revealed activation of caspase3, a marker of apoptosis at 24 h postinfection.

Conlusion: Our findings firstly demonstrate an oncolytic effect of CpHV‐1 in neoplastic cell lines in terms of caspase activation and apoptosis modulation suggesting CpHV‐1 as a potential candidate in oncolytic virotherapy.

Keywords: Apoptosis , Caprine Herpesvirus 1, oncolytic virotherapy

17th International Congress of Virology 446

Late breaking abstract

Viruses and Cancer

PO866

Detection of OncoE6protein by newly developed OncoE6 cervical test and molecular characterization of Human Papilloma virus (HPV) by PCR from VIA clinic patient attending at Mymensing Medical college & hospital ,Bangladesh.

S. Abedin 1,*

1MYMENSING MEDICAL COLLEGE, DHAKA, Bangladesh

Objectives: To detect OncoE6protein both VIA positive and VIA negative cases by OncoE6 cervical test.

Methods: With all universal safety precautions a total of 150 endocervical swabs were collected from VIA outdoor clinic and colposcopy clinic of obstetrics and Gynaecology department of MMCH between March 2016 to February 2017. Laboratory work was done in the department of Microbiology, Mymensingh medical college.OncoE6 strip test is an immunochromatographic test based on the detection of HPV oncoE6protein in cervical swab samples. The swab specimen was treated with lysis solution and conditioning solution. Then the specimen solution was clarified by centrifugation. After that the sample solution was transferred into Detector mAb vial, wash solution vial and finally into developing solution vial. The test unit was then placed on a reading guide. Positive result was indicated by the appearance of purple coloured test line.

Results: OnE6 cervical test was done on 150 cases. Among them 60 (40%) were VIA positive and 90 (60%) were VIA negative. From this VIA positive cases 12 (20%) were OnE6 cervical test positive and from VIA negative cases 3 (3.33%) were positive by this OnE6 cervical test. From 60 VIA positive cases 10(10%) were HPV-16 and 2 (3.33%) were HPV-18 by this test. From VIA negative cases 3 were only HPV-16 by this test and histopathologically, out of 09 cervical carcinoma cases 08 were positive by this OnE6 cervical test. Among 18 PCR positive cases 15 (83.33%) were positive by this test.

Conlusion: Based on the findings of the present study, it may be concluded that early screening with OncoE6 cervical test will be helpful to minimize the overtreatment as well as the colposcopy referral. By this test we are able to indentify high risk cancer causing oncoprotein from VIA positive and VIA negative cases. So it may be used as primary screening to aid colposcopy and to identify real disease in low resource setting country like Bangladesh.

Keywords: OncoE6protein, CIN (Cervical Intraepithelial Neoplasia), VIA (Visual Inspection with Acetic Acid).

17th International Congress of Virology 447

Abstract Submission

Viruses and Cancer PO867

HPV-16 E1 expression is elevated in cervical cancer F. Baedyananda 1,*, A. Chaiwongkot 1, P. Bhattarakosol 1 1Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Objectives: High-risk human papillomavirus (HPV) infections cause transformation of the host cells and cervical carcinoma by down-regulating and inhibiting host regulatory proteins such as p53 and pRb by overexpressing the viral oncoproteins E6 and E7. However, the E1 protein which is the only enzyme encoded by HPV has also been shown to cause DNA instability leading to integration of the virus into the host genome and triggering carcinogenic events. The purpose of this study was to determine the expression level of E1 in HPV-16 positive patients across all clinical stages. Methods: Total RNA was extracted from thirty-nine HPV-16 positive patients across normal, precancerous and cancer stages of cervical carcinoma. Patient RNA samples were then subjected to cDNA synthesis. HPV-16 E1 mRNA level was detected for each patient by droplet digital PCR. Results: A significant increase in E1 expression in correlation with disease progression between SCC samples and normal samples (p < 0.05) and CIN 2/3 and normal samples (p < 0.01) was found. The average relative E1 mRNA level in clinical samples was 0.18 for Normal, 0.41 for CIN 1, 0.65 for CIN 2/3 and 0.79 for SCC. Conclusion: In this study, we characterized the expression of E1 mRNA in clinical samples ranging from normal, precancerous and cancer stages. We were able to determine that E1 is expressed at significantly different levels in normal patients and cancer patients. Further studies in a larger cohort, as well as the functional roles of E1 are warranted, and could lead to targeted treatment of HPV16 infection. Collectively, we have characterized, for the first time, the expression profile of HPV16-E1 in different stages of cervical cancer disease progression.

