NSV 2018, Verona – Abstract Book

ITALY

Abstract Book

Table of Content

Oral Presentations…………………….. Page 3

Poster Presentations………………….. Page 96

Abstract Author Index…………………. Page 336

NSV 2018, Verona – Abstract Book

ORAL PRESENTATIONS

BREAKING AND ENTERING - viral entry Abstract final identifier: 1

HUMAN LEUCOCYTE ANTIGEN DR (HLA-DR) HOMOLOGS ARE CROSS-SPECIES ENTRY RECEPTORS FOR BAT INFLUENZA VIRUSES Silke Stertz* 1, Umut Karakus1, Thiprampai Thamamongood2, Kevin Ciminski2, Wei Ran2, Sira C. Günther1, Davide Eletto1, Benjamin G. Hale1, Adolfo Garcia-Sastre3, Martin Beer4, Martin Schwemmle2 1University of Zurich, Zurich, Switzerland, 2Medical Center University of Freiburg, Freiburg, Germany, 3Icahn School of Medicine at Mount Sinai, New York, United States, 4Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany

Abstract: In 2012 and 2013 two novel influenza A viruses were discovered in South American bat species. Remarkably, the hemagglutinin of these bat influenza viruses was found not to bind the canonical influenza virus receptor, sialic acid, or any other glycan, despite high sequence and structural homology with conventional influenza A virus hemagglutinins. Using transcriptomic profiling of susceptible versus non-susceptible cells, in combination with genome-wide CRISPR/Cas9-based screening, we identified the MHC-II complex HLA-DR as proteinaceous receptor for bat influenza viruses. CRISPR/Cas9- mediated knockout of HLA-DR rendered susceptible cells completely resistant to bat influenza but not conventional influenza virus infection, whereas ectopic expression of HLA-DR in non-susceptible cells conferred susceptibility to bat influenza virus infection. Expression of HLA-DR homologs from three different bat species, pigs or chickens also conferred susceptibility. Notably, infection of mice with bat influenza virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant to infection. Collectively, our data identify HLA-DR homologs from multiple species as receptors for bat influenza virus, suggesting the potential for broad vertebrate tropism.

BREAKING AND ENTERING - viral entry Abstract final identifier: 2

NECTINS TRANSFER CYTOPLASM BETWEEN CELLS AND CAN SPREAD MEASLES VIRUS TO NEURONS Alex Generous1, Oliver Harrison2, Regina Troyanovsky3, Mathieu Mateo1, Chanakha Navaratnarajah1, Ryan Donohue1, Christian Pfaller1, Alina Sergeeva2, Indrajyoti Indra3, Theresa Thornburg4, Irina Kochektova4, Matthew Taylor4, Sergey Troyanovsky3, Barry Honig2, Lawrence Shapiro2, Roberto Cattaneo* 1 1Molecular Medicine, Mayo Clinic, Rochester MN, 2Biochemistry and Molecular Biophysics, Columbia University, New York, 3Dermatology, Northwestern University, Chicago, 4Microbiology and Immunology, Montana State University, Bozeman, United States

Abstract: We discovered a process mediating intercellular transfer of cytoplasmic materials. Cells expressing the adherens junction protein nectin-1 (N1) take in plasma membrane patches and cytoplasmic materials from cells expressing other nectins. This process is most active during cell adhesion, 2-8 hours post-co-culture. It is most efficient from cells expressing nectin-4 (N4) towards cells expressing N1. And, it depends on the N1 cytoplasmic tail: its deletion prevents transfer, while its exchange with the N4 cytoplasmic tail reverses transfer direction. We term the process nectin-elicited cytoplasm transfer (NECT). Nectin family proteins serve as receptors for positive strand RNA viruses like poliovirus, large DNA viruses like herpes simplex virus, and negative strand RNA viruses such as measles virus (MeV). MeV infections can cause subacute sclerosing panencephalitis (SSPE), a rare but lethal disease. Because of SSPE and of other neurological diseases caused by MeV and related animal viruses, a neuronal receptor has been postulated but no consensus candidate has emerged. We show that N4-expressing, MeV-infected epithelial cells transmit infection to axons of N1-expressing primary neurons that cannot be infected by MeV particles. Thus NECT can spread MeV infections to neurons, circumventing the need for a neuronal receptor. We have evidence that NECT functions through an endocytic pathway, and seek to understand how and where MeV ribonucleocapsids escape it.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 3

STRUCTURE OF FULL-LENGTH INFLUENZA HAEMAGGLUTININ Donald J. Benton* 1, Andrea Nans2, Lesley J. Calder2, Davide Corti3, Steven J. Gamblin1, Peter B. Rosenthal2, John J. Skehel1 1Structural Biology of Disease Processes Laboratory, 2Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom, 3-, Humabs BioMed SA, Bellinzona, Switzerland

Abstract: Influenza A virus Haemagglutinin (HA) is the surface glycoprotein responsible for receptor binding and membrane fusion. Previous structural characterisation of HA has been limited to a soluble ectodomain which does not contain membrane proximal or transmembrane regions. We present structures, determined by cryo-EM, of full-length detergent- solubilised HA in isolation and complexed with a Fab fragment from an infectivity neutralising, H1 subtype-specific antibody, that recognises the ecto-domain-transmembrane domain junction. The structures of the ectodomain compare favourably in resolution with those previously determined by x-ray crystallography. Our description of the previously undetermined parts of the molecule may be important in relation to the immune recognition of the membrane proximal region of HA in antibody binding and antibody induction.

BREAKING AND ENTERING - viral entry Abstract final identifier: 4

STRUCTURAL BASIS OF LOW-DENSITY LIPOPROTEIN RECEPTOR RECOGNITION BY VSV GLYCOPROTEIN Laura Belot1, Nikolic Jovan1, Pierre Legrand2, Hélène Raux1, Yves Gaudin1, Aurelie Albertini* 1 1Institut de Biologie Intégrative de la Cellule, CNRS, 2Synchrotron SOLEIL, GIF SUR YVETTE, France

Abstract: Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. VSV G mediates both virus attachment to its receptor and fusion of the viral envelope with the endosomal membrane. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. We demonstrate that VSV G is able to independently bind two distinct cystein-rich (CR) domains (CR2 and CR3) of LDL-R with similar affinities (Kd ~ 5µM). The biological relevance of this interaction was demonstrated by the ability of both CR2 and CR3 to inhibit VSV infection. We obtained two crystal structures of G in its pre-fusion conformation in complex with CR2 and CR3 revealing that the binding sites of CR2 and CR3 on G are identical. CR domains recognition by VSV G involves basic residues pointing toward the calcium-coordinating acidic residues present in each CR. Mutations of two of these residues abolish G ability to bind to LDL-R without impairing G fusion activity. This demonstrates that it is possible to decouple G fusion activity and receptor recognition. We also show that although VSV can use alternative receptors of the LDL-R family, G mutants affected in their CR domain binding site cannot rescue a recombinant VSV lacking the G gene. Those data indicate that the only receptors of VSV are members of the LDL-R family and that G has specifically evolved to interact with their CR domains. This work provides structural insights on the interaction between G and host cell receptors and paves the way for the design of recombinant G with an altered tropism.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 5

DELINEATING INTERACTION OF FILOVIRAL GP WITH ITS ENDOSOMAL RECEPTOR NPC1 BY IN SITU PROXIMITY LIGATION Eva Mittler* 1, Rohit K. Jangra1, Tanwee Alkutkar1, Kartik Chandran1 1Microbiology and Immunology, ALBERT EINSTEIN COLLEGE OF MEDICINE, New York, United States

Abstract: Filoviruses are emerging zoonotic pathogens that cause outbreaks of lethal hemorrhagic fever in sub-Saharan Africa. The development of effective countermeasures against these agents is hindered by our limited understanding of filovirus-host molecular interactions required for viral entry and infection. For host cell entry, virions traffic to late endosomes/lysosomes (LE/LY), where the viral surface glycoprotein GP gains access to multiple essential host factors: GP is proteolytically processed by cathepsin B/L, and a cleaved form of GP (GPCL) binds to the critical intracellular receptor Niemann-Pick C1 (NPC1). Mechanistic studies of this indispensable binding step have been limited to a truncated, soluble form of a single domain in NPC1, domain C, as robust cell-based assays assessing interaction of GPCL with full-length NPC1 in its native context have been lacking. Here, we developed an in situ assay to monitor GPCL:NPC1 binding in infected cells. Subcellular visualization of this interaction at single molecule resolution was based on the principle of DNA-guided, antibody- + mediated in situ proximity ligation. GPCL:NPC1 interaction was restricted to the lumina of NPC1 LE/LY, and was blocked by disruption of GP’s proteolytic cleavage or GPCL:NPC1 interface formation. Testing the effect of FDA-approved small molecule inhibitors on proximity ligation revealed that drug treatments significantly disrupted virus entry and GPCL:NPC1 binding by distinct mechanisms. In summary, our in situ proximity ligation assay allows us to monitor GPCL:NPC1 engagement in intact cells. We are employing it to define the cellular and viral requirements for this interaction and to delineate the mechanisms of action of small molecules with anti-filovirus activity.

BREAKING AND ENTERING - viral entry Abstract final identifier: 6

HOST FACTOR REQUIRED FOR IN VIVO PATHOGENICITY OF NEW WORLD HANTAVIRUSES Rohit K. Jangra* 1, Andrew S. Herbert2, Lucas T. Jae3, Rong Li4, Lara M. Kleinfelter1, Megan M. Slough1, Eva Mittler1, Ana I. Kuehne2, James Pan5, Sarah L. Barker5, Ariel S. Wirchnianski1, Sushma Bharrhan1, Anna Z. Wec1, J. Maximilian Fels1, Melinda Ng1, Nicolás A. Muena6, Nicole D. Tischler6, Sachdev S. Sidhu7, Jason Moffat5, 7, Zhongde Wang4, Thijn R. Brummelkamp3, John M. Dye2, Kartik Chandran1 1Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 2United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, United States, 3Netherlands Cancer Institute, Amsterdam, Netherlands, 4Department of Animal, Dairy and Veterinary Sciences, Utah State University,, Logan, UT, United States, 5Donnelly Centre and Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada, 6Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile, 7Canadian Institute for Advanced Research, Toronto, ON, Canada

Abstract: Hantaviruses are rodent-borne RNA viruses that cause a severe cardiopulmonary syndrome (HCPS) in humans in the New World. Currently, no vaccines or therapeutics are available against these viruses. Host factors that mediate hantavirus infection and disease in vivo remain unknown. Using a genetic screen in human haploid cells, we identified a cadherin-superfamily protein, previously associated with hereditary respiratory disease, as an entry receptor for HCPS- causing Andes and Sin Nombre viruses. This cadherin is highly expressed in pulmonary endothelial cells, which are major hantavirus targets in vivo, and its genetic depletion substantially reduced infection. Cadherin directly engaged the viral glycoprotein via its first extracellular cadherin (EC1) domain to mediate virus-cell attachment and internalization, and viral infection was sensitive to soluble EC1 and EC1-targeting monoclonal antibodies. Finally, Cadherin–knockout Syrian hamsters, generated by CRISPR/Cas9 genome engineering, were strongly protected from lethal Andes virus challenge, highlighting its pathogenicity relevance and therapeutic potential. [RKJ, ASH, LTJ & RL contributed equally]

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 7

ANCHORING THE SPRING: THE ROLE OF TRANSMEMBRANE DOMAIN INTERACTIONS IN VIRAL FUSION PROTEIN FUNCTION Rebecca E. Dutch* 1, Chelsea Barrett1, Stacy Webb1 1Molecular and Cellular Biochemistry, UNIVERSITY OF KENTUCKY, Lexington, United States

Abstract: Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the Paramyxoviridae family, utilizes the HeV attachment protein (G) and fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a set of large, irreversible conformation changes to drive membrane fusion. We have shown that the transmembrane domain (TM) of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium, and that specific sequences drive this association. This TM-TM association contributes to the stability of the pre-fusion form of the protein, supporting a role for the TM-TM interactions in control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Co-expression of these constructs with HeV F resulted in dramatically reductions in the stability of F protein expression and ablation of fusion activity. In contrast, no effects were observed when the HeV F TM constructs were co-expressed with the non-homologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited viral infection with PIV5, but did not significantly affect infection of human metapneumovirus, a related virus. These findings indicate that TM-TM interactions are a critical stabilizer for the pre-fusion form of viral fusion proteins, and suggest that disruption of these interactions inactivates F protein function, likely by prematurely triggering F protein conformational changes.

BREAKING AND ENTERING - viral entry Abstract final identifier: 8

UNCOVERING A NEW PARAMYXOVIRAL ATTACHMENT GLYCOPROTEIN ROLE BEYOND FUSION TRIGGERING Hector C. Aguilar* 1, I Abrrey Monreal1, Victoria Ortega1, J. Lizbeth R. Zamora1 1Microbiology and Immunology, Cornell University, Ithaca, NY, United States

Abstract: Paramyxoviruses have two glycoproteins that in concert promote viral-cell membrane fusion during viral entry and cell-cell membrane fusion during the pathognomonic syncytia formation. The attachment glycoprotein (HN, H, or G) has two known functions: 1. binding the target cell surface receptor, and 2: subsequently triggering the fusion protein (F) to initiate its own conformational cascade. The F cascade proceeds from pre-fusion to pre-hairpin intermediate (PHI) to post- fusion conformations, executing membrane fusion. Recently, we identified conformational changes in Nipah virus (NiV) G that link receptor binding to F triggering, and identified a role of the NiV G stalk domain in F triggering. Further, we found G and F mutants whose fusogenicity negatively correlates with the avidity of G/F interactions, supporting a model in which dissociation of a G/F bidentate interaction allows membrane fusion. In this study, we found several hypofusogenic mutants in the NiV G stalk that expressed well at the cell surface, bound receptors at wild-type levels, but relatively increased the G oligomeric strength, and bound F with higher avidities. Surprisingly, in a heterologous cell-cell fusion assay, some of these G mutants trapped the membrane fusion cascade at the hemifusion stage, whereby only lipids, but not cytoplasmic contents were mixed. Importantly, these G mutants were capable of F triggering, defined as changing F from pre-fusion to PHI conformations. Combined, these data suggest that G has a role in a later F conformational step beyond the PHI, and that detachment of G from F helps F undergo such conformational step(s) to allow full membrane fusion. This role is novel for the paramyxoviral attachment glycoproteins.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 9

HANTAVIRUS GC HOMOMERIC DIMER AND TRIMER CONTACTS DIRECT SPIKE DISSOCIATION AND MEMBRANE FUSION Eduardo A. Bignon1, Pablo Guardado-Calvo2, Félix Rey2, Nicole Tischler* 1 1Molecular Virology Laboratory, FUNDACIÓN CIENCIA & VIDA, Santiago, Chile, 2Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France

Abstract: Rodent-transmitted hantaviruses are human pathogens that expose (Gn/Gc)4 tetrameric spikes on viral particles. During hantavirus cell entry, these surface glycoproteins mediate receptor binding and membrane fusion. The recent Gc crystal structures from the Orthohantavirus genus confirmed that it is a class II viral fusion protein and the crystals further revealed a homo-dimeric association of Gc at neutral pH and a homo-trimeric assembly at low pH. The Gc dimer interface connects adjacent spikes and consists of four strictly conserved Gc residues, which form a polar network of interactions with inter-chain ionic and hydrogen bonds. These residues are further involved in inter-chain contacts of the low pH Gc homotrimer. Here we aimed to functionally characterize the observed Gc dimer and trimer interface, by assessing the spike stability and fusion phenotype of interface mutants. The substitution of the conserved charged residues lowered or increased the temperature stability of the spikes and changed the pH required for the spike dissociation and fusion activation. Further, all Gc dimer interface contacts were crucial for the membrane fusion activity, and their substitution showed a decreased Gc homotrimer stability, arresting fusion after the trimerization step. Together, our results functionally confirm that the residues forming the Gc homodimer induce spike assembly during the exit of the virus from the cell, while during viral cell entry, these residues direct acid-induced spike dissociation and fusion by stabilizing the post-fusion homotrimer.

BREAKING AND ENTERING - viral entry Abstract final identifier: 10

ANTIGENIC ARRANGEMENT OF THE DYNAMIC HANTAVIRAL SURFACE Ilona Rissanen* 1, 2, Robert Stass1, Sai Li1, Antra Zeltina1, Jussi Hepojoki3, Karl Harlos1, Jayna Raghwani4, Oliver Pybus4, Robert Gilbert1, Juha Huiskonen1, Thomas Bowden1 1Division of Structural Biology, University of Oxford, Oxford, United Kingdom, 2Helsinki Institute of Life Science, 3Department of Virology, University of Helsinki, Helsinki, Finland, 4Department of Zoology, University of Oxford, Oxford, United Kingdom

Abstract: Despite the risks presented by zoonotic hantaviruses, there exists a paucity of knowledge regarding the pathobiology of viral infection, and both the treatment and prevention options remain extremely limited. Early stages of hantavirus infection are regulated by the glycoprotein lattice presented on the hantaviral surface. The two glycoproteins forming the lattice, Gn and Gc, are responsible for host recognition and entry, and are targeted by the neutralizing humoral immune response. Through structural investigation of the Gn glycoprotein from three genetically distinct hantavirus species, we reveal that the fold of the Gn is highly conserved and can form discrete oligomeric configurations. We describe the molecular-level arrangement of the Gn and Gc glycoproteins, as displayed on the hantavirus surface, and address the function of the hantaviral envelope during host cell entry. Our analysis reveals the antigenic determinants of the humoral immune response and provides insights into the structural transitions that the envelope undergoes during endocytosis of the virus into the host cell.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 11

RECEPTOR TYROSINE KINASES ARE PROMISING TARGETS IN LASSA VIRUS ANTIVIRAL THERAPY Chiara Fedeli* 1, Giulia Torriani1, Hector Moreno1, Gert Zimmer2, Gisa Gerold3, Stefan Kunz1 1Institute od Microbiology, CHUV, Lausanne, 2Institute of Virology and Immunology, Mittelhäusern, Switzerland, 3TWINCORE, Institute for Experimental Virology, Hannover, Germany

Abstract: Lassa Virus (LASV) causes a severe viral hemorrhagic fever with high mortality in humans. The main LASV receptor is dystroglycan (DG) in its functional O-glycosilated form. However, glycosylation of DG does not always correlate with LASV tropism in vivo, suggesting alternative receptors. In absence of functional DG, LASV can hijack phosphatidylserine receptors of the Tyro3/Axl/Mer (TAM) family and enter cells via “apoptotic mimicry”. We showed that productive LASV entry via the receptor tyrosine kinase (RTK) Axl involves virus-induced activation of Axl signalling and macropinocytosis. Here we investigated the role of Axl RTK activity for LASV entry as a function of DG modification, using recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rLCMV-LASVGP) as a BSL2 surrogate.Rather unexpected, we found that the specific Axl RTK inhibitor R428 potently inhibits LASV entry in different cell types with EC50 in the mid-nanomolar range, independently of the functional glycosylation of DG. Notably, LASV entry via functionally glycosylated DG into human epithelial cells did not induce Axl activation but was still highly sensitive to R428, suggesting a role of Axl RTK activity as an essential “permissive” signal. Combination of R428 with the hepatocyte growth factor (HGFR) inhibitor EMD1214063 resulted in a pronounced synergistic anti-viral effect, indicating non-redundant roles of these RTKs in LASV entry. Used in combination with ribavirin, a nucleoside analogue used to treat human Lassa fever in the clinic, the RTK inhibitors showed additive anti-viral effects. Our studies provide a rationale to target RTKs in combinatorial therapy against human Lassa fever.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 12

STRUCTURE AND FUNCTION OF PHENUIVIRIDAE CAP-SNATCHING ENDONUCLEASES Juan Reguera* 1, 2, Sana Lessoued2, Gabriel Bragagnolo2, Maria Mate2 1INSERM, 2AFMB, Aix Marseille University/CNRS, Marseille, France

Abstract: Segmented negative stranded viruses (sNSVs) perform transcription initiation by cap snatching. By using a viral endonuclease they snatch short capped nucleotides from cellular mRNAs to subsequently use them for priming transcription. In the last two years the crystal structures of the cap snatching endonucleases of several highly pathogenic viruses have been reported, broadening our understanding of the diversity of the cap snatching mechanism and some basic common features that allow the transcription of this large group of animal, plant and human pathogens. Here we present the structural and functional characterization of the Toscana cap-snatching endonuclease, the first reported for a Phenuivirdae, a family of Bunyavirales order including highly pathogenic arboviruses such as Rift Valley Fever Virus. The structure reveals new unexpected features differing from cap snatching endonucleases of other related virus such Influenza, Lassa, Hantaan or La Crosse, while maintaining some basic core folding features and enzymatic activity. Our results confirm that the in vitro endonuclease activity of cap snatching endonucleases is dependent of the presence of a catalytic histidine in the active site, validating the proposed classification of these enzymes as His+ and His-. This classification is critical for the design of strategies for antiviral discovery targeting cap snatching. Thus, the results of our study pave the way for the discovery of specific antivirals targeting the viral transcription of Orthophlebovirus. Our study evidences the large folding diversity among sNSVs cap snatching endonucleases and shows the potential for discovery of antivirals with broad spectra for sNSVs.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 13

MECHANISM OF POLYMERASE REGULATION BY A 5’ HOOK-LIKE STRUCTURE IN THE GENOMIC RNA OF MACHUPO VIRUS Jesse D. Pyle* 1, Sean P. J. Whelan* 1 1Harvard Medical School, Boston MA USA

Abstract: The atomic structures of the influenza (Orthomyxoviridae) and La Crosse virus (Peribunyaviridae) RNA- dependent RNA polymerases (RdRPs) revealed that the 5’ terminal 10-nucleotides of the viral genome RNA (vRNA) are bound as a hook like structure proximal to the RdRP active site. This 5’ hook RNA appears to stabilize the catalytic residues of the RdRP. For influenza virus, mutagenesis of the element results in changes in gene expression but the underlying mechanism remains to be determined. Using an in vitro biochemical assay for RdRP function, we probe the requirement for a similar 5’ hook structure in arenaviruses, which like the orthomyxoviruses and peribunyaviruses have segmented negative- sense RNA genomes. Working with the purified 250 kDa large polymerase (L) of Machupo virus (MACV), we demonstrate that the 5’ terminal 12-nucleotides of the vRNA stimulate RdRP activity >6-fold. The stimulation is specific to the MACV RNA sequence, and requires quasi-complementary interactions between the 5’ and 3’ vRNAs. The presence of the 5’ hook also renders the polymerase dependent on magnesium as the divalent cation – in common with other polymerases – further validating the importance of this RNA ligand in polymerase function. A 5’ hook-like structure is thus a shared requirement for function in polymerases of segmented negative-strand RNA viruses.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 14

STRUCTURAL INSIGHTS INTO THE MECHANISMS OF INFLUENZA A VIRUS RNA REPLICATION Haitian Fan* 1, Itziar Serna Martin1, Jeremy Keown2, Narin Hengrung3, Robert P. Rambo4, Jonathan M. Grimes2, Ervin Fodor1 1Sir William Dunn school of pathology, 2Division of Structural Biology, University of Oxford, Oxford, 3The Francis Crick Institute, London, 4Diamond Light Source Ltd, Didcot, United Kingdom

Abstract: Transcription and replication of the influenza A virus single-stranded viral RNA (vRNA) genome is carried out by the viral RNA-dependent RNA polymerase, composed of the PB1, PB2, and PA subunits. Transcription of vRNA into mRNA is primed by short capped RNA fragments derived from host capped RNAs by the cap-snatching activity of the viral polymerase and occurs in association with host RNA polymerase II. On the other hand, replication of vRNA is a primer independent process and proceeds through a complementary RNA (cRNA) replicative intermediate. However, the molecular mechanisms of vRNA replication, which has been reported to require polymerase dimerisation, remain largely unknown. Here we report the high-resolution crystal structures of polymerases from human A/NT/60/1968 (H3N2) and avian A/duck/Fujian/01/2002 (H5N1) influenza viruses in their apo forms. In solution, both polymerases form dimers of heterotrimers and analysis of the crystal structure revealed a dimerisation interface which has been confirmed by mutagenic studies as well as small-angle X-ray scattering (SAXS). Dimerisation is mediated mostly by the PA C-terminal domain but involves also regions of PB1 and PB2. Furthermore, a nanobody, a single-domain antibody, raised against the viral polymerase, was found to inhibit polymerase dimerisation. Cryo-EM and crystal structures of the nanobody-polymerase complex confirmed that that nanobody binds near the dimerisation interface. Inhibition of polymerase dimerisation by nanobody resulted in reduced polymerase activity in ribonucleoprotein reconstitution assays. We propose that the polymerase dimer we have identified could play a role in RNA genome replication.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 15