Keywords: carcinogenesis, cervical cancer , HPV-16 E1

17th International Congress of Virology 448

Viruses and MicroRNA

17th International Congress of Virology 449

Abstract Submission

Viruses and microRNA

PO869

High-Throughput Sequencing Analysis of MicroRNA Profile in MDCK Cells upon Infection by Seasonal Influenza A Viruses.

S. Saengchoowong 1 2,*, W. Poomipak 3, K. Praianantathavorn 4, Y. Poovorawan 5, Q. Zhang 1, S. Payungporn 4

1Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom, 2Graduate Division, Faculty of Medicine, 3Research Affairs, Faculty of Medicine, 4Department of Biochemistry, Faculty of Medicine, 5Center of Excellence in Clinical Virology, Department of Paediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Objectives: Influenza is a highly contagious respiratory disease affecting humans and animals. Currently, the pandemic H1N1 2009 (pH1N1) and seasonal H3N2 viruses are the major influenza A subtypes circulating in humans worldwide. In addition to embryonated chicken eggs, Madin-Darby Canine Kidney (MDCK) cell line has been recently utilized for viral vaccine seed production. MicroRNAs, small non-coding RNAs, play a vital role in host-pathogen interaction. In this study, we have investigated the profile of cellular microRNA expression in MDCK cell upon pH1N1 and H3N2 virus infection.

Methods: MDCK cells were infected with either influenza A/Thailand/104/2009 (H1N1) or A/Thailand/CU-H1817/2010 (H3N2) virus at multiplicity of infection (MOI) of 0.01. The cells were collected for microRNA extraction at 6, 12, and 24 hours post infection (hpi). The libraries of uninfected and infected groups were prepared using NEBNext ® Multiplex Small RNA Library Prep Set and performed on the Illumina® MiSeq Platform. MicroRNAs that were expressed greater than 2-fold following infection (compared to uninfected control) were validated further by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). MiRBase, RNAhybrid, and BioEdit were then used for the prediction of microRNA targets.

Results: The result demonstrated that cfa-miR-340, the most markedly upregulated microRNA, was expressed at 12 hpi in both pH1N1 and H3N2 infection groups. Moreover, cfa-miR-146b and cfa-miR-215 were upregulated at various time points in H3N2 infection group. Specifically, cfa-miR-146b was over-expressed at 6 and 12 hpi, whereas the expression of cfa- miR-215 was increased at 12 and 24 hpi. Furthermore, in silico analysis revealed that the seed sequence of cfa-miR-340 could bind to viral polymerase basic 1 (PB1) gene, which was conserved between both subtypes used in this experiment and viral vaccine seed strains. Therefore, cfa-miR-340 might affect the replication of seasonal influenza A subtypes via targeting PB1 gene.

Conclusion: This information may help us better understand the interaction between seasonal influenza A viruses and host cells, which may lead to strategies to improve the production of cell-based influenza vaccines.

Keywords: Influenza A virus, MicroRNA, Next-generation sequencing

17th International Congress of Virology 450

Late breaking abstract

Viruses and microRNA

PO870

Role of endothelial miRNAs in mediating dengue virus pathogenicity and endothelial permeability

S. Banerjee*

Objectives: Poor prognosis of dengue haemorrhagic fever(DHF) and dengue shock syndrome(DSS), which results from increased vascular permeability, continues to be a public health problem. MicroRNAs have been shown to exhibit diagnostic potential for viral infections and may also be involved in regulating endothelial dysfunction and vascular permeability.

Methods: We have developed an in vitro model for studying endothelial permeability post dengue infection in human umbilical vein endothelial cells (HUVEC). RNASeq analysis was carried out to study the differential miRNA expression profiles in HUVEC post dengue infection. In silico target prediction algorithms, luciferase reporter assay and protein expression studies were used to identify the putative targets of the miRNAs.

Results: 273 miRNAs were differentially expressed upon dengue infection. miR-X was observed to be significantly downregulated and upon overexpression resulted in a decrease in endothelial permeability. Angiopoietin2 (Ang2) was found to be a predicted target of miR-X , which was further validated by various functional assays. We also observed that addition of recombinant Ang2 increased endothelial permeability despite overexpressing miR-X. miR-X overexpression also resulted in a decrease in viral titres. No putative binding sites for miR-X was found within the DENV2 genome. miR-X also targets TLR-2 and IL-6 and we hypothesise that the observed antiviral effect is due to the regulation of host immune response.

Conlusion: We have shown that miR-X targets Ang2 and regulates endothelial permeability. Further work is required to elucidate the antiviral effect of miR-X and study its prognostic or diagnostic potential in clinical cases of dengue.

Keywords: Dengue virus, hemorrhage, miRNA

17th International Congress of Virology 451