TRANSLATIONAL REGULATION OF BORNA DISEASE VIRUS Akiko Makino* 1, Yutaro Yamamoto1, Yuya Hirai2, Keizo Tomonaga1 1KYOTO UNIVERSITY, Kyoto, 2Osaka Dental University, Osaka, Japan

Abstract: Borna disease virus (BoDV), which belongs to the order Mononegavirales, establishes a persistent infection in the nucleus. Because BoDV replicates in the nucleus, the viral translation is separated from its transcription in space and time. This feature indicates that BoDV may control the quality and quantity of translating mRNAs to suppress virus production and maintain the persistent infection in the nucleus. However, translational regulation of BoDV has not been understood in detail. To elucidate translational regulation of BoDV, we performed screening of shRNA library using BoDV-infected cells and identified insulin-like growth factor 2 (IGF2) as a host factor associated with viral particle production. Knockdown of IGF2 enhanced translation activity and particle production of BoDV, and overexpression of IGF2 in IGF2-knockdown cells reduced BoDV translation. On the other hand, treatment of recombinant IGF2 protein to BoDV-infected cells had no effect on the viral production. These results suggest that not IGF2 protein, but its mRNA plays a role in the regulation of BoDV translation. We therefore assessed the involvement of IGF2 mRNA binding proteins (IGF2BPs), which regulate IGF2 mRNA translation. Overexpression of IGF2BP2 enhanced the BoDV translation and particle production, whereas it decreased expression level of viral mRNAs in infected cells, like IGF2-knockdown. The RNA recognition motif and RhoGAP domains of IGF2BP2 were dispensable for its enhancing effect on viral translation. We also showed that IGF2BPs were co-immunoprecipitated with mRNAs and N protein of BoDV in infected cells. These results suggest that BoDV mRNAs undergo translational regulation of IGF2BP2 in competition with IGF2BPs-binding mRNAs, such as IGF2, for regulation of viral particle production in infected cells.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 16

VIRAL N6-METHYLADENOSINE PROMOTES REPLICATION AND GENE EXPRESSION OF PNEUMOVIRUSES Miaoge Xue1, Boxuan Zhao2, Mijia Lu1, Scott Zhang2, Xueya Liang1, Zhike Lu2, Yuanmei Ma1, Anzhong Li1, Mark Peeples3, Chuan He2, Jianrong Li* 1 1Department of Veterinary Biosciences, THE OHIO STATE UNIVERSITY, Columbus, 2Department of Chemistry, The University of Chicago, Chicago, 3Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, United States

Abstract: N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Likewise, viral RNAs may acquire m6A methylation during replication within these cells. The m6A modification is installed by host m6A methyltransferases and is reversible by RNA demethylases. The biological functions of m6A are mediated through m6A binding proteins that specifically recognize and bind the methylated adenosine on RNA. Here, we show that genome, antigenome, and mRNAs of human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), two medically important pneumoviruses, are modified by m6A within discreet regions and that these modifications enhance viral replication and gene expression. Notably, overexpression of the m6A binding proteins, YTHDF1-3, significantly enhanced viral RNA synthesis, translation, replication, and viral release. Knockdown of m6A methyltransferases decreased viral replication and gene expression whereas knockdown of m6A demethylases had the opposite effect. Subsequently, the m6A sites in the viral G protein mRNA, the most abundant m6A modified gene transcript of both RSV and hMPV, were inactivated by mutagenesis. The resultant recombinant RSVs and hMPVs were defective in replication, gene expression, and spread in A549 cells and primary well differentiated human airway epithelial (HAE) cultures. Moreover, a small molecule that inhibited S-adenosyl-L- homocysteine (SAH) hydrolase, thereby reducing the cellular SAH pool and viral RNA m6A, also inhibits viral replication in HAE cells. Collectively, our results identify a novel mechanism for regulating pneumovirus replication and gene expression and identify m6A methylation as a target for controlling infection by pneumoviruses and perhaps other non-segmented negative-sense (NNS) RNA viruses.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 17

PROTEOMIC ANALYSIS IDENTIFIES HUMAN E3 UBIQUITIN LIGASE RBBP6 AS A NEGATIVE REGULATOR OF EBOLA VIRUS INFECTION Christopher Basler* 1, Jyoti Batra1, Judd F. Hultquist2, John VonDollen2, Laura Satkamp2, Gwendolyn M. Jang2, Olena Shtanko3, Manu Anantpadma3, Robert Davey3, Nevan J. Krogan2 1Center for Microbial Pathogenesis, Georgia State University, Atlanta, 2Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, 3Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, United States

Abstract: Ebola virus (EBOV) is a member of Filovirdae family of negative-sense RNA viruses. EBOV infection causes severe hemorrhagic fever in humans, but little is understood as to how this virus exploits the host machinery to replicate successfully. To better define this process, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 193 high-confidence EBOV-human PPIs, including one between the EBOV VP30 protein and the host ubiquitin ligase human retinoblastoma binding protein 6 (RBBP6). EBOV VP30 acts as a regulator of viral RNA synthesis including through interactions with the viral nucleoprotein (NP). RBBP6 is a large multidomain protein that interacts with a variety of host molecules. We mapped the interaction to a 23 amino acid stretch within RBBP6 that was both necessary and sufficient for VP30 binding. Furthermore, a co-crystal structure of RBBP6 peptide bound to VP30 C-terminus solved to 1.5 resolution revealed that RBBP6 usurps the NP binding cleft on VP30. Knockdown of endogenous RBBP6 enhances viral RNA synthesis and increases EBOV infectivity, while over-expression of either RBBP6 or RBBP6 peptide results in strongÅ inhibition. Common between RBBP6 and the region of NP that binds VP30 is a PPxPxY motif. Strikingly, three other VP30 interactors identified by the PPI screen, hnRNPL, hnRNPUL1 and PEG10, possess the same motif as RBBP6 and NP, and can also modulate EBOV RNA synthesis. Cumulatively, these data unexpectedly identified host negative regulators of EBOV replication and point to the VP30-NP interaction as a therapeutic target.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 18

ADVANCED ANALYSES ON THE ROLE OF VP30 AND RNA STRUCTURES IN EBOLA VIRUS TRANSCRIPTION S. Bach* 1, A. Grünweller1, S. Becker2, N. Biedenkopf2, R. K. Hartmann1 1IPC, 2IfV, PHILIPPS-UNIVERSITÄT, Marburg, Germany

Abstract: Ebola virus (EBOV) genome replication requires the polymerase L, NP and VP35. Transcription of viral mRNAs additionally requires transcription factor VP30. The transcription start regions (TSR) of all EBOV genes are embedded in potential 5'-UTR secondary structures of varying stability on the genomic RNA or mRNA level. We used mono- and bicistronic minigenomes to investigate the role of these secondary structures in viral transcription and their dependency on VP30. The TSR of the first NP gene and its complementary spacer sequence separate the replication promoter elements PE1 and 2. Through replacing the NP TSR:spacer hairpin structure with those of the internal EBOV genes, we found that the spacing between PE1 and 2 has to be a multiple of 6 nt (termed rule of six) not only for efficient replication [Weik et al., 2005, J Virol] but also efficient transcription initiation. This suggests that the replication promoter directs replication and transcription. In line with this, exchange of 5'-UTRs at the TSR of the 2nd EBOV gene was permissive toward deviations from the rule of six. We further showed (i) that, for efficient transcription initiation, insertions between PE1 and 2 are limited to 48 nt; (ii) stabilization of 5’-UTR hairpin structures by GC-rich helical extensions eliminated reporter gene activity; (iii) a 12-nt deletion of the NP spacer, which eliminates any potential to form RNA structures at the TSR in (-) and (+) RNAs, still allowed transcription and, remarkably, did not abolish VP30 dependency of transcription. We propose that a major function of VP30, which primarily binds ssRNA [Schlereth et al., 2016, RNA Biol], is to induce and support a conformational switch of the RNA:L/35 complex required for transcription initiation.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 19

REPURPOSING CANONICAL ANTIVIRAL RESPONSES TO PROMOTE TRANSLATION OF INFLUENZA VIRUS MESSENGER RNAS Vy G. Tran1, Mitch P. Ledwith1, Thiprampai THAMAMONGOOD2, Adoflo Garcia-Sastre3, Martin Schwemmle2, Adrianus C. Boon4, Michael S. Diamond4, Andrew Mehle* 1 1Medical Microbiology and Immunology, University of Wisconsin Madison, Madison, United States, 2Institute of Virology, Medical Center, University of Freiburg, Freiburg, Germany, 3Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, 4Departments of Medicine, Molecular Microbiology, and Pathology & Immunology, Washington University School of Medicine, St Louis, United States

Abstract: Cells infected by influenza virus mount a large-scale antiviral response and most ultimately initiate cell death pathways in an attempt to suppress viral replication. During a CRISPR knockout screen designed to query post-entry steps during infection, we identified a large class of presumptive antiviral factors, including IFIT2, as important enhancers of influenza virus replication. IFIT2 is an interferon stimulated gene with well-established antiviral function for some viruses. We show here that IFIT2 is instead repurposed by influenza virus to support viral replication. IFIT2 interacts with viral RNAs and the viral nucleoprotein to stimulate viral gene expression. IFIT2 recruits viral mRNAs to translating ribosomes, enhancing production of viral proteins and ultimately replication of the virus. IFIT2 also stimulates virally-induced apoptosis. Cells lacking IFIT2 fail to undergo apoptosis and survive infection. Remarkably, genetic and chemical ablation of apoptosis further reduced viral yield. Our results suggest that influenza virus has evolved to exploit the antiviral and apoptotic cellular environment, redirecting these classically antiviral events into pro-viral effectors. Moreover, it is highly unlikely that the repurposing of antiviral genes is specific to influenza A virus, but rather is generalizable to additional viruses and other antiviral viral proteins defining a new interface between the virus and host.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 20

BINDING TO ANP32 IS REQUIRED BUT NOT SUFFICIENT FOR INFLUENZA A VIRUS POLYMERASE ACTIVITY Jason Long* 1, Wendy Barclay1, Bhakti Mistry1, Ecco Staller1, Jocelyn Schreyer1 1Virology, Imperial College London, London, United Kingdom

Abstract: Influenza pandemics are sparked by zoonotic infections from animal influenza A viruses in humans. Thankfully, these events are rare due to species specific barriers that the virus encounters when it switches host species. Host protein ANP32 underlies the important host barrier that prevents avian influenza polymerases functioning in human cells.

Avian influenza polymerases have learnt to utilise avian ANP32A which encodes a 33aa insertion between the LRR and LCAR domains of the protein that is absent in mammals, flightless birds and ANP32B from birds and mammals. Mutations in PB2, most famously E627K, enable the polymerase to utilise the shorter ANP32 forms in human cells.

We have shown that the influenza polymerase heterotrimer binds to ANP32 proteins in the absence of RNA, and that this binding is required but not sufficient for activity. We have mapped the minimal requirements of ANP32 proteins to support influenza polymerase and conducted experiments to understand at what stage of virus replication the interaction occurs.

Understanding how the polymerase interacts with essential host proteins may enable the identification of novel small molecule inhibitors and help map mutations required by the polymerase to overcome the species barrier, informing surveillance and disease control.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 21

INCOMPLETE GENOMES OF INFLUENZA A VIRUS DRIVE ABUNDANT REASSORTMENT Anice C. Lowen* 1, Nathan T. Jacobs1, Kara L. Phipps1, John Steel1 1Department of Microbiology and Immunology, Emory University, Atlanta, United States

Abstract: Evolution of influenza A virus (IAV) poses a continued threat to public health by allowing evasion of immunity and emergence of pandemic strains. The segmentation of the IAV genome contributes to this evolution by facilitating diversification through reassortment. Using genetically tagged parental viruses to ensure unbiased detection of all progeny genotypes, we found that reassortment is a routine feature of IAV infection, even in vivo. In cell culture, where MOI can be more tightly controlled, reassortment levels greatly exceeded predictions based on the random distribution of two eight- segmented parental viruses into cells. Modeling suggested that high reassortment might be due a dependence on co- infection for productive infection. In agreement with this prediction, single cell analysis of cells infected at low MOI revealed that each of the segments of A/Panama/2007/99 (H3N2) virus was present with a frequency of ~0.56. Thus, Pan/99 viral genomes in singly infected cells were complete with a frequency of only 0.568=0.01. Strain variation in reassortment phenotype allowed identification of the polymerase and nucleoprotein genes as viral determinants of genome completeness. Importantly, comparison of a given strain in multiple cell lines revealed that incomplete genome levels are cell type dependent. This host-dependent phenotype suggests that segments are lost after delivery of the viral genome to the cell, through interaction with host factors. Rather than dead-end products, however, incomplete viral genomes appear to be complemented frequently through co-infection. In summary, our data suggest that inefficiencies early in the life cycle often render IAV genomes incomplete; that IAV infection is typically mediated by a multi-particle infectious unit; and that abundant reassortment results.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 22

DIFFERENCES BETWEEN TRANSCRIPTION AND REPLICATION INITIATION MECHANISMS OF THE PNEUMO- AND PARAMYXOVIRIDAE

Afzaal Shareef1, Paul Jordan2, Michael Mawhorter1, Tessa Cressey1, Sarah Noton1, Kartikeya Nagendra1, Jerome Deval2, Rachel Fearns* 1 1Microbiology, Boston University School of Medicine, Boston, 2Alios Biopharma, Johnson and Johnson, South San Francisco, United States

Abstract: The polymerase of the non-segmented negative strand RNA viruses (nsNSVs) initiates both transcription and genome replication from the same promoter region. We have previously shown that the polymerase of respiratory syncytial virus (RSV), a member of the Pneumoviridae, accomplishes this by initiating RNA synthesis from two sites on its promoter: position 1U to begin replication and position 3C to begin transcription. Here we used purified RSV polymerase in an in vitroRNA synthesis assay to study the mechanism by which this occurs. We show that initiation at 1U and 3C occurred independently of each other and that the polymerase preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. We then used a similar assay system to investigate if other nsNSV promoters and polymerases have similar properties. The polymerase of human metapnuemovirus, another pneumovirus, also initiated at positions 1U and 3C of its promoter. In contrast, the polymerase of human parainfluenza virus 3 (HPIV3), a paramyxovirus, only initiated at position 1U of its own promoter. However, evidence indicates that the HPIV3 polymerase could initiate internally on an RSV promoter sequence. These findings suggest that the pneumo- and paramyxoviruses have different initiation mechanisms, but that these differences are largely driven by differences in the promoter sequences, rather than by differences in polymerase structure.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 23

NEXT GENERATION SEQUENCING REVEALS NOVEL ASPECTS OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION AND THE SWITCH BETWEEN ACUTE AND PERSISTENT INFECTIONS. Richard Randall* 1, Elizabeth Wignall-Fleming2, Dan Young1, Elizabeth M. Randall1, Andrew Davison2, Steve Goodbourn3 1UNIVERSITY OF ST ANDREWS, St Andrews, 2University of Glasgow, Glasgow, 3St George's, London, United Kingdom

Abstract: Following a high moi, maximum amounts of viral mRNA are observed between 12 – 18h p.i., when the viral mRNA contributes 4-8% of the total mRNA of the cell. Maximum levels of virus genomes/ antigenomes are observed at 24h p.i. Whilst the ratio of genomes to antigenomes in infecting virus is ~ 9:1, by 6 h p.i. the number of antigenomes exceeds the number of genomes, but this ratio reverts in favour viral genomes by 12h p.i. As virus replication begins before 6h p.i., virus replication must occur before there are high overall levels of NP within infected cells. Immunofluoresence suggests a model in which virus transcription and replication initially occurs at a limited number of local foci within an infected cell, before the virus spreads to initiate new foci of replication throughout the cell. At later times p.i., whilst >90% of the cells survive the infection, virus transcription and replication become represses such that the level of virus mRNAs contribute approximately 0.1% of the total mRNA. Repression of virus replication at late times p.i. is linked to the phosphorylation status of the P protein. Thus, virus replication is not switched off, and infected cells die, following infection with a recombinant virus with a single amino acid substitution of serine to phenylalanine at position 157 on P. We speculate that in vivo, during acute phases of virus infection variants of PIV5 will be selected for in which virus replication cannot switched off. However, as the adaptive immune response develops, variants in which virus replication can be repressed will be selected, leading the establishment of prolonged/ persistent infections.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 24

STRUCTURE GUIDED TUNING OF VESICULAR STOMATITIS VIRUS RNA DEPENDENT RNA POLYMERASE FIDELITY Louis-Marie Bloyet* 1, Sean P. Whelan1 1Microbiology and Immunobiology, Harvard Medical School, Boston, United States

Abstract: Reflecting the error prone nature of RNA replication, RNA viruses exist as a quasispecies – a swarm of sequences around a core consensus sequence. Experimental evidence supports the hypothesis that quasispecies diversity influences viral pathogenesis by – for example – providing variants necessary to overcome intrinsic host barriers to infection. This has led to efforts to rationally attenuate RNA viruses through manipulation of their intrinsic polymerase error rates. Reducing error rates diminishes quasispecies diversity, restricting the ability of the virus to overcome barriers. Increasing polymerase error rates pushes the virus swarm past a point of recovery through a process termed “error catastrophe”. Mutations in other viral polymerases that influence error rate map to the active-site region, the nucleoside triphosphate (NTP) entrance channel, and other regions that likely operate through an allosteric mechanism. Guided by the structure of the vesicular stomatitis virus (VSV) large polymerase protein, we generated a panel of 25 mutants and screened them in a minigenome assay which identified 6 functional mutants. We introduced each of those into an infectious cDNA clone of VSV and recovered 4 of 6 variants and used frequency of escape to a neutralizing antibody, and incorporation of incorrect nucleotides in an in vitro transcription assay to compare polymerase error rates. Substitution F514Y in the template entrance channel reduced polymerase fidelity 2-fold, whereas K535R in the NTP entrance channel increased fidelity. Catalytic site region substitutions N763K and E766D reduced fidelity 1.5 and 25-fold respectively. Substitutions that influence polymerase error rate by directly impacting the active site and NTP entrance channel may aid in rationally attenuating other mononegavirales.

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 25

TO V, OR NOT TO V? CYCLIC SELECTION OF GENOMIC QUASISPECIES WITH ALTERNATIVE CODING CAPACITIES IN DUAL-TROPIC MEASLES VIRUS Ryan C. Donohue* 1, Christian K. Pfaller1, Roberto Cattaneo1 1Molecular Medicine, MAYO CLINIC, Rochester, United States

Abstract: RNA virus genomes are quasispecies differing by one or more nucleotides from the consensus sequence. Genomic diversity is instrumental for virus escape from immune selection, and for adaptation to new hosts. It is less clear whether it also contributes to virus adaptation to different tissues within a host. Towards answering this question, we operated with measles virus (MeV), which replicates first in immune cells and then in respiratory epithelia. We mimicked its infection by adapting the virus cyclically to human lymphocytic (Granta-519) and epithelial cells (H358) or exclusively in either cell line. MeV adapted to Granta-519 cells replicated poorly on H358 cells. Thus, we performed deep-sequencing genome analyses. Strikingly, adaptation to Granta-519 cells reproducibly resulted in genomic variants with different combinations of four mutations within an 11-nucleotide region of the phosphoprotein gene. This sequence mediates polymerase slippage and insertion of a guanosine in the corresponding mRNA, resulting in expression of an interferon antagonist (V protein) in place of a polymerase co-factor (P protein). Granta-519-adapted MeV produces minimal amounts of edited transcripts, while parental and H358-adapted viruses produce edited and non-edited transcripts at a 1:1 ratio. Granta-519-adapted MeV expresses much more P than V protein, while the other viruses express P and V at similar levels. We have rescued recombinant viruses carrying individual mutations and confirmed a shift the P to V expression ratio towards enhanced P production. Thus, we discovered a mechanism that may accelerate the speed of MeV replication in lymphocytic cells. It is important to assess whether tissue-specific quasispecies adaptation occurs within infected hosts.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 26

INFLUENZA A VIRUS GENOMIC HETEROGENEITY REGULATES SUPERINFECTION POTENTIAL AND THE CELLULAR RESPONSE TO INFECTION

Chris Brooke* 1 1University of Illinois, Urbana, United States

Abstract: The vast majority of influenza A virus (IAV) virions express highly variable, incomplete subsets of viral genes, and cannot undergo productive replication without complementation. Despite being the primary product of IAV infection, the role that these semi-infectious particles (SIPs) play in shaping viral dynamics and the host response to infection remains poorly understood. Here, we used a novel single virion infection approach to reveal that SIPs play a direct role in determining the frequency of superinfection. We show that superinfection susceptibility is determined by the total number of viral genes expressed within a cell, independent of their identity. IAV particles that express a complete set of viral genes potently inhibit superinfection, while SIPs that express incomplete subsets of viral genes do not. As a result, viral populations that contain more SIPs undergo more frequent superinfection. Finally, we used single cell RNAseq to dissect how viral genomic heterogeneity within IAV populations shapes the host transcriptional response to infection. Altogether, these studies reveal viral genomic heterogeneity as a major factor that governs both superinfection susceptibility and the overall cellular response to infection.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 27

NUCLEAR IMPORT OF THE RABIES VIRUS P PROTEIN IS REQUIRED FOR INHIBITION OF RIG-I-LIKE RECEPTOR- MEDIATED TYPE I INTERFERON INDUCTION Marco Wachowius1, Verena Pfaffinger1, Maximilian F. Eizinger1, Karl-Klaus Conzelmann* 1 1Max von Pettenkofer Institute Virology and Gene Center, LMU Munich, München, Germany

Abstract: The rabies virus phosphoprotein P plays essential roles in various aspects of the cytoplasmic viral life cycle, including viral RNA synthesis and RNA encapsidation, as well as in counteracting the antiviral immune response. Although P is accumulating almost exclusively in the cytoplasm, it is long known to undergo nucleocytoplasmic shuttling, as directed by various conserved nuclear import and export signals. However, the biological relevance of nuclear shuttling has remained unclear up to now. We here provide experimental evidence that nuclear import of P is absolutely required for inhibition of RIG-I-dependent IFN induction both upon plasmid transfection and in the context of virus infection. Deletion of nuclear import signals of P proteins from a vaccine strain and a virulent clinical isolate rendered the proteins defective in preventing IRF3 phosphorylation and transcription of IFN-β. Recombinant RABV carrying shuttling-defective P effectively induced IFN. The defect was cured by addition of an ectopic NLS from SV40, and an extra NLS even improved the IFN inhibitory capacity of wt P protein. Mechanisms involved in priming of RABV P for IFN inhibition are being analyzed.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 28

NOVIRHABDOVIRUS NV PROTEINS ACT AS ANTAGONISTS OF THE HOST ANTIVIRAL DEFENSE Stéphane Biacchesi* 1, Emilie Mérour1, Laury Baillon1, Didier Chevret1, Michel Brémont1 1INRA, Jouy en Josas, France

Abstract: The non virion (NV) protein expression is critical for pathogenesis of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in trout. We further described that a single amino acid change in VHSV NV found in field isolates highly attenuated in trout had a tremendous impact on the virulence, underlying the major function of this protein. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomics that allowed us identifying several NV-associated cell partners. We showed that both NV proteins specifically interact with a serine/threonine protein phosphatase, PPM1B, which has been shown in mammals to negatively regulate the host antiviral response via dephosphorylating TBK1. We demonstrated that NV and PPM1B efficiently counteract RIG-I- and TBK1-dependent IFN and ISG promoter induction in fish cells and, hence, the establishment of an antiviral state. We further showed that PPM1B dephosphorylates TBK1, as previously observed for its mammalian orthologs, and that the expression of VHSV NV re-located PPM1B in close vicinity of mitochondria, a subcellular compartment important for the recruitment and the activation of TBK1, and led to TBK1 dephosphorylation. In contrast, IHNV NV did not lead to TBK1 inactivation meaning that IHNV NV has another mechanism of action. These data bring new insights on the function of the NV expressed by two major pathogens for aquaculture and ecosystems worldwide. Although both NV share a same function, their mechanism of action seems to be distinct. All together, these findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1B to subvert innate immune response.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 29

MECHANISMS OF IMMUNE EVASION BY RSV NON-STRUCTURAL PROTEINS Daisy W. Leung* 1 1Pathology & Immunology, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, St. Louis, United States

Abstract: Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections, morbidity, and mortality in the pediatric, the elderly, and immunocompromised populations worldwide. Despite decades of intensive research, prophylactic and therapeutic treatment options are limited and in need of improvement. RSV encodes for two nonstructural (NS) proteins that are unique among non-segmented negative strand viruses and are important, multifunctional immune antagonists. NS1 and NS2 are involved in host immune suppression, including inhibition of Type I interferon (IFN) induction and signaling, as well as inhibition of the NF-kB pathway and apoptosis. However, limited information on the mechanistic aspect of how these proteins function or their therapeutic potential for antiviral and vaccine development has not been fully explored. Here we describe our recent novel structural findings on NS1 and NS2, which now provides a framework to mechanistically dissect the function of these well described, but poorly defined viral proteins. We also provide insights to target NS1 and NS2 for therapeutic development to limit RSV-associated morbidity and mortality.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 30

A BARCODED LIBRARY UNVEILS NS1-DRIVEN INFLUENZA VIRUS TROPISM Raquel Muñoz-Moreno* 1, Carles Martínez-Romero1, Asiel Arturo Benitez1, Christian Forst2, Daniel Blanco-Melo1, Raffael Nachbagauer1, Vinod Balasubramaniam1, Ilseob Lee1, Sadaf Aslam1, Maryline Panis1, Ignacio Mena1, Juan Ayllón1, David Sachs2, Florian Krammer1, Benjamin R. Tenoever1, Adolfo García-Sastre1, 3, 4 1Microbiology, Icahn School of Medicine at Mount Sinai, 2Genetic and Genomic Sciences, 3Mount Sinai School of Medicine, 4Institute of Global Health and Emerging Pathogens, Icahn School of Medicine at Mount Sinai, New York, United States

Abstract: Influenza NS1 protein is a key virulence factor that inhibits type I interferon (IFN), thus allowing the virus to replicate efficiently in host cells. In this work, a PR8 backbone has been used to generate a library of recombinant viruses, each expressing a different NS1. This approach allowed us to obtain a representative library of recombinant PR8 viruses containing NS1 sequences encompassing the known NS1 evolutionary landscape from different viral strains, countries, years and hosts. Different sequences were collected from the Influenza Research Database (IRD) based on different structural and functional Sequence Feature Variant Types (SFVT). The NS1 library was designed by using a modified split NS segment where we inserted a 22-nucleotide barcode (BC) that allows for the monitoring and quantitation of the different recombinant NS1 viruses through the Illumina Miseq deep sequencing platform after infecting different systems. Equivalent levels of each unique virus were combined to obtain the NS1-barcoded library. Egg allantoic fluid, MDCK supernatants and lungs collected from mice previously infected with the NS1 library were analyzed. The study revealed the emerging groups of NS1s that are best adapted, the ones that tend to disappear as the infection progresses as well as the impact of the host antiviral pathways in determining the fitness of influenza A viruses according to their specific NS1 sequences.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 31

MARBURG VIRUS PROTEIN VP30 REGULATES THE IRE1/XBP1-DEPENDENT UNFOLDED PROTEIN RESPONSE TO ENSURE EFFICIENT VIRAL REPLICATION Cornelius Rohde* 1, Stephan Becker1, Verena Krähling1 1Institute of Virology, Philipps University, Marburg, Germany

Abstract: Too many unfolded proteins impose stress on the endoplasmic reticulum (ER) leading to the activation of three distinct signaling cascades, referred to as unfolded protein response (UPR). The most conserved among them is triggered by IRE1. IRE1 is activated upon ER stress resulting in XBP1 mRNA splicing and the translation of the transcription factor XBP1s (XBP1 spliced). XBP1s is transported into the nucleus to bind cis-acting UPR elements (UPRE) and thereby enhances the expression of many genes to restore ER homeostasis. Under non-UPR conditions XBP1 mRNA is not spliced, resulting in the translation of XBP1u (XBP1 unspliced). XBP1u recruits its own mRNA to the ER membrane to facilitate IRE1-mediated splicing. The aim of our study was to examine if and how Marburg virus (MARV) interacts with the IRE1-dependent UPR. IRE1 activation can be detrimental or beneficial for viral replication. Here we show that the ectopic expression of MARV GP induced UPR via the IRE1-XBP1 axis while MARV infected cells did not show hallmarks of IRE1-dependent UPR activation. These seemingly contradictory results could be reconciled by the finding that MARV VP30 counteracted the GP-activated UPR. In addition, VP30 also regulated UPRE activation upon stimulation by other potent UPR inducers. Co-immunoprecipitation studies revealed that VP30 interacts with XBP1u; this interaction was RNA-dependent. Using IRE1 knock-out cells and artificial stimulation of UPR we could show that the growth of MARV is influenced by activation of UPR. These results support the idea that tight regulation of the IRE1-dependent UPR by MARV VP30 via interaction with XBP1u is needed for an efficient viral propagation.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 32

SUBVERSION OF LYMPHOCYTE RESPONSE AND MODULATION OF GENE EXPRESSION AND SIGNALING BY EBOLA VIRUS Patrick Younan1, Mathieu Iampietro1, Ndongala Lubaki1, Rodrigo Santos1, Palaniappan Ramanathan1, Andrew Nishida2, Mukta Dutta2, Michael Katze2, Fabian Gusovsky3, Richard Koup4, Alexander Bukreyev* 1 1University of Texas Medical Branch, Galveston National Laboratory, Galveston, 2University of Washington, Seattle, 3Eisai Inc., Andover, 4National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States

Abstract: Fatal outcomes of Ebola virus (EBOV) infections are preceded by a “sepsis-like” syndrome and lymphopenia despite T cells being resistant to EBOV infection. To determine the effects of EBOV interferon-inhibiting domains (IIDs) on cell-mediated responses, we used a panel of recombinant strains of EBOVs with point mutations disabling the VP24 and/or VP35 IIDs. The viruses were used for infection of human dendritic cells (DCs) co-cultured with T cells. We found that IIDs block activation and proliferation of T cells as a result of their role in suppressing maturation of DCs and limiting the formation of immunological synapses. We demonstrated that IIDs block phosphorylation of TCR adapters and downstream signal molecules. Similarly, we showed that IIDs suppress activation of B and NK cells. Furthermore, we demonstrated that EBOV binds to CD4+ T cells through interaction of GP with TLR4 leading to T cell death. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the upregulation of interferon signaling, pattern recognition receptors and NFκB pathways. Transcriptome analysis and specific inhibitors identified apoptosis and necrosis as mechanisms of the T cell death. Lastly, TLR4 antagonist Eritoran protected mice from lethal EBOV challenge by alleviating the “cytokine storm”. Thus EBOV IIDs cause a global suppression of cell-mediated responses, including T, B and NK cells, as a consequence of the deficient DC maturation. In addition, EBOV GP subverts the immune response by triggering lymphopenia through direct and indirect mechanisms and also causes the “cytokine storm”.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 33

EPIGENETIC CONTROL OF INFLUENZA VIRUS MEDIATED BY H3K79 HISTONE METHYLATION: ROLE IN INTERFERON-INDUCED ANTIVIRAL RESPONSE

Laura Marcos-Villar1, 2, Juan Diaz-Colunga1, Noelia Zamarreño1, Ana Falcon1, 2, Amelia Nieto* 1, 2 1Biologia Molecular y Celular, CENTRO NACIONAL DE BIOTECNOLOGIA-CSIC, 2CIBERES, Madrid, Spain

Abstract: Studying possible epigenetic changes elicited by influenza virus infection, we identified a marked increase on methylation of lysine 79 of histone 3 (H3K79). There is a histone methylase named Dot1L, which exclusively methylates H3K79 residue and an inhibitor specific for this histone methylase. The use of this specific inhibitor or the silencing of Dot1L methylase, increase influenza virus replication. In addition, in conditions of Dot1L downregulation there are decreased nuclear translocation of NF-kB complex, and IFN-b, Mx1 and ISG56 expression. In agreement with a role of H3K79 methylation controlling the antiviral signaling, influenza virus replication was unaffected in IFN pathway-compromised Dot1L-inhibited cells. Dot1L down-regulation did not cause significant changes on viral replication of an influenza virus lacking NS1 protein (delNS1), which is unable to counteract the antiviral response. In addition, delNS1 infection caused a lower increase on H3K79 methylation compared with the wild type virus. These results indicate that epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens. Accordingly, the analysis of cellular RNAs expression modified by influenza virus replication in Dot1L down-regulated cells, showed a reduced expression of genes that have a pivotal role controlling the antiviral response.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 34

THE INFLUENZA A VIRUS HOST SHUTOFF RNASE PA-X USURPS HOST MRNA PROCESSING MECHANISMS Denys Khaperskyy1, Brittany Porter1, Lea Gaucherand2, Craig McCormick1, Marta Gaglia* 2 1Microbiology and Immunology, Dalhousie University, Halifax, Canada, 2Molecular Biology and Microbiology, TUFTS UNIVERSITY SCHOOL OF MEDICINE, Boston, United States

Abstract: The influenza A-encoded RNase PA-X modulates innate immune responses. PA-X deficiency exacerbates lung inflammation during infection, but this stronger response fails to clear the virus, instead causing increased morbidity and mortality. A mechanistic understanding of PA-X effects on the cell transcriptome is required to determine how PA-X modulates inflammatory responses. We previously showed that PA-X selectively degrades transcripts that are made by host RNA Pol II and undergo canonical 3’ end processing, and that Pol II transcripts have varying sensitivity to PA-X. We hypothesize that PA-X binds mRNA processing factors to access substrates, and that the differential sensitivity of RNAs reflects differential engagement of this machinery. We are using two approaches to test this hypothesis: transcriptome-wide profiling of PA-X targets and proteomic analysis of PA-X-interacting proteins. By comparing host RNA levels in cells infected with wild-type and PA-X-deficient viruses or overexpressing PA-X, we discovered that RNA splicing is a determinant of susceptibility to PA-X. We found a negative correlation between exon number and RNA levels. Also, intronless RNAs were largely resistant to PA-X. Using proximity-dependent biotinylation, we determined that the PA-X C-terminal domain interacts with several cellular proteins involved in RNA metabolism, particularly splicing and alternative polyadenylation. Together, our transcriptomic and proteomic data support a model in which PA-X interacts with cellular mRNA processing pathways to target subsets of host RNAs. This mechanism of action sets PA-X apart from other viral host shutoff RNases that selectively target mRNAs based on interactions with actively translating mRNAs in the cytoplasm.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 35

TRIM28/KAP1 IS A PKR-CONTROLLED NEGATIVE REGULATOR OF THE INNATE IMMUNE RESPONSE TO HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES Tim Krischuns1, Vanessa Gerlt1, Carolin Nordhoff1, Joschka Willemsen2, Marco Binder2, Sebastian Schloer3, Stephan Ludwig1, Linda Brunotte* 1 1Institute of Virology Muenster (IVM), Westfaelische Wilhelms-University Muenster, Muenster, 2German Cancer Research Centre (DKFZ), Division virus-associated carcinogenesis, Heidelberg, 3Institute of Medical Biochemistry (IMB), Westfaelische Wilhelms-University Muenster, Muenster, Germany

Abstract: TRIM proteins are well known for their multifaceted immune regulatory functions mainly brought by the highly conserved N-terminal ubiquitin-ligase domain. In contrast to most other TRIM family members TRIM28/KAP1 is a transcriptional co-repressor involved in many cellular processes such as DNA-damage response, cell cycle regulation, cancer development, reactivation of retroviruses and also immune regulation. TRIM28 co-repressor activity is alleviated by stress-induced phosphorylation at serines 473 and 827 leading to the expression of repressed genes. Our experimental data provide the first evidence that TRIM28 is a negative regulator of the innate immune response and selectively phosphorylated during infection with the highly pathogenic avian influenza A virus strains A/FPV/Bratislava/79 (H7N7) and a A/Thailand/1(KAN-1)/2004 (H5N1) but not by the human prototype H1N1 strain A/Puerto Rico/8/1934. Interestingly, phosphorylation is induced by viral RNA, however, in a RIG-I-independent manner. In contrast, we could demonstrate that TRIM28 phosphorylation is largely dependent on protein kinase R (PKR) and induction of the stress kinase p38 and its downstream effector MSK1 during viral infection, as evidenced by using CRISPR/Cas9-technology and specific kinase inhibitors. Elevated levels of Type-I-IFNs as well as prototypic pro-inflammatory cytokines in HPAIV-infected TRIM28- KO-cells corroborate the role of TRIM28 as a negative regulator of the innate immune response. We hypothesize that PKR/p38/MSK1-mediated phosphorylation of TRIM28 provides a novel RIG-I-independent signaling pathway contributing to elevated cytokine expression during HPAIV infection. In conclusion, our data indicate an extended role of PKR and TRIM28 as modulators of the innate immune response towards influenza A viruses beyond todays understanding.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 36

H5N1 INFLUENZA A VIRUS PB1-F2 RELIEVES HAX-1-MEDIATED RESTRICTION OF AVIAN VIRUS POLYMERASE PA IN HUMAN LUNG CELLS Béryl Mazel-Sanchez* 1, Ines Boal Carvalho1, Filo Silva1, Ronald Dijkman2, Mirco Schmolke1 1Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, 2Institute of Virology and Immunology, University of Bern, Bern, Switzerland

Abstract: Highly pathogenic IAV from avian hosts were first reported to directly infect humans 20 years ago. However, these are rare events and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB1-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This 90-amino-acid- short peptide does not harbour an enzymatic function. We thus identified host factors interacting with H5N1 PB1-F2, which could explain its importance for virulence. PB1-F2 binds to HCLS1 associated protein X1 (HAX-1), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammalian adapted H1N1 IAV is resistant to HAX-1 imposed restriction while the PA of an avian origin H5N1 IAV remains sensitive. We also showed HAX-1 sensitivity for PAs of A/Brevig Mission/1/1918 (H1N1) and A/Shanghai/1/2013 (H7N9), two avian origin zoonotic IAV. Inhibition of H5N1 PA by HAX-1 can be alleviated by PB1-F2 through direct competition. Accordingly, replication of PB1-F2 deficient H5N1 IAV is attenuated in presence of high amounts of HAX-1. Mammalian adapted H1N1 and H3N2 viruses do not display this dependence on PB1- F2 for efficient replication in presence of HAX-1. We propose that PB1-F2 plays a key role in zoonotic transmission of avian H5N1 IAV into humans.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 37

IDENTIFICATION OF A NEW INTERFERON-INDUCIBLE PROTEIN INHIBITING ENDOSOME MEDIATED VIRAL ENTRY THROUGH REGULATION OF THE VACUOLAR H+-ATPASE

Tomas Doyle1, Olivier Moncorgé2, Darja Pollpeter1, Boris Bonaventure2, Marine Tauziet2, Michael H. Malim1, Caroline Goujon* 2 1King's College , London, United Kingdom, 2IRIM, CNRS, Montpellier, France

Abstract: Type 1 interferon (IFN) induces an antiviral state through the regulation of numerous IFN-stimulated genes (ISGs), with some acting broadly while others display distinctive substrate specificity. Following IFN treatment, influenza A virus (IAV) infection is largely inhibited, and both the MX1 GTPase and the integral membrane protein IFITM3 contribute substantially to suppression. However, these ISGs do not fully account for the IFN-induced restriction of IAV, and we have identified a new ISG playing an important role. CRISPR/Cas9 knock-out of this gene partially rescues IAV infection in the presence of IFN. Conversely, the ectopic expression of this gene potently inhibits IAV infection. Importantly, VSV-G- and rabies-pseudotyped lentiviruses are also inhibited by expression of this gene, in contrast to lentiviruses bearing their natural Env glycoproteins, suggesting that this factor could prevent endosome-mediated entry of viruses. Step-wise dissection of IAV entry showed that this gene does not prevent acid-induced conformational changes in the viral haemaglutinin but inhibits fusion of the viral and endosomal membranes. Critically, combinations of knock-out and overexpression experiments have shown that IFITM3, which also inhibits viral entry, and our gene of interest are functionally independent. Ectopic expression of this gene globally increases the pH of intracellular vesicles and the activity of lysosomal proteases. Finally, interactions with several cytoplasmic subunits of the vATPase and the product of our gene provide support for a model in which IFN- mediated regulation of the vacuolar H+ ATPase (vATPase) restricts IAV infection.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 38

DEFINING HOST RESTRICTION FACTORS THAT MODULATE RESPIRATORY VIRUS ENTRY AND EXIT FROM INFECTED CELLS. Patrick Reading* 1, 2, Sarah Londrigan1, Fernando Villalon-Letelier1 1Department of Microbiology and Immunology, The University of Melbourne, 2WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Australia

Abstract: Acute respiratory tract infections are important causes of morbidity and mortality worldwide, particularly in infants and the elderly. Influenza A virus (IAV), human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are major causes of viral respiratory disease. Respiratory viruses infect airway epithelial cells, resulting in virus amplification and spread. Viruses such as IAV and RSV also infect cells of the immune system, such as airway macrophages (AMΦ), however virus replication is generally blocked in these cells. Recent studies in our laboratory used RNA-seq to investigate differences in expression of host factors between AMΦ and airway epithelial cells, in the presence or absence of IAV infection, in an attempt to identify putative restriction factors that may block virus infection. Based on these results we have focused on particular gene families where certain members were expressed at high levels in AMΦ, but not in epithelial cells. We have used overexpression and/or knockdown approaches to screen families of membrane-associated RING-CH (MARCH) ubiquitin ligases, interferon-inducible transmembrane (IFITM)-family proteins and T-cell immunoglobulin and mucin (TIM)- domain family proteins for antiviral activity against IAV, RSV and HMPV. Preliminary data indicates that overexpression of MARCH8 (but not other MARCH-family proteins) does not alter IAV entry, but does inhibit virus release from infected cells. Moreover, MARCH8 expression also inhibited replication of other respiratory viruses such as RSV. Current studies in the laboratory aim to define the mechanisms underlying the antiviral activity of MARCH8, as well as defining the role of endogenous MARCH8 in limiting respiratory virus infection.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 39

TRIM25 TARGETS EBOLA VIRUS RIBONUCLEOPROTEIN TO SENSITIZE IT TO ZAP-MEDIATED RESTRICTION Rui P. Galao* 1, Harry Wilson1, Kristina Schierhorn1, Franka Debeljak1, Chad Swanson1, Stuart Neil1 1Infectious Diseases, King's College London, London, United Kingdom

Abstract: As part of the early host antiviral defence, RNA viruses are sensed by pattern recognition receptors whose activation triggers a signalling cascade that ultimately results in the expression of type-I interferons (IFN-I), and consequent upregulation of hundreds of IFN-stimulated genes (ISGs). Although Ebola virus (EBOV) has evolved strategies to counteract antiviral responses, we observed that IFN-I treatment of target cells impacted the propagation of EBOV transcription and replication competent virus-like particles (trVLP). Using a well-characterized library of human ISGs we identified TRIM25 as potently restricting EBOV trVLP replication, and its deletion by CRISPR/Cas9 markedly alleviated viral sensitivity to IFN-I. TRIM25 is an E3-ubiquitin ligase known to ubiquitinate the RNA sensor RIG-I to facilitate its interaction with MAVS, thus modulating downstream signalling of the IFN- response. Here, we uncovered an antiviral mechanism for TRIM25 that requires MDA5 and MAVS, but is independent of RIG-I and downstream pro-inflammatory signalling through canonical sensing of cytoplasmic viral RNA. Instead, TRIM25 is recruited to incoming viral particles after cell entry and interacts with the EBOV ribonucleoprotein complex, leading to ubiquitination of the viral nucleoprotein (NP), its own auto-ubiquitination and to a considerable reduction of NP-associated viral genome levels. This is turn sensitizes the virus to the Zinc Antiviral Protein (ZAP), the potency of which can be modulated by the CG dinucleotide content of the trVLP genome. These findings suggest that TRIM25 couples cytoplasmic RNA sensing to direct restriction of EBOV via ZAP. The uncovered antiviral mechanism indicates that TRIM25 may act as a direct antiviral mediator, suggesting that the full scope of TRIM25 antiviral roles is still to be fully defined.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 40

INHIBITION OF THE CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS-ENCODED DEUBIQUITINASE BLOCKS VIRAL REPLICATION Florine E. Scholte* 1, Brian L. Hua1, Stephen R. Welch1, Laura K. McMullan1, Stuart T. Nichol1, Scott D. Pegan2, Christina F. Spiropoulou1, Éric Bergeron1 1Virus Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, 2Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, United States

Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus (Nairoviridae) that can cause a fatal hemorrhagic syndrome. CCHFV has a broad geographic distribution, and no licensed vaccines or effective antiviral treatments are available. The CCHFV L segment encodes a multifunctional protein, containing the viral RNA-dependent RNA polymerase (RdRp), and an ovarian tumor-like (OTU) cysteine protease. The OTU domain can remove ubiquitin (Ub) and ubiquitin-like interferon-stimulated gene-15 (ISG15) conjugates. Ub and ISG15 are involved in post-translational regulation of many signaling pathways, including innate immune responses. Therefore, CCHFV can manipulate the immune responses by reversing Ub/ISG15 conjugation, and we demonstrated that the OTU domain suppresses RIG-I-mediated type I interferon responses during infection. Disrupting OTU activity should block OTU-mediated immune suppression, and consequently enhance immune responses to CCHFV. To address the therapeutic potential of OTU inhibition, we used a synthetic Ub variant (UbV-CC4) previously shown to form high affinity complexes with CCHFV-OTU. We demonstrate that UbV-CC4 completely reversed the immunosuppressive activity of overexpressed CCHFV-OTU, and blockage of OTU during viral infection resulted in a >4-log reduction in wild-type CCHFV infectious titers. In contrast, CCHFV mutants unable to bind Ub were insensitive to UbV-CC4, suggesting that inhibition requires direct binding to OTU. Interestingly, the strong inhibition of wild-type CCHFV replication did not solely rely on enhanced immune responses, as blocking OTU resulted in reduced RdRp activity, likely by affecting L protein levels. Taken together, we demonstrate that targeting CCHFV-OTU robustly inhibits CCHFV replication and is a promising target for antiviral therapies targeting viral RdRps.

NSV 2018, Verona – Abstract Book

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 41

HANTAAN VIRUS DOWNREGULATES CELL SURFACE EXPRESSION OF DEATH RECEPTOR 5 VIA THE 26S PROTEASOME PATHWAY AND INHIBITS TRAIL-MEDIATED INDUCTION OF APOPTOSIS Carles Solà Riera* 1, Shawon Gupta2, Kimia Maleki1, Clas Ahlm3, Hans-Gustaf Ljunggren1, Niklas Björkström1, Jonas Klingstrom1 1Department of Medicine, KAROLINSKA INSTITUTET, Stockholm, Sweden, 2Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany, 3Department of Clinical Microbiology, Umeå University, Umeå, Sweden

Abstract: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is an important factor of our immune system to combat cancer and virus-infected cells. Cytotoxic lymphocytes induce TRAIL-mediated apoptosis to specifically kill targeted cancer and virus-infected death receptor (DR) 4 and DR5 expressing cells. Hantaviruses, order Bunyavirales, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with up to 40% case fatality rates. Increased levels of soluble TRAIL (sTRAIL) have previously been observed in Hantaan virus (HTNV) infected HFRS-patients, and in HCPS-patients. Here we show that HTNV induces TRAIL in endothelial cells, as well as production and secretion of TRAIL by NK cells co-incubated with infected cells. As TRAIL is a potent apoptosis-inducing ligand, we next analyzed the effect of soluble TRAIL on infected cells. HTNV-infected cells strongly resisted TRAIL-mediated apoptosis. When investigating possible mechanisms behind this phenomenon we observed that HTNV causes downregulation of DR5 from the cell surface. Early after infection, an almost complete depletion of cellular DR5 was observed. Production of DR5 was then resumed, including the appearance of the short DR5 isoform. Interestingly, the newly produced DR5 accumulated in lysosomes, not reaching the cell surface. Inhibition of E3 ubiquitin ligase by the chemical SMER-3 blocked the internalization and degradation of DR5 via the 26S proteasome pathway, restoring DR5 to the cell surface. The finding that hantaviruses inhibit TRAIL-mediated apoptosis, together with our previous finding that hantaviruses inhibit cytotoxic granule- mediated apoptosis, shows that these viruses have a strong capacity to inhibit cytotoxic lymphocyte-mediated killing of infected cells.

SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: 42

REPLICATION HETEROGENEITY DRIVES DISTINCT CELLULAR RESPONSES TO INFLUENZA A INFECTION IN VIVO Elizabeth Fay* 1, 2, Louisa Sjaastad3, Jessica Fiege2, 3, Ian Stone3, Matthew Markman3, Marissa Macchietto4, Steven Shen4, Ryan Langlois1, 2, 3 1Biochemistry, Molecular Biology, and Biophysics, 2Center for Immunology, 3Microbiology & Immunology, 4Institute for Health Informatics, University of Minnesota, Minneapolis, United States

Abstract: Influenza A virus (IAV) infects a broad range of cell types within the respiratory tract. The cells initially targeted by the virus in a naïve host are the site of primary replication and virus spread. These cells are difficult to detect using replication competent IAV as the virus rapidly spreads to secondary cells. To overcome this obstacle we utilize a fluorescence expressing single cycle IAV (scIAV) to identify the cells initially infected in the mouse lung. Using this tool, we observed two distinct populations of epithelial cells during the early stages of infection: cells with high virus replication and cells with low virus replication. This suggests that some cells within the lung are innately permissive to virus replication while others are able to blunt replication. We have determined that this phenotype is not due to coinfection of a cell with multiple virions. The level of replication is also not dependent on epithelial cell type. There are distinct cellular pathways that are significantly impacted in each population of infected cells compared to uninfected cells. Additionally, there are subsets of interferon-stimulated genes that are specifically upregulated in each population. These data indicate that different levels of virus replication may activate distinct cellular responses to infection. Fluorescent reporter scIAVs could be used to further elucidate the mechanism of cellular permissibility and the early innate immune response to IAV infection.

NSV 2018, Verona – Abstract Book

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 43

MOLECULAR ANATOMY OF THE INFLUENZA VIRION Naina Nair1, Terry Smith2, Swetha Vijayakrishnan3, Daniel Goldfarb3, Elizabeth Sloan3, David Bhella3, Edward Hutchinson* 3 1Glasgow School of Art, Glasgow, 2University of St Andrews, St Andrews, 3MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom

Abstract: As influenza virions are complex and irregularly shaped, no single method can describe their structure completely. We applied a multidisciplinary approach, combining proteomics, lipidomics, cryo-electron tomography and molecular modelling, to reconcile quantitative data about the virion’s components with their atomic structures and the virion’s overall shape and produce a highly detailed, pseudo-atomic model of the virions of influenza A/WSN/33 virus (WSN).

We first considered the viral envelope. This was relatively crowded, with a tenth of its area occupied by protein transmembrane domains, including low levels of the M2 splice variant M42. Its lipid contained similar levels of phosphatidylcholine and sphingomyelin to cell lysates, with more phosphatidylserine and less phosphatidylglycerol. The envelope’s inner surface area matched the total membrane-binding surface of the viral matrix protein lying beneath it. This area of the bacilliform virions observed budding from cells and the spherical virions often observed in solution was comparable, suggesting that the former can ‘relax’ into the latter.

We next considered the virion interior. This contains a substantial amount of host protein along with the viral genome and the immunosuppressive protein NS1. In budding and bacilliform virions the genome forms a well-ordered array of ribonucleoproteins (RNPs), but we showed that when virions collapse into spheres this must force the RNP array to become disordered.

Integrating qualitative and quantitative data in this way clarified and corrected our understanding of the molecular anatomy of influenza virions. This multidisciplinary approach is applicable to many other complex structures, including other enveloped virions.

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 44

ASSEMBLY AND STRUCTURE OF MEASLES VIRUS NUCLEOCAPSID PARTICLES Sigrid Milles1, Malene R. Jensen1, Ambroises Desfosses2, Guy Schoehn1, Irina Gutsche1, Rob W. Ruigrok* 1, Martin Blackledge1 1IBS, CEA-CNRS-Univ Grenoble Alpes, 2IBS, CEA-CNRS-Univ Grenoble Alpes,, Grenoble, France

Abstract: Paramyxoviridae such as Measles, are important human pathogens. Their RNA genomes are packaged into long helical nucleocapsids, comprising thousands of copies of the nucleoprotein (N) binding the entire genome. The N-RNA complex provides the template for replication and transcription by the viral polymerase complex and as such presents a promising target for viral inhibition. Elucidation of mechanisms regulating transcription and replication have been hampered by the inability to controllably assemble nucleocapsids (NCs). We recently demonstrated self-organization of N into NC-like particles upon addition of RNA to N, chaperoned by a peptide from the viral phosphoprotein (P).1 This offers a tool for investigating NC assembly, as well as establishing a platform for biotechnological applications. NMR and fluorescence spectroscopy reveal biphasic assembly kinetics. Assembly is seen to strongly depend on RNA-sequence, and we use cryo- electron microscopy to determine atomic resolution structures of NCs assembled on different RNA sequences to understand this phenomenon. Full length, tetrameric P comprises a 300 amino acid intrinsically disordered N-terminal domain whose role is poorly understood. An integrated structural dynamics study is used to delineate its importance in NC assembly

1 – Milles, Jensen, Communie, Maurin, Schoehn, Ruigrok, Blackledge. Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence. Angew Chemie Intl Edition, 128, 9502–9506 (2016)

NSV 2018, Verona – Abstract Book

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 45

ULTRASTRUCTURE OF THE INFLUENZA VIRUS RIBONUCLEOPROTEIN COMPLEXES PRODUCING VIRAL RNAS Masahiro Nakano* 1, Keiko Shindo1, Yukihiko Sugita2, Yukiko Muramoto1, Yoshihiro Kawaoka3, Matthias Wolf4, Takeshi Noda1 1Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 2Institute for Protein Research, Osaka University, Osaka, 3Institute of Medical Science, University of Tokyo, Tokyo, 4Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

Abstract: The influenza A virus genome is composed of eight segmented single-stranded negative-sense RNAs (vRNAs). Each vRNA is encapsidated by multiple nucleoproteins (NPs) and an RNA-dependent RNA polymerase to form a ribonucleoprotein complex (vRNP). The vRNP shows a twisted rod-shaped configuration, where an NP-vRNA strand is folded back and coiled on itself to form a double-stranded helix. The vRNA is either transcribed into mRNA or replicated into complementary RNA (cRNA) while complexed with RNPs. However, the configuration of vRNPs while performing these functions remains unknown. Here, we first isolated vRNPs from influenza A virions and confirmed by RT-qPCR that the vRNPs were able to produce both mRNA and cRNA in vitro. Then, we analyzed the configuration of vRNPs during an in vitro RNA synthesis reaction by using high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Two different vRNP configurations associated with newly synthesized RNAs were observed. The vRNPs associated with structured RNA had a twisted rod-shaped configuration similar to vRNPs not undergoing RNA synthesis. In contrast, vRNPs associated with a looped RNA were deformed and did not retain their helical configuration. In addition, it was demonstrated that the looped RNA was double-stranded, and composed of a template vRNA and a progeny cRNA. Our findings provide important insights into viral RNA synthesis by vRNPs and suggest mechanisms for transcription and replication of the influenza virus genome.

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 46

NEGRI BODIES ARE VIRAL FACTORIES WITH PROPERTIES OF LIQUID ORGANELLES Jovan Nikolic1, Cécile Lagaudrière-Gesbert1, Romain Le Bars1, Nathalie Scrima1, Danielle Blondel1, Yves Gaudin* 1 1INSTITUT DE BIOLOGIE INTÉGRATIVE DE LA CELLULE, CNRS, Gif sur Yvette, France

Abstract: Replication of Mononegavirales occurs in viral factories which form inclusion in the host-cell cytoplasm. For rabies virus (RABV), those inclusions are called Negri Bodies (NBs). NBs concentrate both viral nucleoproteins (N) and phosphoproteins (P). They also constitute the site of synthesis of viral RNAs (messengers, antigenomes and genomes). We have demonstrated that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase was confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Co-expression of RABV N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for NB-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.

NSV 2018, Verona – Abstract Book

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 47

NEW TECHNIQUES TO VISUALIZE THE MAMMARENAVIRUS (LCMV) LIFE CYCLE REVEAL A RAB5C-POSITIVE COMPARTMENT AS A POTENTIAL SITE FOR GENOME REPLICATION AND VIRAL PARTICLE PRE-ASSEMBLY Emily A. Bruce* 1, Benjamin R. King1, 2, Christopher M. Ziegler1, Philip L. Eisenhauer1, Daniel Zenklusen3, Jason Botten1 1Medicine, University of Vermont, Burlington, 2Institute for Systems Genetics, Department of Cell Biology, New York University School of Medicine, New York City, United States, 3Departement de Biochimie et Médecine Moléculaire , Université de Montréal , Montréal, Canada

Abstract: While arenaviruses, including lymphocytic choriomeningitis mammarenavirus (LCMV), are known to bud at the plasma membrane, relatively little is known about the late stages of the viral life cycle, including the process of viral particle assembly. We have developed a fluorescence in situ hybridization (FISH) assay and a recombinant LCMV that encodes an HA-tagged matrix protein (Z) to permit the robust detection of genomic RNAs and Z protein respectively, in individual cells. At the peak of viral particle production, we show that the viral S segment genomic RNA, along with viral nucleoprotein, coalesces into a perinuclear foci that is located near the cellular microtubule organizing center and colocalizes with the small GTPase Rab5c and the viral glycoprotein (GPC). In addition, we report that during the peak of infection, the LCMV matrix protein Z is also found concentrated in a perinuclear compartment. In cells stably expressing GFP-Rab5C, the matrix protein colocalizes with GFP-Rab5c in both perinuclear foci and cytoplasmic puncta. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA, glycoprotein, and matrix prior to their transport to the plasma membrane, where new particles will bud. As Rab5c is required for efficient LCMV production and is redistributed during the late stages of LCMV infection, we propose that Rab5c plays a role in coordinating the assembly and/or trafficking of viral components to the site of budding.

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 48

KEY LCMV-HOST INTERACTIONS REQUIRED FOR DEFECTIVE INTERFERING PARTICLE PRODUCTION AND THE HIGHLY DYNAMIC STATE OF VIRAL REPLICATION AND TRANSCRIPTION DURING PERSISTENCE. Christopher M. Ziegler 1, Benjamin R. King2, Emily A. Bruce 1, Philip L. Eisenhauer1, Marion E. Weir 3, David J. Shirley 4, Joseph P. Klaus5, Dimitry N. Krementsov 6, Aubin Samacoits7, Christophe Zimmer7, Daniel Zenklusen8, Florian Mueller7, Bryan A. Ballif9, Jason Botten* 1 1Medicine, University of Vermont, Burlington, 2Cell Biology, New York University School of Medicine, New York, 3 Biology, University of Vermont, 4Data Science, Ixis, Burlington, 5Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 6Medical Laboratory and Radiation Sciences, University of Vermont, Burlington, United States, 7Unité Imagerie et Modélisation, Institut Pasteur, Paris, France, 8Departement de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Canada, 9Biology, University of Vermont, Burlington, United States

Abstract: Lymphocytic choriomeningitis arenavirus (LCMV) causes disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. To better understand how rodent-borne viruses like LCMV maintain a persistent infection without compromising the fitness of the host rodent, we investigated (i) the host and viral machinery needed for the production of defective interfering (DI) particles and (ii) the dynamics of virus genome replication and transcription at the single cell level during persistence. Our studies show that LCMV uses divergent pathways to create infectious virus particles versus DI particles. In particular, LCMV’s only late domain, PPXY, and a functional ESCRT pathway are critical for the production of DI particles, but not for infectious virus. Further, phosphorylation of the PPXY motif appears essential for DI particle production, suggesting that reversible phosphorylation may regulate the rate of DI particle production independent of standard virus. Finally, our smFISH studies suggest that most LCMV-infected cells, despite supporting high levels of virus replication and transcription in the first days following infection, ultimately clear infection as evidenced by a progressive loss of viral RNA and antigen. Further, during persistence, the majority of cells appear to exhibit repeating cyclical waves of viral transcription and replication followed by clearance of viral RNA. Thus, our data suggest that infected cells spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population. Collectively, our studies support a model whereby LCMV spread can be restricted through multiple mechanisms, including (i) host kinase- driven DI particle production and (ii) the establishment of short-lived, transient infections in permissive host cells.

NSV 2018, Verona – Abstract Book

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 49

RESTRICTED INTRANEURONAL TRANSPORT OF FIELD RABIES VIRUS GLYCOPROTEIN Verena te Kamp* 1, Tobias Nolden2, Sabine Nemitz1, Michael Christen1, Luca Zaeck1, Stefan Finke1 1FRIEDRICH-LOEFFLER-INSTITUT, Greifswald - Insel Riems, Germany, 2Vira Therapeutics, Innsbruck, Austria

Abstract: Rabies virus (RABV) is a neurotropic virus that enters the central nervous system (CNS) through infection of peripheral neurons and subsequent trans-synaptic spread to next-order neurons. Beside the common knowledge of exclusively retrograde RABV transport we have shown glycoprotein G dependent anterograde transport of newly replicated virus in axons of peripheral dorsal root ganglion (DRG). However, it is so far unknown whether anterograde transport is limited to peripheral sensory neurons due to neuron specific sorting mechanisms or whether virus strain/isolate specific sequences contribute to the directionality of virus transport in infected neurons. We compared the distribution of different RABV G proteins in peripheral DRG and CNS neurons. G protein of cell culture adapted RABV exhibited efficient surface transport with accumulation at the plasma membranes of cell soma and dendritic/axonal sites. By contrast, infection with RABV field isolate led to accumulation of G in the cell body with only few G particles at plasma membranes of DRG and hippocampus neurons. This indicates that retention and limited surface transport of G protein is characteristic for field virus. By use of chimeric RABV it could be shown, that G protein retention was mediated by the ectodomain. However, adaptation of field virus replication to cell culture by sequential passages led to the identification of three amino acid replacements required for efficient surface transport, counteracting G protein retention. Based on these data we propose a model, in which limited and highly regulated transport of RABV G protein to synaptic budding sites is characteristic for pathogenic field viruses and therefore an important factor in RABV pathogenesis.

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 50

NUCLEOTIDE RESOLUTION MAPPING OF INFLUENZA A VIRUS NUCLEOPROTEIN-RNA INTERACTIONS REVEALS THE LANDSCAPE OF VIRAL RNA FEATURES REQUIRED FOR REPLICATION Graham Williams1, Dana Townsend1, Kristine Wylie1, Preston Kim1, Gaya Amarasinghe1, Sebla Kutluay1, Adrianus Boon* 1 1WASHINGTON UNIVERSITY, Saint Louis, United States

Abstract: Influenza A virus nucleoprotein (NP) associates with all eight negative-sense genomic RNA segments during virus replication. Although the positioning of protein components within viral ribonucleoprotein complexes (RNPs) is defined, the native interaction of NP with the viral RNA (vRNA) and the mechanisms by which a complete multi-segment genome assembles remain elusive. Here, we applied photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP bound short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding was reduced at specific locations within the viral genome including regions previously identified as required for viral genome segment packaging. Synonymous, structural mutations in these low-NP binding regions impacted genome packaging and resulted in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions had no effect. Finally, we demonstrate that the sequence conservation of low-NP binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.

NSV 2018, Verona – Abstract Book

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 51

FINE MAPPING OF INFLUENZA A VIRUS INTRA AND INTERSEGMENT RNA INTERACTIONS Naoki Takizawa* 1 1Laboratory of Virology, INSTITUTE OF MICROBIAL CHEMISTRY (BIKAKEN), Tokyo, Japan

Abstract: Influenza A virus genome consists of eight single-stranded negative-sense viral RNA (vRNA) segments and one set of eight segments is packaged together into a single virus particle. The vRNA is complexed with viral RNA polymerase complexes and nucleoproteins (NP) to form a viral ribonucleoprotein complex (vRNP) and 3′ and 5′ sequences of vRNA are annealed to form a hairpin structure. Classically, the interaction between NP and vRNA is considered to be a uniform and to melt secondary structure of vRNA. However, it has recently been shown that NP dose not bind vRNA uniformly and direct RNA-RNA interactions between segments are required for packaging of the eight vRNA segments. Although electron microscopy studies revealed the architecture of vRNP and configuration of vRNPs in virion, the intra and intersegment vRNA interaction sites are not fully identified. To elucidate intra and intersegment vRNA structures more detail, we employed the mapping RNA interactome in vivo (MARIO) like method for mapping the intra and intersegment proximal RNA sites and the psoralen analysis of RNA interactions and structures (PARIS) like method for mapping the intra and intersegment vRNA interactions. The genome-wide contact maps were generated by both method and intra and inter vRNP architectures were reconstituted form the contact map. While intrasegment architecture of vRNP can be reconstituted from both MARIO and PARIS experiments, intersegment architecture and 7+1 configuration of vRNPs in the virion can be reconstituted only from PAIRS experiment. Our data reveal the segment contact networks by direct RNA-RNA interactions and the role of intersegment interactions for genome packaging will be discussed.

BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: 52

VISUALIZING THE INTRACELLULAR ASSEMBLY OF INFLUENZA VIRAL RNA USING CONFOCAL AND LIGHT SHEET LIVE CELL MICROSCOPE

Seema Lakdawala* 1 1University of Pittsburgh, Pittsburgh, United States

Abstract: Influenza A viruses (IAV) contain eight negative-sense RNA gene segments that replicate in the nucleus, export to the cytoplasm as subcomplexes and travel along Rab11A recycling endosomes to the plasma membrane for budding. However, the role of host cytoskeletal proteins in IAV assembly are still poorly understood. To study viral RNA (vRNA) transport and assembly dynamics within a single cell, we utilized two imaging techniques: 1) multi-color fluorescent in situ hybridization and 2) a custom light-sheet microscope with isotropic resolution along the x, y and z directions. Treatment of cells with nocodazole, a microtubule (MT) depolymerizing drug, did not alter the release of infectious virus but decreased the co-localization of Rab11A and vRNA, suggesting the presense of a Rab11A and MT independent transport mechanism. To determine whether the transport dynamics of vRNA were altered in the absence of MT we used a virus encoding a GFP- tagged polymerase (PA-GFP) protein, as a surrogate for vRNA. Nocodazole treatment only slightly lowered the speed and straightness of PA-GFP movement, yet the speeds were still fast enough to indicate directed movement. This observation demonstrates that MT are not critical for vRNA transport. Additionally, we used cell lines stably expressing a GFP-tagged Rab11A protein to characterize the movement of Rab11A during IAV infection. We observed that nocodazole treatment drastically inhibited the movement of Rab11A. In contrast, IAV infection significantly altered the straightness, speed and arrest coefficient of Rab11A movement in infected cells. In this study, we have used multiple microscopy tools to provide novel insights into the transport and assembly processes of IAV vRNA and have described the presence of a Rab11A independent transport mechanism.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 53

HA STABILITY GOVERNS INFLUENZA A VIRUS REPLICATION, VIRULENCE, HOST RANGE, AND PANDEMIC POTENTIAL Charles J. Russell* 1, Guohua Yang1, Marion Russier2 1Infectious Diseases, St. Jude Children's Research Hospital, Memphis, United States, 2Max Planck Institüt für Biochemie, Martinsreid, Germany

Abstract: Various hosts differ in extracellular and intracellular pH. The influenza A virus HA protein couples changes in pH to activation, but HA acid stability varies by subtype and host species. Seasonal influenza viruses have HA proteins that are generally more stable (pH 5.0-5.6) while exotic viruses like H5N1 and H7N9 are less (pH 5.5-6.2). To dissect the biological impact of HA stability, we generated A/TN/560-1/09 (H1N1) variants including wild-type (pH 5.5), Y17H (6.0), and R106K (5.3). We experimentally infected ferrets, swine, and mice. In ferrets, Y17H lost airborne transmissibility until acquiring mutations that stabilized the HA protein to pH 5.3. This parallels the human 2009 pandemic, during which early isolates had moderate stability (pH 5.5-5.6) and later isolates were stabilized (pH 5.2-5.4). Swine can serve as a bridging host between avians and humans. We found naturally occurring swine isolates range in activation pH from 5.1-6.0. Experimental infections showed swine tolerate a wide range of HA activation pH for growth and transmission (at least pH 5.3-5.8), overlapping the ranges of avian and human viruses. This suggests swine are an ideal host for humanizing HA receptor-binding specificity and HA stability and emphasizes the need for swine surveillance. In mice, the destabilized Y17H variant was attenuated due to susceptibility to extracellular inactivation and diminished antagonization of type I interferon responses in dendritic cells. In addition to being a pH sensor that triggers membrane fusion at the right time and place, our findings show the HA protein needs to shift its stability so that influenza viruses can adapt to unique extracellular and intracellular environments in different host species.

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 54

IMPACT OF OBESITY ON INFLUENZA VIRUS; PATHOGENESIS, VIRAL POPULATIONS AND TRANSMISSION Stacey Schultz-Cherry* 1, Victoria Meliopoulos 1, Brandi Livingston1, Rebekah Honce1, Erik Karlsson2, Elodie Ghedin3 1Infectious Diseases, St Jude Children's Research Hospital, Memphis, United States, 2Pasteur Institute, Phnom Penh, Cambodia, 3New York University, New York City, United States

Abstract: Obesity is a known risk factor for increased disease severity during influenza virus infection. Using diet-induced and genetically obese mouse models our group and others have shown that this increased disease severity is due to enhanced acute lung injury and respiratory distress syndrome leading to pneumonia. We found that the virus spreads more rapidly into the deep lung of obese hosts and persists longer in infected animals; often up to 18 days post-infection. Preliminary data from a human cohort studies suggests that similar trends may be seen in infected people. To better understand how prolonged viral persistence or “shed” impacts the viral populations within obese hosts, we developed a diet-induced obese (DIO) ferret model. Young male ferrets placed on a defined high fat diets gained more weight, had increased visceral fat accumulation, and higher circumference or “BMI” as compared to ferrets fed a regular diet after 6 to 8 weeks. As seen with the obese mouse models, DIO ferrets were more susceptible to influenza infection and had enhanced clinical disease, increased viral titers and spread throughout the lungs as well as prolonged shed. Surprisingly, we found that a virus that was unable to transmit in lean ferrets could transmit even by respiratory routes in one hundred percent of DIO ferrets. Next generation sequencing highlighted a significant change in the viral population of obese animals that may lead to the generation of minor viral variants that are not only capable of enhanced spread but also increased transmission. Studies are underway to define these populations and determine if obese hosts are “super-spreaders” of influenza virus.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 55

MULTIROUTE MORBILLIVIRUS ENTRY: DISEASE INFORMS VACCINE DELIVERY Linda J. Rennick1, Sham Nambulli 1, Natasha L. Tilston-Lunel1, Rory D. de Vries2, Rik L. de Swart2, W. Paul Duprex* 1 1Microbiology, Boston University School of Medicine, Boston, United States, 2Viroscience, Erasmus Medical Center, Rotterdam, Netherlands

Abstract: Morbilliviruses represent some of the most transmissible pathogens on the planet. Attaining an understanding of primary pathogenesis and tropism is critical for the development of novel countermeasures. To extend standard reverse genetics approaches we developed a pipeline for the de novo synthesis of morbillivirus genomes based on sequences obtained directly from clinical material. To dissect multiroute morbillivirus entry we generated virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) and measles viruses (MV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets and macaques. Animals simultaneously received three viruses expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intranasal (IN) or intra-tracheal (IT) inoculation. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections for MV and CDV. Multicolor fluorescence in situ hybridization was used to determine if RNA persisted in vivo following virus clearance, seroconversion and disappearance of clinical signs of disease. We show both morbilliviruses can use multiple entry routes in parallel, that co-infection of cells during viral dissemination in the host is common, that cell-to-cell spread in vivo is the norm and that airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in donor animals. Intranasal infection was inefficient, paralleling what is seen when rMV vaccines are delivered using this delivery route in macaques.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 56

INTERFERON LAMBDA PREVENTS THE SPREAD OF RESPIRATORY VIRUSES FROM THE UPPER RESPIRATORY TRACT TO THE LUNGS AND RESTRICTS VIRUS TRANSMISSION IN MICE Daniel Schnepf* 1, Jonas Klinkhammer1, Liang Ye1, Marilena Schwaderlapp1, Hans H. Gad2, Rune Hartmann2, Dominique Garcin3, Tanel Mahlakõiv1, Peter Stäheli1 1Institute of Virology, Medical Center University of Freiburg, Freiburg im Breisgau, Germany, 2Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 3Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland

Abstract: Host factors restricting the spread of viruses from the upper respiratory tract to the lungs and limiting virus transmission to the next host are poorly characterized. Previous infection studies in which influenza A viruses (IAVs) were administered directly to the lungs of mice suggested a surprisingly minor role of type III interferon in the defense against respiratory viruses. We analyzed the contribution of type I (IFN-α/β) and type III interferon (IFN-λ) using a mouse model in which the virus is selectively administered to the upper airways, mimicking a natural respiratory virus infection. Mice lacking functional IFN-λ receptors (Ifnlr1-/-) no longer restricted influenza or Sendai virus dissemination from the upper airways to the lungs. Ifnlr1-/- mice shed significantly more infectious virus particles via the nostrils and transmitted respiratory viruses much more efficiently to naïve contacts compared with wild-type mice or mice lacking functional type I IFN receptors. Prophylactic treatment with IFN-α or IFN-λ inhibited initial virus replication in all parts of the respiratory tract, but only IFN-λ conferred long-lasting antiviral protection in the upper airways and blocked virus transmission. Thus, IFN-λ has a decisive and non-redundant function in the upper airways that greatly limits transmission of respiratory viruses to naïve contacts.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 57

BREACHING THE OVINE PLACENTAL BARRIER BY RIFT VALLEY FEVER VIRUS Judith Oymans* 1, 2, Paul Wichgers Schreur1, Lucien van Keulen1, Jet Kant1, Jeroen Kortekaas1, 2 1Department of Virology, Wageningen Bioveterinary Research, Lelystad, 2Laboratory of Virology, Wageningen University, Wageningen, Netherlands

Abstract: Rift Valley fever virus (RVFV), an arbovirus of the order Bunyavirales, causes severe disease in ruminants and occasionally humans. Outbreaks, which are thus far mainly confined to the African continent, are characterised by new- born fatalities and abortion storms in ruminants. The capacity of RVFV to transmit from mother to foetus is well recognized, however the exact route, susceptible cells and mechanisms triggering abortion are unknown. We have infected pregnant ewes with RVFV and studied the dissemination of virus at 4, 6 and 7 days post infection (dpi) in both ewe and foetal tissues. We found that the pregnant ewes became viremic at 1 dpi and that viremia peaked at 3 dpi. The foetuses of the ewes which were necropsied at 4 dpi were still alive, while nearly all foetuses of ewes necropsied at 6 and 7 dpi had succumbed to the infection. At 7 dpi, two out of three remaining ewes aborted. Using real-time PCR and immunohistochemistry, we showed that maternal epithelial cells of the placenta are primary target cells of RVFV. Moreover, we observed that RVFV infects foetal trophoblasts when the placental barrier is breached. Interestingly, in half of the dead foetuses viral RNA and viral antigen were detected in liver and brain samples, while in all placentas both the maternal epithelium and the foetal trophoblast layer were infected. This implies that abortion can be caused directly by infection of the foetus, or indirectly by damage to the placenta. Furthermore, these results suggest that infection of the maternal epithelium is the first and most important step in crossing the placental barrier. How this barrier is breached is subject of ongoing research. Graphical Abstract:

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 58

MECHANISMS AND CONSEQUENCES OF CELLULAR SURVIVAL FROM INFLUENZA VIRUS INFECTION Jessica K. Fiege* 1, Ryan A. Langlois1 1Microbiology and Immunology, University of Minnesota, Minneapolis, United States

Abstract: Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects a variety of cells in the respiratory tract causing damage to the lungs. CD8+ T cells are the primary immune cell population responsible for IAV clearance. CD8+ T cells must balance controlling the spread of infection without causing excessive pathology. We have developed an IAV expressing Cre recombinase which permanantly labels infected cells in a Cre- inducible reporter mouse. Using this system, we have identified reporter+ epithelial cells that survive both lytic IAV replication and CD8+ T cell-mediated clearance, and are henceforth termed survivor cells. How survior cells are able control IAV infection and elude the immune response has yet to be elucidated. We are investigating mechanisms by which survivor cells evade killing by CD8+ T cells. Additionally, the role of survivor cells in lung injury and is still unknown. We have observed proliferation of survivor cells, and at later time points after IAV clearance, a larger percentage of survivor cells have divided when compared to their uninfected counterparts. Fluorescent microscopy imaging revealed clusters of survivor cells in the lung in the weeks after IAV clearance, suggesting survivor cells are proliferating to assist in lung recovery. Together these data indicate that CD8+ T cells spare previously infected cells to minimize lung injury and aid in long-term pulmonary recovery.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 59

CHARACTERIZATION OF EBOLA VIRUS DEFECTIVE GENOMES IN THE TESTES OF PERSISTENTLY INFECTED NON-HUMAN PRIMATES Beata Boczkowska1, Taylor Weary* 1, Kendra Alfson1, Jennifer Delgado1, Gloria Rodriguez1, Anthony Griffiths1 1Virology & Immunology, TEXAS BIOMEDICAL RESEARCH INSTITUTE, San Antonio, United States

Abstract: Persistent infection of individuals that survive Ebola virus disease has been reported but occurs at an unknown frequency. Interestingly, there is evidence of sexual transmission via semen from survivors suggesting the involvement of testes, which are an immune privileged site. It is important to understand the molecular basis of persistence to identify improved diagnostics and therapies. Deep sequencing of nucleic acids harvested from testes during the acute phase of EBOV infection showed an enrichment of reads in the trailer region of the genome, which for other viruses has been shown to be characteristic of defective genomes. Given the association of defective viral genomes and persistence, we deep sequenced nucleic acids harvested from the testes of drug treated animals that survived EBOV infection. This is a model used by other groups to study persistent EBOV disease. Sequencing showed some evidence of enrichment of the trailer region but coverage was poor. We amplified this region of the genome by PCR and deep sequenced the product. This permitted identification of defective viral genomes in the testes of >90% of surviving animals. Further characterization identified the break and reinitiation points of copy-back defective genomes. These data suggest that defective viral genomes may play a role in EBOV persistence.

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 60

STUDIES INTO THE MECHANISM OF MEASLES-ASSOCIATED IMMUNE SUPPRESSION DURING AN OUTBREAK OF MEASLES IN THE NETHERLANDS Brigitta M. Laksono1, Rory D. de Vries1, Pieter L. Fraaij2, Menno C. van Zelm3, Wilhelmina L. Ruijs4, Marion P. Koopmans1, Albert D. Osterhaus1, Rik L. De Swart* 1 1Viroscience, 2Pediatrics, 3Immunology, ERASMUS MC, Rotterdam, 4Centre for Infectious Disease Control, National Institute of Public Health and the Environment, Bilthoven, Netherlands

Abstract: Measles is associated with immune suppression, leading to increased susceptibility to opportunistic infections. Based on observations in experimentally infected non-human primates, we previously hypothesised that immune suppression is caused by infection and depletion of CD150+ lymphocytes, referred to as “immune amnesia”. In 2013, a large measles outbreak among unvaccinated individuals in the Dutch Orthodox Protestant community provided a unique opportunity to test this hypothesis in measles patients. We performed an observational cohort study in unvaccinated children aged 4-17 years. Nose- and throat swabs and a blood sample were collected from acute measles patients (cohort A), or paired blood samples were collected from healthy children before and after measles (cohort B). 26 children were included in cohort A, of whom 23 had laboratory-confirmed measles and 14 were sampled before onset of rash. Phenotyping of MV- infected cells in PBMC, by combining lineage markers with intracellular staining of MV nucleoprotein, identified MV-infected B-cells and memory T-cells between five days before and two days after onset of rash. 90 children were included in cohort B, resulting in collection of 77 paired blood samples from children with lab-confirmed measles and 35 paired PBMC samples suitable for multi-colour flow cytometry. We detected a significant reduction in peripheral memory B-cells and increased frequencies of regulatory T-cells and transitional B-cells after measles. These findings support the hypothesis that measles causes immune amnesia.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 61

VIRAL ENTRY PROPERTIES REQUIRED FOR FITNESS IN HUMANS REVEALED THROUGH RAPID GENOMIC CHANGE DURING VIRAL ISOLATION Cyrille Mathieu* 1, Sho Iketani1, Ryan C. Shean2, Marion Ferren1, Negar Makhsous2, Dolly B. Aquino 2, Bert Rima3, Matteo Porotto1, Alexander Greninger2, Anne Moscona1 1Center for Host-Pathogen Interaction, Microbiology & Immunology, Pediatrics, Physiology & Biophysics, Columbia University Medical Center, New York, 2Laboratory Medicine, University of Washington, Seattle, United States, 3Centre for Experimental Medicine, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom

Abstract: Human parainfluenza viruses cause a large burden of respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, parainfluenza 3 (HPIV3) evolves in culture and properties required for fitness in vivo differ when compared to cultured strains. In our genome-wide survey of HPIV3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates, non-synonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins– the receptor binding hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV3 primary culture isolates and 23 HPIV3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization site, a site critical for activation of viral fusion during entry. Alterations in HN dimer interface residues known to differ between cultured and clinical strains occurred rapidly, with H552 and N556 being critical residues for adaptation. A novel cluster of residues at a different face of the HN dimer interface also emerged and imply a role in HPIV3-mediated fusion in vivo. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity, and engineered viruses bearing alterations at these sites reveal the critical nature of these residues for growth in airway. These results highlight the notion that even brief exposure to immortalized cells may affect key viral properties, and underscore the balance of features of the HN-F complex and the sites at the HN dimer interface required for fitness in humans.

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 62

GENETIC DETERMINANTS OF SEVERE RESPIRATORY SYNCYTIAL VIRUS INFECTIONS IN INFANTS Martin Wetzke1, Sibylle Haid2, Chris Lauber3, Daniel Todt2, 4, Lars Kaderali5, Robert Geffers6, Ehsan Vafadarnejad7, Bettina Wiegmann8, Eike Steinmann2, 4, Emmanuel Saliba7, Thomas F. Schulz9, Gesine Hansen1, Thomas Pietschmann* 2 1Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 2Experimental Virology, TWINCORE, Hannover, 3Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, TU Dresden, Dresden, 4Molecular and Medical Virology, Ruhr-University Bochum, Bochum, 5Bioinformatics, University of Greifswald, Greifswald, 6Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, 7Single Cell Analysis, HIRI, Würzburg, 8Cardiothoracic, Transplantation and Vascular Surgery, 9Virology, Hannover Medical School, Hannover, Germany

Abstract: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants. Disease severity varies widely among children and ranges from mild upper respiratory symptoms to severe bronchiolitis. Genetic factors governing disease severity are incompletely defined. 101 children aged between 0-2 years and suffering from severe acute RSV infections were subjected to whole exome sequencing (WES). Since interferon-regulated immune responses are critical for the defense of RSV infections and the course of the disease, we focused on 5142 genes that either trigger expression of interferons, or contribute to interferon signaling or are controlled by interferons. In total 30,039 variants mapped to these genes. Heterozygosity and homozygosity counts in our cohort as well as in an ethnically matched sub-cohort of the Exome Aggregation Consortium (ExAC) were used to calculate the significance of association of variants with severe RSV infection. Collectively, 218 coding polymorphisms mapping to 84 genes were significantly associated with severe RSV infection. Associated genes expressed in primary human airway epithelial cells were silenced and the impact on RSV infection was quantified. Moreover, their expression upon RSV infection of air-liquid interface cultures of human airway epithelial cells was quantified with single cell resolution. More than six novel viral restriction or dependency factors were identified including proteins involved in cellular ER-stress response and regulation of ER-associated protein degradation (ERAD), in inflammatory cytokine signaling and a protein kinase activated by double-stranded RNA which mediates the effects of interferon in response to viral infection. This integrated approach provides a new paradigm for discovery of genetic traits and protein functions affecting the course and outcome of infectious diseases.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 63

THE EGYPTIAN ROUSETTE GENOME, PROVEN HOST FOR MARBURG VIRUS, REVEALS UNEXPECTED FEATURES OF BAT ANTIVIRAL IMMUNITY Gustavo Palacios* 1, Jonathan Towner2, Stephanie Pavlovich3, Mariano Sanchez-Lockhart1, Tom Kepler3 1Center for Genome Sciences, USAMRIID, Frederick, 2Viral Special Pathogens Branch, CDC, Atlanta, 3Boston University, Boston, United States

Abstract: Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection rather than enhanced potency of antiviral defences may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans. Keywords: genome

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 64

BIPHASIC VASCULAR BREAKDOWN AND INFLUX OF NEUTROPHILS INTO THE BRAIN DURING RIFT VALLEY FEVER VIRUS ENCEPHALITIS Amy Hartman* 1, Michael Kujawa1, Joseph Albe1 1Center for Vaccine Research, UNIVERSITY OF PITTSBURGH, Pittsburgh, United States

Abstract: Rift Valley Fever Virus (RVFV) is a vector-borne infection endemic to Africa. However recent outbreaks in the Arabian Peninsula have expanded its potential range. RVFV causes a number of clinical outcomes in people, including hemorrhagic fever and encephalitis with associated morbidity and mortality. Encephalitis is an understudied clinical outcome in people. We use a rat model to better understand the neuropathogenesis of this viral infection. After aerosol infection with the pathogenic ZH501 strain of RVFV, Lewis rats develop a uniformly lethal encephalitic disease with neurological symptoms. The pathogenic events between infection and lethal disease are not well defined. An important question is the timing of breakdown of the brain vasculature in relation to other pathogenic events such as virus invasion and replication, infiltration of immune cells, and inflammatory cytokine expression. We found that early, transient opening of the blood brain barrier in the olfactory bulb occurs concomitantly with the detection of virus in the brain. Using in vivo imaging (IVIS), widespread vascular breakdown in the brain, however, was a late event that corresponded temporally to high levels of virus, expression of matrix metalloproteinase 9 (MMP-9), and an influx of immune cells, which were primarily neutrophils with some macrophages and lymphocytes. There was also evidence of microglia activation. Intracellular staining for viral glycoprotein showed infected cells were neurons, macrophages, and neutrophils. In addition to limiting virus replication, prevention of vascular leakage and infiltration of leukocytes late in infection will likely be an important component for prevention of lethal neurological disease.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 65

CARDIAC DISORDERS AND SUDDEN DEATH CAUSED UPON HEART INFECTION BY HUMAN PATHOGENIC INFLUENZA A VIRUS Jasmina Vasilijevic1, David Filgueiras-Rama2, 3, 4, Jose Jalife2, 3, 5, Sami N. Noujaim6, Celia Gutiérrez1, Noelia Zamarreño1, Jose M. Alfonso2, Alejandro Bernabé2, Christian P. Cop2, Daniel G. León2, Daniel Calle2, 7, Manuel Desco8, 9, 10, Jesús Ruiz- Cabello11, 12, Amelia Nieto1, 12, Ana Falcon* 1, 12 1Molecular and Cellular Biology, National Center for Biotechnology (CNB-CSIC), 2Myocardial Pathophysiology, National Center for Cardiovascular Research (CNIC), 3Cardiovascular Diseases, Center fo Biomedical Research (CIBER), 4Cardiac Electrophysiology Unit, Hospital Clínico San Carlos, Madrid, Spain, 5Center for Arrhythmia Research, University of Michigan, Ann Arbor, 6Morsani College, University of South Florida, Tampa, United States, 7Instituto de Investigación Sanitaria, Hospital Gregorio Marañón, 8Imaging, National Center for Cardiovascular Research (CNIC), 9Bioengineering and Aerospace Engineering, University Carlos III of Madrid, 10Mental Health, Center fo Biomedical Research (CIBER), Madrid, 11CIC biomaGune, IKERBASQUE, Basque Foundation for Science, San Sebastian, 12Respiratory Diseases, Center fo Biomedical Research (CIBER), Madrid, Spain

Abstract: Influenza A virus (IAV) infection has been associated with important cardiovascular complications such as myocarditis, heart failure and acute myocardial infarction, besides respiratory alterations. Sudden death has also been described in infected patients, even without apparent respiratory damage, which could be attributed to heart failure. However, direct infection of cardiac tissue by IAV has been rarely detected. This has led to the assumption that cardiac pathology is derived from lung damage and inflammation caused by the infection. We have explored the relationship between structural and functional cardiac damage and the infective capacity in heart tissue of mice by viruses of different pathogenicity. Heart damage was evaluated by sequential electrocardiograms, cardiac magnetic resonance imaging, connexin43 protein expression and ATP levels in infected versus control animals. We demonstrate that the viruses with higher pathogenicity replicate best in cardiac cell cultures. Moreover, differences in pathogenicity of human viruses did correlate directly with the capacity to replicate in the heart, cause electrical conduction disorders and induce sudden death in infected animals. The data offer a new paradigm toward better treatment(s) of IAV-related heart disease and indicates that direct heart infection should be considered clinically in addition to the indirect effects derived from respiratory pathology. Graphical Abstract:

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 66

ABERRANT VIRAL RNAS LINK AVIAN AND PANDEMIC INFLUENZA VIRUS VIRULENCE TO ERRONEOUS RNA POLYMERASE ACTIVITY Aartjan Te Velthuis* 1, 2, Joshua Long1, David Bauer1, Rebecca Fan3, Hui-Ling Yen3, Jane Sharps4, Marian Killip1, Jurre Siegers5, Maria Jose Oliva-Martin4, Rick Randall6, Emmie de Wit7, Debby Van Riel5, Leo Poon3, Ervin Fodor1 1Pathology, UNIVERSITY OF OXFORD, Oxford, 2Pathology, UNIVERSITY OF CAMBRIDGE, Cambridge, United Kingdom, 3University of Hong Kong, Hong Kong, China, 4UNIVERSITY OF OXFORD, Oxford, United Kingdom, 5Erasmus Medical Centre, Rotterdam, Netherlands, 6University of St Andrews, St Andrews, United Kingdom, 7NIH, Hamilton, United States

Abstract: Infections with highly pathogenic influenza viruses result in a dysregulation of the innate immune response and severe disease. To investigate the molecular mechanism underlying this process, we analysed human lung cells and lung tissues of mice and ferrets infected with the 1918 H1N1 pandemic virus or H5N1 strains. In all experiments, we found a new type of influenza RNA that is <125 nt in length and generated from viral genome segments through a copy-choice mechanism that maintains the 5' and 3' terminal sequences of the genome segments, but deletes internal sequences. We call these RNAs mini viral RNAs (mvRNAs). mvRNAs were preferentially bound by RIG-I over other viral RNAs and extremely potent inducers of the immune response. Moreover, they were only formed by influenza viruses that were not adapted to humans, such as zoonotic avian or pandemic viruses. We believe that these results provide an important advance in our understanding of the molecular basis of influenza virus lethality and that this new RNA is a biomarker for highly pathogenic influenza virus infections in humans.

NSV 2018, Verona – Abstract Book

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 67

INFLUENZA A VIRUS M2 PROTEIN INTERACTS WITH CELLULAR NA,K-ATPASE: DOES IT HAVE A PATHOPHYSIOLOGICAL ROLE IN INFLUENZA PNEUMONIA?

Jessica Schulze* 1, Christin Peteranderl2, Irina Kuznetsova3, Stephan Pleschka3, Susanne Herold2, Thorsten Wolff1 1Unit 17 Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, 2Department of Internal Medicine II, Universities of Giessen and Marburg Lung Center, 3Institute of Medical Virology, Justus Liebig University, Giessen, Germany

Abstract: Influenza A Virus (IAV) infections of the lower respiratory tract can induce viral pneumonia resulting in acute lung injury (ALI/ARDS) with fatal outcome. Characteristics of an IV-induced pneumonia are an alveolar epithelial cell (AEC) damage and accumulation of edema fluid in the alveolar compartment impairing gas exchange. Depending on a sodium gradient established by the basolateral Na,K-ATPase (NKA) and the apical epithelial sodium channel (ENaC) edema fluid is removed from the alveolar space under normal conditions. However after IV-infection a decreased alveolar fluid clearance was observed. In primary AEC it was shown that an IAV-infection leads to a mistargeting of the NKAα1-subunit to the apical cell membrane, but to a reduced NKA expression in the non-infected neighbouring cells. Co-immunoprecipitation studies identified the viral M2 protein as a binding partner of NKAα1. To study the pathophysiological implications of this virus-host interaction we characterized the NKA binding site in the viral M2 protein. In a mutational approach we were able to identify three amino acids in the cytoplasmic tail abutting the transmembrane domain as critical for NKAα1 binding. A recombinant seasonal IAV with impaired NKAα1 binding was slightly attenuated for replication in vitro and ex vivo. Currently the impact of this virus on NKAα1 relocalization and edema clearance is investigated in a polarized Calu 3 cell model. In addition to established roles in intracellular genome release and virus budding our data suggest a further function of the IV M2 protein in relocalizing the cellular NKAα1, which is likely to contribute to pathophysiological effects in IV infection. Investigating the impact of the M2/NKA interaction on the impaired edema clearance could help to better understand this outcome of an IV infection in future.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 68

IN VIVO IMAGING CAPTURES THE PATHOPHYSIOLOGICAL CHANGES AND DYNAMICS OF IMMUNE CELLS IN INFLUENZA VIRUS-INFECTED MOUSE LUNG Hiroshi Ueki* 1, I-Hsuan Wang1, Satoshi Fukuyama1, Hiroaki Katsura1, Tiago J. D. S. Lopes1, 2, Matthias Gunzer3, Gabriele Neumann2, Yoshihiro Kawaoka1, 2 1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States, 3Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg–Essen, Essen, Germany

Abstract: Influenza virus is a respiratory pathogen that causes pandemics and seasonal epidemics. The pathophysiological changes and dynamics of immune cells in influenza virus-infected lungs are poorly understood. In this study, we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity (backbones of H5N1 A/Vietnam/1203/2004 or A/Puerto Rico/8/34 [H1N1; PR8]). This approach allowed us to monitor and correlate several parameters and pathophysiological changes including the spread of infection, pulmonary permeability, perfusion speed, the number of recruited neutrophils, and neutrophil motion in the lungs of live mice. Several pathophysiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared to those in mice infected with a PR8 strain. Time-lapse imaging analysis also revealed that neutrophil movement in the pulmonary capillaries was interlaced with slow (=<50 µm/s) and rapid (>50 µm/s) motions under naïve conditions. In contrast, influenza virus-infected lung showed a temporal increase in pulmonary neutrophil numbers, and neutrophil movement changed to longer durations of the slow motion after the climax of neutrophil recruitment. We also made real- time observations of cell-cell interactions with morphological changes between infected cells and neutrophils or alveolar macrophages infiltrating the alveoli of infected lungs. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infection.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 69

STOPPING MEASLES IN ITS TRACKS - EFFICACY OF AN ORALLY BIOAVAILABLE ANTIVIRAL AGAINST MEASLES VIRUS IN SQUIRREL MONKEYS Kevin Wittwer1, Kristin Pfeffermann1, Danielle Anderson2, Sabine Santibanez3, Annette Mankertz3, Richard K. Plemper4, Veronika von Messling* 1, 2 1Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany, 2Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore, 3Infectious Diseases, Robert-Koch-Institut, Berlin, Germany, 4Biomedical Sciences, Georgia State University, Atlanta, United States

Abstract: Despite ongoing elimination efforts, measles virus (MeV) continues to circulate, and reduced compliance with vaccine recommendations has led to a resurgence of cases and even outbreaks in industrialized countries. Therapeutic options are thus urgently needed. Towards this we assessed the potential of an orally bioavailable small molecule inhibitor of the viral polymerase in squirrel monkeys. These New World monkeys are highly susceptible to MeV infection and reproduce disease severity and clinical signs seen in patients. Animals were treated twice daily in a prophylactic regimen starting the day before or therapeutically at different times after infection with a recent German MeV field isolate. The drug was generally well-tolerated and reached high plasma concentrations. Prophylactic treatment effectively blocked infection, and treatment start in the incubation period greatly reduced or even prevented clinical disease. Treatment after onset of clinical signs had no effect on the course of disease, but stopped virus shedding from the respiratory tract and may thereby reduce transmission. While robust antibody titers were detected in the therapeutic groups, antibody levels in prophylactically treated animals were variable. These results demonstrate promising efficacy and support further clinical development of this candidate drug.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 70

REMDESIVIR (GS-5734) PROTECTS NONHUMAN PRIMATES AGAINST PATHOGENIC FILOVIRUSES Travis Warren* 1, Alison Hogg2, Robert Jordan2, Laura Gomba1, Jessica Weidner1, Veronica Soloveva1, Pamela Wong2, Iva Trantcheva2, Scott Sellers2, Darius Babusis2, Danielle Porter2, Roy Bannister2, Richard Mackman2, Tomas Cihlar2, Sina Bavari1 1US Army Medical Research Institute of Infectious Diseases, Frederick MD, 2Gilead Sciences Inc, Foster City CA, United States

Abstract: The W. Africa Ebola virus (EBOV) outbreak highlighted the lack of approved therapeutics and vaccines for filovirus infection. Remdesivir (RDV) is a monophosphoramidate prodrug of a modified adenosine nucleoside analog with potent in vitro antiviral activity against multiple pathogenic RNA virus families including filoviruses, coronaviruses, and paramyxoviruses. Here, we present key findings from nonhuman primate efficacy studies, in which animals were infected with pathogenic filoviruses and treated with various intravenously administered regimens of RDV.

Key Filovirus Antiviral Efficacy Findings in Nonhuman Primates Virus Challenge RDV Regimen(s)* Treatment Survival(# survivors/# Route Initiation per group) EBOV Makona/2014 IM 10 mg/kg for 12 days 3 days PI Vehicle: 0 (0/6) RDV: 100% (6/6) EBOV Kikwit/1995 IM (A) 10 mg/kg loading dose + 5 mg/kg for 4 days PI Vehicle: 16.7% (1/6) 11 days (A) RDV: 100% (6/6) (B) 5 mg/kg for 12 days (B) RDV: 83.3% (5/6) EBOV Kikwit/1995 Mucosal 10 mg/kg loading dose+ 5 mg/kg for 11 4 days PI Vehicle: 16.7% (1/6) (aerosol) days RDV: 66.7% (4/6)

EBOV Kikwit/1995 IM (A) 10 mg/kg loading dose+ 5 mg/kg for 5 days PI Vehicle: 20% (1/5) 11 days (A) RDV: 50% (3/6) (B) 7.5 mg/kg loading dose+ 5 mg/kg for (B) RDV: 66.7% (4/6) 11 days

MARV Angola/2005 IM 10 mg/kg loading dose+ 5 mg/kg for 11 5 days PI Vehicle: 0% (0/6) days RDV: 83.3% (5/6)

*, all regimens are qd IV. PI, Post infection; IV, intravenous; IM, Intramuscular. Phase 1 studies with RDV in normal human subjects have been completed. The safety and efficacy is currently being investigated in a Phase 2 study in male survivors.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 71

THE MECHANISM OF RESISTANCE TO FAVIPIRAVIR IN INFLUENZA Daniel Goldhill* 1, Pinky Langat1, Robert Fletcher1, Angie Lackenby2, Aartjan te Velthuis3, Wendy Barclay1 1Imperial College, 2Public Health England, London, 3Cambridge University, Cambridge, United Kingdom

Abstract: The evolution of drug resistance is a major problem for currently licensed antivirals for influenza. New antivirals with a high barrier to resistance are needed to treat influenza. Favipiravir is a novel antiviral that has undergone phase III clinical trials and is licensed in Japan to treat pandemic influenza. Favipiravir is a nucleoside analogue which causes mutations. Until now, no resistance to favipiravir has been reported for influenza viruses despite several passaging studies. In this study, we experimentally evolved a prototypical pH1N1 influenza A virus in the presence of favipiravir and identified the first mutations causing drug resistance for influenza. We showed that a single mutation in the polymerase subunit PB1 gave resistance to favipiravir but at a cost to polymerase activity and viral fitness. A mutation in PA compensated for the cost in polymerase activity and restored viral fitness. We used structural modelling, next generation sequencing and in vitro assays to demonstrate the mechanism by which resistance was conferred. We showed that resistant influenza polymerase does not incorporate favipiravir and this prevents favipiravir from causing mutations. We showed that this resistance mechanism was conserved across different subtypes of influenza and propose that this mechanism could be generalised to other viruses.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 72

IDENTIFICATION OF CALCIUM CHANNEL BLOCKER AS AN ANTI-SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS (SFTSV) COMPOUND Shuzo Urata* 1, 2, Jiro Yasuda1, 2 1NAGASAKI UNIVERSITY, INSTITUTE OF TROPICAL MEDICINE, 2National Research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki, Japan

Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV), which has a high mortality rate. SFTSV belongs to Phenueviridae, Bunyavirales. Currently, no licensed vaccines or therapeutic agents have been approved for treating SFTSV infected patients. Our aim is to identify effective compounds which can be used to treat SFTSV infected patients. Out of approximately 650 compounds from an FDA-approved compound library, 6 compounds, which belong to L-type calcium channel blocker, exhibited strong anti-SFTSV effects. We further examined to rule out the target of the calcium channel blocker, Manidipine, on SFTSV replication and propagation. With a time-of addition experiment, we found that it targets replication/transcription step of SFTSV. We also found that this anti-viral effect by Manidipine is not restricted only to SFTSV, but also adopted to other negative strand RNA viruses which belong to different virus families. With the same concentration, the inhibitory effect on infectious SFTSV production by Manidipine was higher than that of T-705 (favipiravir). Inhibiting the activation of calcineurin, a downstream effector of calcium channel, by FK506 and Cyclosporin A, also reduced SFTSV propagation. These results showed that calcium signaling is important for SFTSV replication and could be a target to combat SFTSV. Since calcium channel blockers have been used as a medication to decrease blood pressure in patients with hypertension in clinic, it might be possible to apply these calcium channel blockers to treat SFTS patients. These results also enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 73

BUNYAVIRUS VACCINE DEVELOPMENT BOOSTED BY NOVEL INSIGHTS INTO GENOME REPLICATION AND PACKAGING Paul Wichgers Schreur* 1, 2, Lucien van Keulen1, Jet Kant1, Sandra van de Water1, Nadia Oreshkova1, 2, Judith Oymans1, 3, Jeroen Kortekaas1, 2, 3 1Department of Virology, Wageningen Bioveterinary Research, 2BunyaVax, Lelystad, 3Laboratory of Virology, Wageningen University, Wageningen, Netherlands

Abstract: Bunyaviruses have evolved to replicate in both insects, ticks and mammals. An important step in the bunyavirus life-cycle is the packaging of the tri-segmented RNA genome into maturing virions. Using single-molecule RNA fluorescence in situ hybridization (FISH) we showed that the zoonotic Rift Valley fever virus (RVFV), in contrast to multi-segmented viruses like influenza virus, uses a non-selective genome packaging strategy to produce progeny. Remarkably, the overall efficiency of genome packaging was higher in virus grown in insect cells compared to mammalian cells, suggesting that genome-segment incorporation may differ between hosts. We took advantage of the non-selective nature of RVFV genome packaging and designed two-segmented replicons that lack the M-genome segment but are able to infect and replicate for a single round in vivo when used as a vaccine. These RNA particles are currently developed into a vector platform suitable for counteracting emerging viruses and cancer. In addition, the flexibility in RVFV genome packaging enabled the rescue of four-segmented variants (RVFV-4s). RVFV-4s is based on the splitting of the M-genome segment into two M-type segments either encoding the Gn or the Gc glycoprotein. RVFV-4s is currently used as a RVFV vaccine and induces a strong protective immune response in young sheep. The vaccine was also shown to be safe in pregnant ewes and protected the animals from RVFV induced abortion. Altogether, these studies show that by exploring fundamental aspect of bunyavirus replication and genome packaging effective vaccines can be developed. Graphical Abstract:

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 74

CONVERGENT IMMUNOLOGICAL SOLUTIONS TO ARGENTINE HEMORRHAGIC FEVER VIRUS NEUTRALIZATION Antra Zeltina1, Stefanie A. Krumm2, Mehmet Sahin3, Daniel D. Pinschewer3, Katie Doores* 2, Thomas Bowden1 1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, 2Department of Infectious Diseases, King's College London, London, United Kingdom, 3Division of Experimental Virology, Department of Biomedicine, University of Basel, Basel, Switzerland

Abstract: New World arenaviruses cause substantial public health concerns and economic dangers when transmitted from their rodent reservoirs to humans. Transmission is facilitated by specific interactions between viral attachment glycoprotein, GP1, a key target for neutralizing antibodies (nAb), and the cell surface human transferrin receptor (hTfR1). We present the structural basis for how the mouse-derived nAb OD01 disrupts this interaction by targeting the receptor- binding surface of GP1 from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina, and describe the elicitation of nAbs using a rLCMV system displaying arenaviral GPs and their isolation via B-cell sorting. Comparison of our structure with that of a previously reported nAb complex (JUNV GP1– GD01) reveals overlapping epitopes but highly distinct antibody-binding modes. Both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP1 and mimics the host TfR1 contacts. These data provide a molecular-level description of how antibodies, derived from different germlines, arrive at equivalent immunological solutions to virus neutralization.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: 75

STRUCTURAL BASIS FOR RECOGNITION OF THE CENTRAL CONSERVED REGION OF RSV G BY NEUTRALIZING HUMAN ANTIBODIES

Johannes P. Langedijk* 1, Harrison G. Jones2, Tina Ritschel1, Gabriel Pascual3, Just P. Brakenhoff4, Elissa Keogh3, Polina Furmanova-Hollenstein1, Ellen Lanckacker5, Jay Wadia3, Anthony Williamson3, Dirk Roymans5, Angelique van 't Wout4, morgan Gilman2, Jason McLellan2 1viral vaccines, JANSSEN VACCINES AND PREVENTION, Leiden, Netherlands, 2of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, 3Janssen Prevention Center, Janssen, La Jolla, United States, 4Janssen Prevention Center, Janssen, Leiden, Netherlands, 5Janssen Infectious Diseases, Janssen Pharmaceutica, Beerse, Belgium

Abstract: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections, and yet there remains no effective treatment or vaccine. The virion is decorated with the fusion glycoprotein and the attachment glycoprotein G, which binds to CX3CR1 on human airway epithelial cells. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region have been shown to neutralize RSV and protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated a large panel of broadly reactive human antibodies directed against the central conserved region of RSV G, demonstrated complement-dependent neutralization for both HRSV type A and B and we mapped the epitopes using peptide libraries in full detail and demonstrated several different conserved epitopes covering the complete surface of the central conserved domain. Strong complement-independent neutralization of RSV infection could only be demonstrated in human bronchoepithelial cell cultures. The antibodies protected cotton rats from severe RSV disease. High-resolution crystal structures of two antibodies in complex with the domain revealed two conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with a recently determined structure of fractalkine (CX3CL1) in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. These results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide development of new vaccines and therapies for RSV

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 76

HEADS WIN! HEAD-SPECIFIC B CELLS AND ANTIBODY DOMINATE THE IMMUNE RESPONSE IN A MISMATCHED PRIME-BOOST VACCINE STRATEGY

S Jegaskanda1, 2, S Andrews3, A K. Wheatley2, J W. Yewdell4, A McDermott3, K Subbarao* 1 1Lab of Infectious Diseases, NIAID, NIH, Bethesda, United States, 2Microbiology and Immunology, University of Melbourne, Melbourne, Australia, 3Vaccine Research Center, 4Lab of Viral Diseases, NIAID, NIH, Bethesda, United States

Abstract: Prime-boost vaccination with matched pandemic live-attenuated influenza vaccines (pLAIV) and inactivated subunit vaccine (pISV) establishes long-term immune memory. Here, we asked whether mismatched pLAIV-ISV would generate stem-specific memory B cells (MBCs) and broadly neutralizing antibodies (NAbs). We vaccinated monkeys with H5N1 pLAIV, H5N1 pLAIV-pISV or H5N1 LAIV followed by seasonal trivalent influenza vaccine (TIV); the H1N1 component has a mismatched HA head but conserved stem. We measured serum NAbs and HA-specific B cell responses using HA- specific B cell probes. H5N1 pLAIV elicited both H5+-specific and dual (H5+H1+)-specific B cells, reflecting HA head and stem specificity, respectively in the peripheral blood and mediastinal lymph nodes (LN). The frequency of head-specific MBCs was higher than stem-specific MBCs. Following matched pISV boost, the frequency of both head- and stem-specific B cells increased in the blood, axillary LN and spleen but only H5-specific NAbs were detected. In contrast, mismatched boost elicited a small early expansion of stem-specific MBCs in the periphery but predominantly H5+ or H1+ head-specific B cells in the LN. Stem-specific germinal centre B cells were predominantly found in the spleen and peripheral blood. However, broadly NAbs were not detected. Thus, mismatched boost generated a higher frequency of head- than stem- specific MBCs and did not induce broadly cross-neutralizing Abs. These results have significant implications for vaccine strategies that aim to generate stem-specific NAbs but include the HA head.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 77

THE POTENTIAL OF HUMAN MONOCLONAL ANTIBODIES THAT RECOGNIZE THE INFLUENZA A(H1N1)PDM09 VIRUS HEMAGGLUTININ RECEPTOR-BINDING SITE AS ANTI-INFLUENZA AGENTS. Atsuhiro Yasuhara* 1, Seiya Yamayoshi1, Maki Kiso1, Shinya Yamada1, Yoshihiro Kawaoka1, 2, 3 1Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2School of Veterinary Medicine, University of Wisconsin- Madison, Madison, United States, 3International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Abstract: Antiviral therapy can reduce the burden of seasonal influenza and provides the first line of defense against pandemic influenza before vaccines are available. However, because influenza virus rapidly mutates to escape from neutralizing antibodies, one of the main disadvantages of monoclonal antibodies (mAbs) as antiviral treatments is the potential emergence of escape mutants. Here we isolated three human mAbs from volunteers vaccinated with seasonal influenza vaccine or H5N1 pre-pandemic vaccine, and characterized them by evaluating their in vivo protective efficacy, generating escape mutant viruses, and examining the fitness of the escape mutants in vitro. We found that all three mAbs possessed high in vivo efficacy against lethal infection of mice with A(H1N1)pdm09 virus. Mutant viruses that escaped from these mAbs rarely appeared, and the escape mutations were identified in conserved residues of the receptor-binding site (RBS) of hemagglutinin (HA). Furthermore, the escape mutant viruses showed significantly lower replication in vitro than their parental viruses, indicating that the mutant viruses would be unlikely to dominate. These results suggest that mAbs that recognize the functionally conserved residues within the RBS rarely produce escape mutant viruses. Such mAbs could fulfill the need for anti-influenza agents with a low propensity for the emergence of escape mutants.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 78

COORDINATE DE-IMMUNIZATION OF MEASLES VIRUS ENVELOPE GLYCOPROTEINS DRIVES THE EMERGENCE OF ANTIGENIC VARIANTS Miguel Ángel Muñoz Alía* 1, Claude P. Muller2, Stephen J. Russell1 1Molecular Medicine, Mayo Clinic, Rochester, United States, 2Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette, Luxembourg

Abstract: Measles virus (MeV) is monotypic and in 70 years measles genotypes evading the vaccine-driven polyclonal response against the virus surface glycoproteins H and F have not emerged. The basis of the remarkable antigenic stability in the measles coat remains unknown. We here show that systematic ablation of 30 known epitopes from the 8 known antigenic sites on the MeV H glycoprotein resulted in resistance to neutralization by anti-H reactive polyclonal antibodies in H-immune sera from humans, mice, rabbits and guinea pigs. Viruses incorporating the fully mutated H glycoprotein interacted poorly with MeV receptors SLAM and Nectin-4, leading to virus entry and syncytia formation solely via receptor CD46, due to an increased in binding avidity. MeVs incorporating the fully de-immunized H protein could still be efficiently neutralized by MeV-immune sera not depleted of anti-F antibodies, underscoring the importance of F-reactive antibodies in protective anti-MeV immune responses. Substitution of MeV F for the homologous F from Canine Distemper Virus, resulted in the generation of MeV Stealth, which behaved as a new MeV serotype, showing increased resistance to neutralization by serum from vaccinated humans or from previously MeV infected subjects. All viruses had the Moraten vaccine backbone and were considered BSL2 agents. Our work sheds new light on the mechanisms that constrain the plasticity of the MeV coat, and helps to explain why global vaccine coverage has not led to the emergence of new serotypes. Also, the MeV Stealth here described may aid to close the gap of measles susceptibility in infants, allowing effective vaccination in the presence of transplacentally transferred maternal anti-MeV antibodies during the first few months of life, and may further broaden the applicability and safety properties of MeV-based cancer therapies in the clinic. Graphical Abstract:

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 79

LIVE-ATTENUATED RESPIRATORY SYNCYTIAL VIRUS (RSV) VACCINE CANDIDATE WITH NS2 DELETION AND GENETICALLY STABILIZED TEMPERATURE SENSITIVITY MUTATION IS SAFE, IMMUNOGENIC, AND GENETICALLY STABLE IN RSV SERONEGATIVE CHILDREN Ursula Buchholz* 1, Cindy Luongo1, Ruth Karron2, Peter Collins1 1RNA Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD, 2Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States

Abstract: Respiratory syncytial virus (RSV) is the leading viral cause of pediatric respiratory disease, and there is no vaccine. Non-replicating RSV vaccines are contraindicated for RSV naïve recipients due to the risk of priming for enhanced disease upon subsequent infection with community-acquired RSV. Live intranasal vaccines do not prime for enhanced RSV disease, but development is challenging, because immunogenicity is thought to be inversely correlated with attenuation. We generated the RSV vaccine candidate ∆NS2/∆1313/I1314L by reverse genetics, combining a deletion of the RSV interferon antagonist NS2 with the genetically stabilized temperature-sensitivity mutation ∆1313/I1314L in the L ORF, increasing vaccine safety by limiting replication at higher temperatures of the lower respiratory tract. In a Phase 1 study in 6-24 month-old RSV-seronegative children, ∆NS2/∆1313/I1314L was poorly infectious at a low dose of 105 PFU [10 vaccinees (V)/5 placebo recipients (P)]. At a dose of 106 PFU (20V/10P), 80% and 90% of recipients shed vaccine detected by culture and RT-qPCR, respectively (median peak titers 1.7 log10 PFU/mL; 3.6 log10 copies/mL), and 90% and 80% had ≥4-fold increases in RSV F IgG and neutralizing serum antibody titers, respectively. Antibody titers were durable over the following RSV season, and the vaccine primed for strong anamnestic responses to wild-type RSV. No mutations were detected in vaccine isolates, showing that the vaccine was genetically stable. These results put ∆NS2/∆1313/I1314L into the window between previous over- and under-attenuated NS2 candidates, and on fast track for larger studies.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 80

RESPONSE TO RE-EMERGENCE OF INFLUENZA A(H7N9) VIRUSES David Wentworth* 1, Guaniri Mateu-Petit1, Li Wang1, Xudong Lin1, Adam Johnson1, Terianne Wong1, Jaber Hossain1, Pavani Bondugula1, Yunho Jang1, Joyce Jones1, Sharmi Thor1, Genyan Yang1, Han Di1, Erin Hodges1, Claudia Pappas1, Nicole Brock 1, Xiangjie Sun1, Terrence Tumpey1, Taronna Maines1, James Stevens1, Bin Zhou1, C. Todd Davis1, Vivien Dugan1 1Influenza Division, Centers for Disease Control and Prevention, Atlanta, United States

Abstract: A zoonotic lineage of influenza A(H7N9) viruses first emerged in China in February 2013 and subsequently caused annual epidemic waves resulting in 1,567 human infections (as of 26 Feb 2018). A dramatic increase in zoonotic infections (n=766), geographic dissemination, and genetic diversification of the hemagglutinin (HA) was observed in the 5th wave (2017/2018). The HA evolution generated antigenic variants that escaped neutralization from antisera to existing vaccines and gave rise to a subclade of highly pathogenic viruses. Risk assessment indicated the 5th wave H7N9 viruses posed a considerable pandemic threat, therefore new candidate vaccine viruses (CVVs), against both low pathogenic viruses (i.e., A/Hong Kong/125/2017-like) and high pathogenic viruses (i.e. A/Guangdong/17SF003/2016-like), were recommended at the WHO influenza vaccine consultation meeting in March 2017. Good laboratory practice and reverse genetics approaches were used to engineer multiple candidate vaccines designed to increase HA protein yield and/or breadth of host immune response. Extensive characterization, including antigenic analysis, genetic stability, and ferret pathogenicity studies were rapidly conducted. The IDCDC-RG56B CVV developed against the predominant 5th wave viruses (A/Hong Kong/125/2017-like), was available to vaccine manufacturers worldwide in May 2017. Another CVV (IDCDC-RG56N) that showed increased HA yield and induced cross-reactive antisera to both low- and high pathogenic viruses became available for distribution in November 2017. Finally, human clinical trials to analyze the response to an inactivated vaccine generated from IDCDC-RG56B were initiated in February 2018.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 81

H1N1 VACCINE EFFECTIVENESS IN LIVE ATTENUATED INFLUENZA VACCINE IS DRIVEN BY VIRAL REPLICATIVE FITNESS IN THE HUMAN RESPIRATORY TRACT: SELECTION OF A NEW AND IMPROVED VACCINE CANDIDATE Oliver Dibben* 1, Amy Hawksworth1, Jonathan Crowe1, Robert Lockhart1, Shaun Cooper1, Lydia Ritter1, Kasia Schewe1, Laura Hill1, Andrew Nyborg2, Raburn Mallory2, Helen Bright1 1Flu-BPD, MEDIMMUNE, Liverpool, United Kingdom, 2Clinical Development, MEDIMMUNE, Gaithersburg, United States

Abstract: In the 2013/14 and 2015/16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the pandemic 2009 H1N1 (H1N1pdm09) component of the quadrivalent live attenuated influenza vaccine (Q/LAIV). This resulted in the hypothesis that H1N1pdm09 LAIV strains might possess a replicative defect, relative to clinically efficacious pre-2009, seasonal H1N1 (pre-2009) strains. A broad-based investigation was launched in response to this concern. Initial observations comparing pre-2009 and H1N1pdm09 LAIV strains in single and multi-cycle infectivity assays indicated that H1N1pdm09 LAIV suffered from reduced multi-cycle infectivity. A fully differentiated primary human nasal epithelial cell model then confirmed that the replicative fitness of clinically suboptimal H1N1pdm09 LAIV strains was reduced. To assess the impact of this change in vivo, a novel ferret protection model was developed. Using this model, it was shown that the reduced fitness of suboptimal 2015/16 H1N1pdm09 vaccine strain, A/Bolivia/559/2013 (BOL13), led to a marked reduction in protective efficacy, relative to pre-2009 A/New Caledonia/20/1999. Importantly, BOL13 also showed a pronounced reduction in efficacy in trivalent (T/LAIV) and Q/LAIV formulations when compared to monovalent vaccination. This suggested that incorporation of a poorly fit H1N1pdm09 strain into a T/LAIV or Q/LAIV vaccine could result in inter-strain interference, leading to reduced VE. Using these tools, a new H1N1pdmo09 LAIV strain, A/Slovenia/2903/2015 (SLOV15), with improved replicative fitness and protective efficacy in ferrets, was developed. Finally, the benefits of these improvements were clinically validated by demonstrating significantly improved SLOV15 Q/LAIV shedding and immunogenicity relative to suboptimal BOL13 in US children.

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 82

LASSA VIRUS GLYCO-PROTEIN1 CONFORMATIONAL CHANGES – A POSSIBLE ROLE FOR IMMUNE EVASION Aliza Katz* 1, Ron Diskin1 1Structural Biology, WEIZMANN INSTITUTE, Rehovot, Israel

Abstract: Lassa virus (LASV) of the Arenaviridae family is the causative of fatal hemohrahgic fever (FHF) in West Africa. These membrane coated viruses have a single surface protein complex which is referred to as the glyco-protein complex (GPC). The GPC is arranged as a trimer where each protomer is capped with the Glyco Protein 1 (GP1), a protein responsible for host cell receptor recognition and internalization. The GPC serves as an immunogene once encountered by the immune system. Obtaining a vaccine and treatment against this pathogen is a high priority goal due to its fatality and therefore also harboring the danger of it being exploited as a biological weapon. GP1 of LASV was purified and injected into mice to test immunogenicity along side with GP1 of Junin Virus (JUNV), another pathogenic Arenavirus. Both GP1LASV and GP1JUNV were immunogenic in mice yet only GP1JUNV immunized sera can neutralize pseudo viruses and recognize GP1 in the integral GPC. The structure of GP1LASV was solved by our lab and more recently the GPCLASV became available showing conformational changes between the GP1 component in the loose form and as part of a complex. Based on our results of the GP1 being immunogenic yet not neutralizing or recognizing the GP1 on the GPC, we suggest that the GP1LASV serves as an immunological decoy to raise irrelevant antibodies. Interestingly, soluble GP1LASV was detected in LASV patients supporting our hypothesis of loose GP1 serving as decoy. Elucidating the mechanism of LASV immune evasion will further help eradicate this deadly illness.

NSV 2018, Verona – Abstract Book

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: 83

MEASLES WILD TYPE AND VACCINE VIRUSES: REPLICATION AND IMMUNE RESPONSES IN RHESUS MACAQUES Diane E. Griffin* 1, Wen-Hsuan W. Lin1, Robert J. Adams2 1Molecular Microbiology and Immunology, JOHNS HOPKINS BLOOMBERG SCHOOL OF PUBLIC HEALTH, 2comparative medicine, Johns Hopkins University School of Medicine, Baltimore, United States

Abstract: Measles wild type and vaccine viruses: Replication and immune responses in rhesus macaques Wen-Hsuan W. Lin, Robert Adams and Diane E. Griffin W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205

Infection with wild type measles virus (wt-MeV) is an important cause of childhood mortality and also induces life-long protective immunity. Despite the antigenic similarities between wt-MeV and live-attenuated measles virus vaccine (LAMV), the protective immunity after measles vaccination is not as robust or long-lived. To identify mechanisms that may contribute to the longevity of immune responses induced by wt-MeV infection, we compared virus replication and immune responses after respiratory infection with wt-MeV or LAMV in rhesus macaques. Analysis of viral RNA in various tissues revealed distinct in vivo tropisms between the two strains of virus. Wt-MeV caused efficient hematogenous spread and prolonged presence of viral RNA, while LAMV caused transient localized infection in the respiratory tract. Type I interferon was not induced by either wt-MeV or LAMV, but early after infection with wt-MeV plasma levels of cytokines IL-12 and MIF and chemokines CCL2, CCL11, CCL22 were elevated. Infection with wt-MeV and LAMV induced comparable levels of MeV- specific IFN-g-producing T cells, but significantly higher levels of neutralizing and anti-H antibody and more long-lived plasma cells were observed after wt-MeV infection. Therefore, distinct patterns of in vivo virus replication, efficiency of hematogenous spread and persistence of viral RNA are associated with the altered virulence and the level and longevity of the humoral response.

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 84

MEGATAXONOMY OF NEGATIVE-SENSE RNA VIRUSES: PHYLUM _NEGARNAVIRICOTA_ Jens H. Kuhn* 1 1NIH/NIAID/DCR, INTEGRATED RESEARCH FACILITY AT FORT DETRICK, Frederick, United States

Abstract: (-)ssRNA viruses, informally grouped in “Baltimore class V”, are currently classified in the orders Bunyavirales and Mononegavirales, the unassigned families Arenaviridae, Ophioviridae, and Orthomyxoviridae, and the unassigned genus Deltavirus. Recent metagenomic studies revealed the existence of hundreds of novel (-)ssRNA viruses in highly divergent organisms. These viruses either cluster with the existing taxa (e.g., bunyaviruses in citrus, melon, and kinetoplastids; hantaviruses in fish) or are highly divergent (e.g., chǔviruses, qínviruses, yuèviruses). With exception of hepatitis D virus (genus Deltavirus), all these viruses are homophyletically connected via their RNA-dependent RNA polymerases. This connection led to a proposal to the International Committee on Taxonomy of Viruses (ICTV) to replace the unofficial Baltimore class V with a phylum that unifies all current taxa and contains the necessary ranks for classification of all currently described (-)ssRNA viruses excluding hepatitis D virus. I will outline the phylum as recently proposed, present foreseeable and imminent changes to the phylum based on novel discoveries, and discuss current taxonomic challenges. I will explain the taxonomic process from official proposal writing via Study Group deliberations to ICTV Executive Committee decisions. Finally, I will call on the entire (-)ssRNA virology community to become engaged in this process to ensure that classification and nomenclature of all (-)ssRNA viruses is done swiftly, as accurately as possible on an annual basis, and with maximum input from all experts in a transparent manner.

NSV 2018, Verona – Abstract Book

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 85

ISOLATION, CHARACTERIZATION, AND RAPID ADAPTATION OF TICK-BORNE ARENAVIRUS Hector Moreno Borrego* 1, Alberto Rastrojo Lastras2, Katherine Sayler3, Stefan Kunz1 1Microbiologie, Centre Hospitalier Universitaire Vaudoise (CHUV), Lausanne (Switzerland), Switzerland, 2Department of Virology, Centro de Biología Molecular Severo Ochoa, CBMSO-CSIC, madrid, Spain, 3Department of Wildlife Ecology and Conservation, UF/IFAS, University of Florida UF/IFAS, Gainesville, United States

Abstract: The New World Arenaviruses are diverse family of emerging negative strand RNA viruses comprised of Clades A, B, C, and D (former rec A/B). North American Clade D viruses, including Whitewater Arroyo (WWAV), Tamiami (TAMV), Bear Canyon, Skinner Tank, and Catarina virus, are carried in nature by members of the Sigmodontinae and Neotominae subfamilies of Cricetidae rodents. TAMV was isolated in 1970 from the cotton rat Sigmodon hispidus in Florida and clusters phylogenetically closely with WWAV, which has been associated with human disease. Recent field studies resulted in the first isolation of the New World Arenavirus Tacaribe (TCRV), from host-seeking Amblyomma americanum ticks in Florida. Using next generation sequencing (NGS), we detected genetic traces of TAMV (TAMV-FL) in these tick-derived isolates. The ability of TAMV to prevent super-infection of TCRV but not vice versa, allowed enrichment of TAMV with concomitant extinction of TCRV by serial passaging in human cells, evidenced by NGS and immunofluorescence. The new tick-borne TAMV-FL isolate shares only 85% of homology with available TAMV reference sequence, suggesting strong selection and adaptation, and excluding the possibility of a laboratory contamination. During serial passaging, we further observed rapid de novo selection of two mutations (T569A and G582A) located in the putative interaction region between the TAMV envelope glycoprotein and its cellular receptor, transferrin receptor 1, suggesting rapid human adaptation. In summary, this study provides first evidence for a more dynamic host range of TAMV that may include ticks as transmission hosts and reveals the capacity for rapid human adaptation. Graphical Abstract:

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 86

CHARACTERISATION OF A NOVEL PARAMYXOVIRUS ISOLATED FROM PTEROPID BAT URINE Rebecca Johnson* 1, 2, Mary Tachedjian1, Hans Netter3, Glenn A. Marsh1 1CSIRO Health and Biosecurity, Australian Animal Health Laboratory , Geelong, 2Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, 3Victorian Infectious Diseases Reference Laboratory, Melbourne Health, Melbourne, Australia

Abstract: The role of bats as reservoir hosts for zoonotic pathogens makes the discovery of novel bat-borne viruses necessary to prepare for emerging infectious diseases. Characterisation of these viruses improves our understanding of their zoonotic potential and prevents virus spillover from having a significant public health and economic impact. Previous surveillance of Australian fruit bats has resulted in the identification of multiple paramyxoviruses, therefore pteropid bat urine was analysed for the presence of novel viruses. This led to the isolation of Alston paramyxovirus (AlsPV), which allowed further characterization of the virus to better understand its potential to cause disease.

Whole genome sequencing showed that AlsPV belongs to the genus Rubulavirus and is closely related to Parainfluenza virus 5, the causative agent of respiratory disease in dogs. Experimental infection of ferrets and mice resulted in the shedding of infectious virus in respiratory secretions of ferrets between days 3-10 with a rise in neutralizing antibodies detected after day 10. AlsPV could be isolated from upper respiratory tract tissues and viral RNA could be detected in the brains of all infected ferrets between days 3-10 as well as the brains of two mice at day 21 post infection in the absence of clinical symptoms.

The isolation of AlsPV provides a greater understanding of the viral diversity present in Australian bats. These results show that AlsPV replicates in ferrets at sites relevant to virus transmission in addition to infecting the central nervous system. AlsPV has the potential to cause zoonotic infection, indicating the need to learn more about the transmission of this virus and highlighting the importance of the surveillance of bats as a source of emerging viruses.

NSV 2018, Verona – Abstract Book

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 87

INVESTIGATION OF THE BIOLOGICAL ROLE OF AN ENDOGENOUS BORNAVIRUS-LIKE ELEMENT IN MINIOPTERID BAT GENOMES Mukai Yahiro* 1, 2, Horie Masayuki1, 3, Yuki Koboyashi4, Kojima Shohei1, 2, Maeda Ken5, Tomonaga Keizo1, 2 1Institute for Frontier Life and Medical Sciences, Kyoto University, 2Graduate School of Biostudies, Kyoto University, 3Hakubi Center for Advanced Research, Kyoto University, Kyoto, 4Nihon University Veterinary Research Center, Kanagawa, 5Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan

Abstract: Endogenous bornavirus-like N elements (EBLNs) are DNA sequences derived from the N genes of ancient bornaviruses present in diverse mammals. Interestingly, some EBLNs have been co-opted to encode functional proteins by their hosts. However, the biological significance of EBLNs, especially in non-model organisms, are largely unknown. In this study, we searched EBLNs in bats that potentially encode proteins, and investigated their biological functions. We found that an EBLN in the natal long-fingered bat (Miniopterus natalensis), named miEBLN-1, retained an intact ORF that negative selection has operated. We further showed that miEBLN-1 is expressed as an mRNA in many tissues. These strongly indicate that miEBLN-1 encode a functional protein in the bats. To understand its function, we first evaluated the anti-bornaviral effect of miEBLN-1 because an EBLN in the squirrels inhibits BoDV replication. We revealed that miEBLN- 1 inhibits neither the activity of BoDV polymerase nor BoDV infection. We next performed proteomic analyses to identify interaction partners of miEBLN-1. We found that miEBLN-1 interacts with several RNA-binding proteins, such as MOV10 and IGF2BP3 in an RNA-dependent manner. We are currently investigating the biological significance of these interactions. The bornaviral N protein binds to viral RNA to form the nucleocapsid. Our data suggest that miniopterid bats had acquired a bornaviral N gene and may have utilized its RNA-binding property for their adaptation. Thus, this study would propose a novel concept that mammals can acquire riboviral genes and utilize their original properties for cellular functions.

DAMAGING AND SPREADING – pathogenesis Abstract final identifier: 88

NIPAH VIRUS W PROTEIN MODULATES THE NF-ΚB SIGNALING PATHWAY BY TARGETING A HOST SCAFFOLD PROTEIN

François Enchery1, Claire Dumont1, Aline Linder1, Noemie Aurine1, Louis-Marie Bloyet1, Cyrille Mathieu2, Denis Gerlier1, Chloe Journo1, Branka Horvat* 1 1Interanational center for Infectiology Research-CIRI, Lyon, France, 2Center for Host-Pathogen Interaction, Department of Pediatrics, Columbia University Medical Center, New York, United States

Abstract: Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus responsible for regular outbreaks in South-East Asia and associated with high mortality and human-to-human transmission. NiV produces three nonstructural proteins, C, V and W, known to function as virulence factors. NiV-W was shown to inhibit the chemokine response in vitro and to modulate the inflammatory reaction in vivo, however, the mechanism of its action remains unclear. Here, we report that the NiV-W protein represses IL-1β and TNFα-induced activation of the canonical NF-κB signaling pathway, known as the principal regulator of inflammation. This function requires the C-terminal domain of W, and is dependent on the nuclear accumulation of W. In contrast to wild-type NiV, inhibition of the NF-κB pathway was found to be impaired upon infection with a recombinant W-deficient NiV. Moreover, using a mass spectrometry approach, we identified an intracellular partner of NiV-W, which functions as a cellular scaffold protein playing an important role in intracellular signaling, including in the negative feedback regulation of NF-κB. We further identified a specific mutation within NiV-W that leads to the loss of interaction with this intracellular partner, and, as a consequence, of the capacity of W to inhibit NF-κB. This mechanism gives an additional insight into NiV-W-induced inhibition of chemokine production and leukocyte recruitment. Altogether, our results suggest that the W protein regulates the NF-κB pathway and consequently modulates the NiV-induced inflammatory response, paving the way toward a better understanding of the immunopathogenesis of this severe viral infection.

NSV 2018, Verona – Abstract Book

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 89

AVIAN INFLUENZA VIRUS M SEGMENT GENE EXPRESSION IS DYSREGULATED IN MAMMALIAN CELLS, LEADING TO A BLOCK IN AUTOPHAGY AND RESTRICTED VIRAL GROWTH John Steel* 1, Shamika Danzy1, Ketaki Ganti1, Brenda M. Calderon1, Anice C. Lowen1 1Microbiology and Immunology, EMORY UNIVERSITY, Atlanta, United States

Abstract: Influenza A virus (IAV) M segment encodes matrix and M2 proton channel proteins from alternatively spliced mRNAs, but the contribution of these products to host adaptation remain unclear. To investigate, we generated isogenic viruses that encode avian-, swine, or human-derived M segments. In a guinea pig model, avian M-encoding viruses exhibited poorer infectivity, lower titers, slower growth and less efficient transmission than viruses possessing a mammalian M segment. Avian and mammalian cell culture data revealed growth restriction of viruses with avian M segments specifically in mammalian cells. As decreased replication correlated with increased expression of M2 relative to M1, we determined whether altered fitness was due to over-expression of M2, or to amino acid differences that exist between human- and avian-adapted M1 or M2 proteins, using viruses encoding chimeric M segments. We separated synonymous and non- synonymous changes to produce viruses encoding human M1 and/or M2 proteins on avian vRNA background, or avian M1 and/or M2 proteins on human vRNA background. Growth of viruses in mammalian cells was inversely correlated with M2 expression, irrespective of amino acid composition. Analysis of LC3B lipidation and visualization of autophagosomes in infected cells further suggested that high levels of M2 lead to autophagic block. Our data suggest that adaptive change(s) in the M segment are needed to maintain low expression of M2 in mammalian cells and that, in the absence of such changes, excess M2 limits viral growth by blocking autophagy. These results point to novel roles for regulation of viral gene expression and interaction with cellular autophagy components in the host species range of IAV.

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 90

ASSESSMENT OF THE ZOONOTIC POTENTIAL OF THE BAT INFLUENZA A VIRUS H18N11 Kevin Ciminski* 1, Wei Ran1, Jinhwa Lee2, Marco Gorka3, Ebrahim Hassan1, Anne Pohlmann3, Donata Hoffmann3, Wenjun Ma2, Tony Schountz4, Martin Beer3, Martin Schwemmle1 1Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 2Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, United States, 3Institute of Diagnostic Virology, Friedrich- Loeffler-Institut, Greifswald-Insel Riems, Germany, 4Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, United States

Abstract: Recently two novel influenza A genomic sequences designated as H17N10 and H18N11 were found in New World fruit bats. Although these bat derived influenza A viruses (IAVs) overall resemble classical IAVs, their hemagglutinin (HA) and neuraminidase (NA) surface glycoprotein analogs are strikingly different. Here, we show that H18N11 productively replicates in the Neotropical Jamaican fruit bat (Artibeus jamaicensis) and is transmitted to naïve contact bats, thereby causing transient flu-like symptoms. Sequencing of isolated H18N11 from infected donor and contact bats, confirmed predominant presence of HA and NA wild-type sequences. However, following serial in vitro passaging in non-bat cells we only identified virus variants with enhanced viral growth that harbored mutations in HA and were characterized by a loss of the NA ectodomain. In mice, replication of H18N11 was exclusively confined to the upper airways and resulted similarly in the selection of mutant variants with a truncation in NA and various mutations in HA. We next generated a H18N11 variant (rP11) that was selected upon passaging in vitro, harboring two amino acid mutations in HA (K170R and N250S) plus a premature stop codon in NA (G107X) and infected ferrets to assess the zoonotic potential of this mutant virus. Only limited replication in the upper respiratory tract and lung as well as no transmission to naïve contact ferrets was observed. These findings suggest that although bat influenza A viruses can increase their replication properties by losing the NA head domain, they are only poorly adapted to non-bat mammalian species.

NSV 2018, Verona – Abstract Book

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 91

SELECTIVE PRESSURE ON RESPIRATORY SYNCYTIAL VIRUS BEARING CODON-PAIR DEOPTIMIZED F AND G ORFS GENERATES INTERNAL-DELETION GENOMES THAT PARADOXICALLY RESCUE VIRAL REPLICATION Cyril Le Nouen* 1, Thomas McCarty1, Michael Brown2, Lijuan Yang1, Cindy L. Luongo1, Peter L. Collins1, Ursula J. Buchholz1 1RNA Viruses Section, NIH/NIAID/LID, Bethesda, 2Pacific Biosciences inc., Menlo Park, United States

Abstract: Recoding viral genomes by introducing numerous synonymous but suboptimal substitutions provide new types of live-attenuated vaccine candidates. These should have a low risk of de-attenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. For viruses intended for vaccine use, it is essential to evaluate in depth the stability of attenuation. Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory disease. A vaccine is not available yet. Codon-pair deoptimized (CPD) versions of RSV are attenuated and temperature sensitive (ts). We subjected the ts CPD RSV Min B that contains 619 synonymous mutations in the F and G surface glycoproteins ORFs to serial passage in vitro at progressively increasing temperature. During passage, Min B quickly exhibited a restoration of replication fitness and syncytia formation, the latter a hallmark of RSV F expression. Comprehensive sequence analysis of virus populations by whole-genome deep sequencing, coupled with long-range PCR, revealed the presence of large-deletion (LD) viral genomes that accumulated after only a few passages. The shortest LD viral genomes were about 5 kb (one third of the RSV genome) and encoded the CPD F gene in first genome promoter proximal position. Two representative LD viral genomes were chemically synthetized de novo. Phenotypic analysis revealed that both expressed high levels of F proteins and complemented in trans Min B replication. Thus, the LD viral genomes are a new type of internal-deletion defective genome that enhanced rather than interfered with the replication of Min B. This study provides new insights on the adaptability of genome-scale recoded RNA viruses and describes a novel mechanism of adaptation of RNA viruses.

EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: 92

A NEW OPTICALLY CONTROLLABLE MEASLES VIRUS VECTOR Maino Tahara* 1, Moritoshi Sato2, Yuichiro Nakatsu1, Kenzaburo Tani3, Makoto Takeda1 1Department of Virology III,, National Institute of Infectious Diseases, Musashimurayama, 2Graduate School of Arts and Sciences, 3The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Abstract: Measles virus (MeV) vector can transfer multiple genes into human cells efficiently without affecting the host genome. We recently have developed novel MeV gene transfer vector which is non-transmissible, can transfer multiple genes simultaneously. We could successfully generate induced pluripotent stem cells from human fibroblasts or peripheral blood T cells using this vector. Although MeV as gene transfer vector is clearly beneficial, no method has been developed to control viral growth. We set out to design a method of controlling viral growth. Now, we developed optically controllable MeV. This MeV replicated efficiently when cells were illuminated by blue light. When infected cells were kept in dark, MeV hardly replicated. We believe our new optically controllable MeV vector has the potential to revolutionize gene transfer technologies.

NSV 2018, Verona – Abstract Book

EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: 93

LOSS OF RNAI WAS REQUIRED FOR THE INVENTION OF NEGATIVE-STRANDED RNA VIRUSES Benjamin Tenoever* 1 1Microbiology, ICAHN SCHOOL OF MEDICINE, New York, United States

Abstract: Viruses are thought to be living relics of the ancient world yet there is an unequal distribution of viral groups amongst domains and kingdoms. DNA viruses dominate the early prokaryotic world whereas (+)RNA viruses rose to prominence early after eukaryogenesis. Diversification of (+)RNA viruses resulted in the invention of (-)RNA viruses sometime following the appearance of multicellular life. As the emergence of these viral groups follow a trajectory of changing cellular antiviral systems, we set out to determine if this was responsible for the late evolutionary appearance of (-)RNA viruses. Here we define the capacity of different RNA viruses to cope with RNAi, the defense of early eukaryotes, and monitored their propensity to evade this system. Serial passage of viral populations revealed that, while (+)RNA viruses could rapidly excise genomic material and evade targeting, (-)RNA viruses were neutralized. These data suggest that (- )RNA viruses required a multicellular eukaryotic host that was deficient in RNAi and explains their delayed evolutionary invention and increased prevalence in vertebrates. Graphical Abstract:

POSTER PRESENTATIONS

BREAKING AND ENTERING - viral entry Abstract final identifier: P001

INSIGHT INTO THE FUSION MECHANISM GLEANED FROM AN EBOLA VIRUS GLYCOPROTEIN MUTANT THAT DOMINATED THE 2013-2016 PANDEMIC WE Diehl1, D Mu1, L Odongo2, K Szymanska2, M Cabot2, L Feneant2, B Ganser-Pornillos3, J M. White2, Jeremy Luban* 1 1Mol Med, UMass Med, Worcester, 2Cell Biology, 3Mol Phy & Biol Phys, Univ of Virginia, Charlottesville, United States

Abstract: GP-A82V, a clade-defining mutation from the 2013–2016 pandemic, enhances virion infectivity (Cell 167:1088). Though located near the NPC1 receptor (PDB:5F1B), GP-A82V points away from NPC1. Attempting to find explanation for increased infectivity we noticed that GP-A82V abuts GP-R85, and that GP-R85 is tightly coordinated within a charged pocket. We hypothesized that EBOV fusion to the target cell membrane requires a conformational switch in which GP-R85 is expelled from the charged pocket, permitting it to contact NPC1-D502, a residue required for infectivity. According to this model, GP-A82V would enhance infectivity by destabilizing interactions that retain GP-R85 within the charged pocket. To test our model, 55 mutants were engineered in the GP pocket, 8 of which enhanced infectivity of pseudotyped lentiviral vectors, as least as well as did GP-A82V. Each of the 8 mutants is predicted to destabilize GP-R85 charge interactions, and all increased resistance to inhibitors of EBOV entry, including a cathepsin inhibitor, an NPC1 inhibitor, and a neutralizing antibody. While none of these mutations altered EBOV GP-NPC1 binding affinity, they were better than WT GP at entering cells bearing NPC1-D502 mutants with lowered affinity for GP. These data are consistent with a model in which the charged pocket regulates a GP-R85 conformational switch required for fusion and with GP-A82V destabilizing the pocket. Ongoing attempts to to solve structures of the GP mutants are hoped to provide further evidence in support of our model.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: P002

THE HEMAGGLUTININ-NEURAMINIDASE (HN) HEAD DOMAIN AND THE FUSION (F) PROTEIN STALK DOMAIN OF THE PARAINFLUENZA VIRUSES AFFECT THE SPECIFICITY OF THE HN-F INTERACTION Masato Tsurudome* 1, Morihiro Ito1, Machiko Nishio2 1Medical Sciences, Chubu University, Kasugai, 2Microbiology, Wakayama Medical University, Wakayama, Japan

Abstract: Membrane fusion by the parainfluenza viruses is induced by virus-specific functional interaction between the attachment protein (HN) and the fusion (F) protein. This interaction is thought to be mediated by transient contacts between particular amino acids in the HN stalk domain and those in the F head domain. However, we recently found by chimeric analyses of the HN proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) that replacement of specified amino acids at or around the dimer interface of the HN head domain significantly affected the F protein specificity. We then intended to further investigate this issue in the present study, revealing that the HPIV2 HN protein can be converted to an SV41 HN-like protein by substituting at least three amino acids in the HPIV2 HN head domain with the SV41 HN counterparts in addition to the replacement of the stalk domain. Concomitantly, we also found that the PIV5 F protein can be converted to an SV41 F-like protein by substituting 15 amino acids in the PIV5 F stalk domain as well as the 21 amino acids in the head domain with those of the SV41 F protein. These results indicate that mutations of specified amino acids in the HN head domain and the F stalk domain can modify the specificity of the HN-F interaction, suggesting that the conformations of the HN stalk domain and the F head domain are dependent on the structures of the HN head domain and the F stalk domain, respectively.

BREAKING AND ENTERING - viral entry Abstract final identifier: P003

STRUCTURE-GUIDED IDENTIFICATION OF MORBILLIVIRUSES WITH ZOONOTIC POTENTIAL Nurshariza Abdullah1, Stephen C. Graham2, Jamie Birch1, Jamie Kelly1, Daniel Gonçalves-Carneiro1, Tim J. Mitchell3, Robin N. Thompson4, Katrina A. Lythgoe4, Nicola Logan5, Margaret J. Hosie5, Vassiliy N. Bavro6, Brian J. Willett5, Michael P. Heaton7, Dalan Bailey* 1 1Virus Programme, The Pirbright Institute, Guildford, 2Department of Pathology, University of Cambridge, Cambridge, 3Microbiology and Infection, University of Birmingham, Birmingham, 4Zoology, University of Oxford, Oxford, 5Centre for Virus Research, University of Glasgow, Glasgow, 6Microbiology, University of Essex, Essex, United Kingdom, 7Genetics, Breeding, and Animal Health Research Unit, US Department of Agriculture, NE, United States

Abstract: Morbilliviruses infect a broad range of mammalian hosts including ruminants, carnivores and humans. The recent eradication of rinderpest virus (RPV), as well as active campaigns for measles virus (MeV), have raised significant concerns that other morbilliviruses may emerge in so-called ‘vacated ecological niches’. Seeking to assess the zoonotic potential of non-human morbilliviruses we identified that peste des petits ruminants virus (PPRV) - the small ruminant morbillivirus - is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1 – the immune cell receptor. Using a structure-guided approach, as well as quantitative receptor usage assays, we characterised a single gain-of-function amino acid change, mapping to the receptor-binding domain (RBD) in the PPRV Haemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from cross-protective, human-patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Through structural modelling the mechanistic nature of the PPRV restriction was identified as charge incompatibility and steric hindrance at the protein-protein interaction interface. Significantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV.

NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: P004

NATURAL GLYCAN RECEPTORS FOR INFLUENZA VIRUS IN HUMAN LUNG Lauren Byrd-Leotis* 1, 2, Nan Jia2, Chao Gao2, Jamie Heimburg-Molinaro2, Richard Cummings2, David Steinhauer1 1Department of Microbiology and Immunology, Emory School of Medicine, Atlanta, 2Department of Surgery, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, United States

Abstract: Influenza A viruses bind sialylated N-glycans, with species specificity correlating to the sialic acid linkage conformation. Avian viruses recognize α2,3-linked sialic acid and human and swine viruses prefer α2,6. These observations derive from binding studies on erythrocyte agglutination, synthetic receptor analogs, or undefined surface markers on cells or tissues, and the preferences are predicted to reflect the availability of sialic acid linkage at the site of infection as assayed by lectin binding. Here we present the first examination of the N-glycome of the human lung for identifying the natural receptors for avian and mammalian influenza viruses. We found that the viruses exhibit differential binding profiles, binding to a wide variety of receptors or to a select number. In order to more fully characterize the glycan recognition, we focused on A/Pennsylvania/08/2008 and found that fractions containing bi-antennary sialylated glycans +/- core fucose comprised the highest binding structures. We found many α2,3- and α2,6-linked sialylated glycans bound by virus, but remarkably, all viruses also displayed sialic acid independent binding to phosphorylated, non-sialylated glycans. Our results also suggest that the standard lectin for identifying α2,3-linked sialic acid, MAL-I, can bind to terminal galactose, and therefore MAL-I alone may not be a useful indicator of sialic acid linkage at the site of infection. Moreover, we extended our analysis to include human H3N2 seasonal viruses isolated during the past decades that have progressively lost erythrocyte agglutination capabilities and discovered that these strains also lost binding to sialylated receptors during antigenic drift, while maintaining strong binding to phosphorylated glycans.

BREAKING AND ENTERING - viral entry Abstract final identifier: P005

THE CRITICAL ROLE OF HEMAGGLUTININ (HA) IN TRANSMISSION OF DUCK-ORIGIN H5NX LOW-PATHOGENIC AVIAN INFLUENZA VIRUSES IN CHICKENS Gert Zimmer* 1, Daniel Brechbühl1, Nicolas Ruggli1, Samira Locher1 1INSTITUTE OF VIROLOGY AND IMMUNOLOGY (IVI), Mittelhäusern, Switzerland

Abstract: In wild waterfowl avian influenza viruses (AIV) primarily replicate in the gastrointestinal tract and are shed into the environment at large quantities facilitating transmission to domestic poultry. Continuous circulation of H5 and H7 AIVs in domestic poultry may favour the evolution of highly pathogenic AIVs (HPAIV). To understand the adaptation process of AIV in poultry, we experimentally infected chickens via the intratracheal route with a number of low-pathogenic AIV (LPAIV) H5 and H7 isolates from wild waterfowl. While most H7 LPAIV were transmitted to contact animals without prior adaptation, none of the H5 LPAIV was passed to the sentinels. Interestingly, H5N1 and H5N8 HPAIV isolates were not transmitted to sentinel chickens if the HA proteolytic cleavage site was changed from a polybasic to a monobasic motif. Genetic reassortment of a non-transmittable H5N1 virus with either a transmittable H7N7 virus or a chicken-adapted H5N2 virus revealed that the hemagglutinin (HA) is critical for efficient virus shedding and transmission. Mutations in HA changing the pH threshold of fusion, the proteolytic cleavage site or potential glycosylation sites did not enhance virus transmission. However, recombinant H5N1 harboring the globular HA head domain from a chicken-adapted H5N2 was efficiently transmitted. Mutational analysis demonstrated that adaptations in the receptor-binding pocket (130-loop, 190-helix, and 220-loop) of HA are critical for H5 virus transmission in chickens. These findings suggest that duck-origin H5 viruses exhibit different receptor-binding activities than chicken-adapted viruses. Whether these adaptive mutations have an impact on receptor specificity or receptor affinity is currently under investigation.

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NSV 2018, Verona – Abstract Book

BREAKING AND ENTERING - viral entry Abstract final identifier: P006

TWO POINT MUTATIONS IN THE HANTAAN VIRUS GLYCOPROTEIN COMPLEX AFFORD THE GENERATION OF A HIGHLY INFECTIOUS REPLICATION-COMPETENT RECOMBINANT VSV

Megan Slough* 1, Kartik Chandran1, Rohit K. Jangra1 1Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, United States

Abstract: Hantaviruses are highly prevalent rodent-borne zoonotic viruses. Currently, no FDA-approved, specific antiviral drugs or vaccines are available, and the requirement for high biocontainment laboratories limits hantavirus research. Recombinant vesicular stomatitis viruses (rVSVs) bearing Gn/Gc glycoproteins from New World hantaviruses have proven valuable as tools to investigate hantavirus entry under BSL-2 containment. However, no Old World rVSVs have been described. In our initial experiments, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Old World, Hantaan virus (HTNV), were refractory to rescue and only grew to low titers. However, serial passage of the rescued rVSV-HTNV Gn/Gc in Vero cells markedly increased viral infectivity and spread of infection. This gain in viral fitness correlated with the acquisition of two point mutations; I532K in the cytoplasmic tail of Gn, and S1094L in the stem region of Gc. Re-rescue of rVSV-HTNV Gn/Gc, as well as VSV pseudotyping by trans-complementation, verified the mutations were determinative, and contribute to viral infectivity in a synergistic manner. Mechanistic studies revealed the mutations only modestly enhanced protein production and did not alter Gn and Gc co-localization. Rather, they re-localized HTNV Gn/Gc to the cell surface and significantly enhanced Gn/Gc incorporation into budding VSV particles. Our results suggest an enhancement of cell surface expression of other viral glycoprotein(s), by cognate mutations, could enable the generation of other hard-to-rescue rVSVs. The robust replication-competent rVSV-HTNV Gn/Gc reported herein may have utility as a vaccine candidate.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P007

HANTAVIRUS ENTRY INTO HUMAN RESPIRATORY EPITHELIAL CELLS INVOLVES MACROPINOCYTOSIS Giulia Torriani* 1, Jennifer Mayor1, 2, Gert Zimmer3, Nicole Tischler4, Stefan Kunz1, Sylvia Rothenberger1, 2, Olivier Engler2 1Institute of Microbiology, CHUV/UNIL, Lausanne, 2Spiez Laboratory, Spiez, 3Institute of Virology and Immunology, Mittelhäusern, Switzerland, 4Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile

Abstract: Hantaviruses are emerging rodent-borne viruses of the Bunyaviridae family associated with severe human diseases with high mortality. The current lack of a licensed vaccine and the limited therapeutic options make the development of novel efficacious anti-viral agents to combat hantaviruses an urgent need. The identification of cellular factors hijacked by hantaviruses in order to enter host cells is a promising approach for the development of novel strategies to combat pathogenic hantaviruses. Considering the biosafety restrictions linked to work with live pathogenic hantaviruses and that virus cell attachment and entry are mediated exclusively by the viral envelope, we established a pseudotype platform for Hantaan virus (HTNV) and Andes virus (ANDV). In a first approach, we screened a library of 90 small molecule kinase inhibitors in our hantavirus pseudotype platform using a semi-high-throughput assay. Our screen identified specific sets of cellular kinases required for cell entry of HTNV and ANDV that only partially overlapped, indicating important virus- specific differences. We complemented our unbiased screen with a targeted approach including a panel of well-defined “diagnostic” inhibitors against cellular factors involved in endocytosis. Entry of both, HTNV and ANDV critically depended on sodium-proton exchangers (NHE) and actin, which are major hallmarks of macropinocytosis. HTNV and ANDV further showed differential dependence on known regulatory factors of macropinocytosis.Promising candidate anti-viral drugs are currently followed up and validated using live pathogenic hantaviruses in the new high containment facilities at Spiez Laboratory, Switzerland.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P008

HANTAVIRUSES ENTER INTO HUMAN AIRWAY EPITHELIUM USING PHOSPHATIDYLSERINE RECEPTORS TIM-1 AND AXL Jennifer Mayor* 1, 2, Giulia Torriani1, Gert Zimmer3, Nicole Tischler4, Stefan Kunz1, Sylvia Rothenberger1, 2, Olivier Engler2 1Institute of Microbiology, CHUV/UNIL, Lausanne, 2Spiez laboratory, Spiez, 3Institute of Virology and Immunology, Mittelhäusern, Switzerland, 4Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile

Abstract: Hantaviruses are emerging human pathogens leading to serious diseases and represent a challenge for public health. Currently, there are no effective antivirals or licensed vaccines against hantaviruses. Viral attachment and entry represent the first steps in virus transmission and are promising targets for anti-viral therapeutic intervention. We investigated the largely unknown receptor used in human airway epithelium of the Old World Hantaan virus (HTNV) and New World Andes virus (ANDV). We established and validated a hantavirus pseudotype system based on replication competent and propagation deficient recombinant vesicular stomatitis virus as a suitable BSL2 surrogate model to study hantavirus entry into the host cell. We hypothesized that hantaviruses may use apoptotic mimicry to invade human respiratory epithelial cells and examined the role of the major classes of cellular phosphatidylserine (PS) receptors, the T- cell immunoglobulin and mucin (TIM) molecules and receptor tyrosine kinases of the Tyro3/Axl/Mer (TAM) family. Using antibody perturbation and other techniques, we found that cell entry of ANDV critically depended on Axl and TIM-1, HTNV depended mainly on TIM-1.In line with previous studies, HTNV, but not ANDV was able to use the abundant glycosaminoglycan heparan sulfate as co-receptor. In sum, our studies demonstrate for the first time that hantaviruses use PS receptors and hence apoptotic mimicry to invade human airway epithelium, which explains why these viruses can easily break the species barrier.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P009

STRUCTURAL BASIS FOR DIFFERENTIAL EPHRIN-MEDIATED HOST-CELL ENTRY PATHWAYS OF HENIPAVIRUSES Rhys Pryce* 1, Kristopher Azarm2, Robert P. Rambo3, Benhur Lee2, Thomas A. Bowden 1 1Division of Structural Biology, University of Oxford, Oxford, United Kingdom, 2Department of Microbiology , Icahn School of Medicine at Mount Sinai, New York, United States, 3Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom

Abstract: The capability of a paramyxovirus to specifically attach to a human cell surface receptor is a key determinant of cross-species spillover. Here, using a multi-faceted macromolecular crystallography and X-ray foot-printing mass spectrometry approach, we dissect the molecular aspects of host cell attachment for the recently identified Cedar henipavirus (CedV). The CedV attachment glycoprotein (CedV-G) is one of only two antigens displayed on the viral envelope; responsible for viral attachment to cellular membranes it is the primary determinant of host species and cell type tropism. We show that CedV-G displays the canonical six-bladed β-propeller fold utilised by the closely related and highly pathogenic henipaviruses, Hendra (HeV) and Nipah (NiV). Furthermore, we identify the putative ephrin receptor binding site and demonstrate that it is both conserved and compatible with ephrin recognition. XF-MS analysis of CedV-G-ephrinB2 complexes supports a model for a largely conserved receptor binding mode across henipaviruses. These data provide a structural template for understanding the interplay between receptor tropism and pathogenicity within this genus of important human pathogens.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P010

SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN NIEMANN-PICK C1 INFLUENCE ENTRY OF FILOVIRUSES INTO CELLS Tatsunari Kondoh* 1, Michael Letko2, Vincent Munster2, Rashid Manzoor1, Junki Maruyama1, Wakako Furuyama1, Hiroko Miyamoto1, Asako Shigeno1, Daisuke Fujikura1, Yoshihiro Takadate1, Reiko Yoshida1, Manabu Igarashi1, Heinz Feldmann2, Andrea Marzi2, Ayato Takada1 1Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan, 2Laboratory of Virology, Division of Intramural Research, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, United States

Abstract: Niemann-Pick C1 (NPC1), a host cell receptor involved in the envelope glycoprotein (GP)-mediated entry of filoviruses into cells, has been believed to be a major determinant of cell susceptibility to filovirus infection. It is known that proteolytically digested Ebola virus (EBOV) GP interacts with two protruding loops in domain C of NPC1. Using previously published structural data and the single nucleotide polymorphisms (SNPs) database, we identified ten naturally occurring, missense SNPs in the loop regions of human NPC1. To investigate the potential effects of these SNPs on filovirus infection, we generated NPC1 knockout Vero E6 cell lines and newly introduced human NPC1 with SNP substitutions. Their susceptibility was then examined by using vesicular stomatitis viruses (VSVs) pseudotyped with filovirus GPs and infectious EBOV. Although none of the SNPs tested here completely abolished viral infectivity, some of the substitutions resulted in reduced susceptibility to filoviruses as indicated by the lower infectivity and smaller plaque/focus sizes of the viruses. In particular, substitutions at amino acid positions 424, 425, 502, and 508 (P424A, S425L, D502E, and D508N) in NPC1 reduced the entry of VSV pseudotyped with GPs from multiple filovirus species. Interestingly, P424A/D508N and S425L/D502E substitutions seemed to be important for the reduced entry of ebolavirus and marburgvirus, respectively. Taken together, we conclude that human NPC1 SNPs may likely affect host cell susceptibility to filoviruses.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P011

DUAL-REGULATORY ROLE OF THE MORBILLIVIRUS ATTACHMENT PROTEIN HEAD-TO-STALK LINKER MODULE IN MEMBRANE FUSION TRIGGERING Michael Herren* 1, Neeta Shrestha1, Marianne Wyss1, Andreas Zurbriggen1, Philippe Plattet1 1Division of Experimental and Clinical Research, University of Bern, Bern, Switzerland

Abstract: Host cell entry by morbilliviruses (e.g. Measles virus (MeV) or Canine Distemper Virus (CDV)) is coordinated by two interacting envelope glycoproteins; a tetrameric attachment (H) protein and a trimeric fusion (F) protein. The ectodomain of H-tetramers consists of stalk, connector and head domains that adopt “F-triggering-permissive” native structures. The precise functional role of the C-terminal modules of the H stalk domain (termed “linker” and “connector”), although hypothesized to assume flexible structures to sustain putative receptor-induced structural rearrangements, remains largely unexplored. To gain mechanistic insights, we conducted a thorough “non-conservative” mutagenesis-scan analysis of the MeV and CDV H-linker/connector domains. Our data provide evidence that substituting a hydrophobic residue encompassed within the linker module (H-I146) into amino acids without hydrophobic side chains translated into the assembly of covalent H-tetramers, which were fusion triggering-deficient despite proper intracellular trafficking, receptor- binding and F-binding bioactivities. Remarkably, we next demonstrated that covalent H-tetramerization per se was not the only mechanism preventing F-activation: the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, retained limited fusion promotion activity, and, conversely, charged H-I146 mutants additionally carrying alanine-substitution of natural cysteines, therefore unable to form covalently-linked tetramers, were fusion activation-defective. In summary, our data suggest a dual-regulatory role of isoleucine 146 of the morbillivirus H-stalk linker module: it secures the formation of natural non-covalent dimer-of-dimers and contributes to receptor-induced F-triggering activity.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P012

EBOLA VIRUS ENTRY AND EGRESS IN POLARIZED EPITHELIAL CELLS Manasi Ajit Tamhankar* 1, Dawn M. Gerhardt2, Richard S. Bennett2, Nicole Murphy2, Peter B. Jahrling2, Lisa E. Hensley2, Jean L. Patterson1 1Virology & Immunology, TEXAS BIOMEDICAL RESEARCH INSTITUTE, San Antonio, 2Division of Clinical Research, Integrated Research Facility/ NIAID, Frederick, MD, United States

Abstract: Currently, no FDA-approved vaccines or treatments are available for Ebola virus disease, and therapy remains largely supportive. Ebola virus (EBOV) has broad tissue tropism and can infect a variety of cells including epithelial cells. Epithelial cells differ from most other cell types by their polarized phenotype and barrier function. In polarized cells, the apical and basolateral membrane domains are demarcated by tight junctions. Polarized cells also have specialized sorting machinery, which results in a difference in composition of the two membrane domains. These specialized functions of sorting can have important consequences for viral infections. Differential localization of a viral receptor can restrict virus entry to a particular membrane while, polarized sorting can lead to a vectorial virus release. To elucidate the characteristics of EBOV entry and egress in polarized cells, we first characterized the polarized Caco-2 model on semipermeable transwells using measurement of transepithelial resistance and microscopy. Our data in Caco-2 cells indicate that EBOV preferentially infects from the basolateral route, and this preference may be influenced by the extent of polarity. Infection and egress occurs without changes in cellular permeability. Further, our data shows that polarized distribution of heparan sulfate, a known viral attachment factor, may be responsible for causing the basolateral preference shown by the virus during entry in Caco-2 cells. Treatment with iota-carrageenan, which blocks virus interaction with cellular heparan sulfate significantly reduced cell susceptibility to infection. Our results, thus, also show the potential of heparan sulfate as a therapeutic target during EBOV infection

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BREAKING AND ENTERING - viral entry Abstract final identifier: P013

BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF VSV (NEW JERSEY STRAIN) GLYCOPROTEIN Abbas Abou Hamdan1, Laura Belot1, Aurélie Albertini1, Yves Gaudin* 1 1INSTITUT DE BIOLOGIE INTÉGRATIVE DE LA CELLULE, CNRS, Gif sur Yvette, France

Abstract: Vesiculoviruses entry into host cells is mediated by the unique viral glycoprotein G, anchored in the viral membrane. First, G is involved in receptor recognition. Then, after endocytosis of the viral particle, G mediates the fusion between the viral and endosomal membranes. The fusion is triggered by a low pH-induced conformational change from a trimeric pre- to a trimeric post-fusion conformation. For the moment, only crystal structures of VSV Indiana and Chandipura virus glycoproteins have been determined. In this study, we have characterized the ectodomain of glycoprotein of VSV New Jersey strain, which shares 52% amino- acid sequence identity with that of VSV Indiana. The ectodomain was generated by thermolysin-limited proteolysis of viral particles and was shown to interact with CR2 and CR3 of LDL-R receptor. Using Electron Microscopy, we showed that this ectodomain undergoes a similar structural transition from a pre- to a post fusion form when the pH is lowered. At low pH, we observed rosette-like shapes in which the post-fusion trimers associate via their hydrophobic fusion loops. We performed different crystal conditions screen that allows us to obtain several crystal forms at different pH conditions probably corresponding to distinct structural conformations of the ectodomain. The resolution of those crystalline structures is under way.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P014

INFLUENZA VIRUS ENTRY VIA INTERPLAY BETWEEN PLATELET-DERIVED GROWTH FACTOR RECEPTOR BETA AND GM3 GANGLIOSIDE: A VALID ROUTE FOR HOST-TARGETED ANTIVIRAL INTERVENTION Pieter Vrijens1, Els Vanstreels1, Sam Noppen1, Seppe Cambier1, Roberto Ronca2, Paola Chiodelli2, Manon Laporte1, Evelien Vanderlinden1, Sandra Liekens1, Annelies Stevaert* 1, Lieve Naesens1 1Rega Institute for Medical Research, KU Leuven - University of Leuven, Leuven, Belgium, 2Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy

Abstract: The possible resistance of influenza virus against the existing antiviral drugs calls for new therapeutic targets such as host cell factors associated with virus replication. We here evaluated a diverse panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney (MDCK) cells. The receptor tyrosine kinase (RTK) inhibitor Ki8751 displayed robust anti-influenza A and B activity and was selected for mechanistic investigations. Ki8751 efficiently blocked the post-binding internalization of influenza virus in different cell lines including four Chinese hamster ovary (CHO) cell lines with abundant expression of platelet-derived growth factor β (PDGFRβ). For two other RTKs, i.e. fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2, no association with influenza entry was seen. The significantly higher virus uptake in CHO-K1 compared to CHO-wild type cells indicated stimulation by GM3 ganglioside yet suppression by GM1 ganglioside. This GM3/PDGFRβ-controlled virus internalization involved PDGFRβ phosphorylation combined with receptor desialylation by the viral neuraminidase. It coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. The PDGFRβ-Erk loop was fully suppressed by Ki8751. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signaling pathway to penetrate the cells, providing an opportunity for host cell-targeting antiviral intervention.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P015

VIRAL ENTRY AND REPLICATION OF BAT-DERIVED MUMPS VIRUS Nadine Krüger* 1, Sarah Hüttl1, Kathleen Voigt2, Georg Herrler2, Claes Örvell3, Markus Hoffmann4 1Division of Virology, Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, 2Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany, 3Division of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 4Infection Biology Unit, Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung, Göttingen, Germany

Abstract: Mumps is a highly contagious childhood disease with usually mild symptoms caused by mumps virus (MuV), a member of the family Paramyxoviridae. In rare events, mumps can result in complications like encephalitis or meningitis. In 2012, the genome of a virus with close phylogenetic relatedness to human MuV was detected in African fruit bats (batMuV). However, in the absence of an infectious isolate, all recent efforts to characterize batMuV were based on directed expression of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, or chimeric MuVs harboring batMuV F and HN. Although these studies provided initial insights in the functionality of the batMuV glycoproteins, important aspects such as the host range, replication competence or virulence of batMuV remained elusive. Here, we generated recombinant authentic batMuV and analyzed this virus regarding its ability to replicate in different mammalian cells and host cell factors that are required for viral entry. Our data show that batMuV can replicate in cells of human, non-human primate and bat origin. The replication of batMuV most likely occurs in cytoplasmic inclusion bodies the formation of which is mediated by the interaction of the nucleo- and phosphoprotein. The proteolytic cleavage of batMuV F by cellular furin, as well as the binding of batMuV HN to sialic acids expressed on the surface of target cells is a necessity for viral entry. Infection can be inhibited by cross-neutralization of antisera directed against human MuV strains.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P016

A GENOME-WIDE GENETIC SCREEN UNCOVERS A ROLE FOR HEPARAN SULFATE IN LCMV ENTRY Mark J. Bakkers* 1, Sarah Hulsey Stubbs1, Sean P. Whelan1 1MBIB, Harvard Medical School, Boston, United States

Abstract: Lymphocytic Choriomeningitis Virus (LCMV) the prototypic arenavirus is associated with fatal infection in human transplant recipients, pregnancy termination and birth defects. As an infection model, studies of murine LCMV discovered and shaped our understanding of MHC restriction, T cell memory development and exhaustion, and persistent viral infections. Over 20 years ago, it was recognized that alpha-dystroglycan (α-DG) is the receptor for LCMV and the related Lassa fever virus (LASV), yet both viruses infect cells in the absence of α-DG. Using haploid genetic screens, we previously found that LASV, but not LCMV, undergoes a pH-triggered receptor switch during its internalization to engage LAMP1 to facilitate infection. To further interrogate the host requirements for LCMV entry, we performed a genome-wide CRISPR screen in the human lung cell line A549 using recombinant vesicular stomatitis virus (VSV) in which the endogenous glycoprotein was replaced by that of LCMV. Cells that survived infection were enriched for gRNA’s targeting α-DG and heparan sulfate (HS) biosynthesis pathways, implicating both in LCMV entry. Using gene-edited cells in which we inactivated the host gene DAG1 to block all α-DG expression, B4GALT7 to block HS biosynthesis, or both, we confirmed the use of HS by LCMV. A closely related virus isolated from a cluster of transplant recipients, Dandenong, does not use HS for entry. Guided by atomic structures we performed a mutagenic analysis of the respective glycoproteins to define the residues in LCMV GP that confer binding to HS. This study demonstrates that LCMV attaches to the cell surface via α-DG and HS. We also hypothesize that a further internal "LAMP1-like" molecule is required for cell entry during LCMV and Dandenong infection.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P017

CHARACTERIZATION OF HANTAVIRUS ENTRY THROUGH LIVE-CELL IMAGING Lara M. Kleinfelter* 1, Jennifer S. Spence1, Rohit K. Jangra1, Kartik Chandran1 1Albert Einstein College of Medicine, Bronx, United States

Abstract: Hantaviruses are global emerging pathogens that cause hemorrhagic fever with renal syndrome in the Old world and hantavirus cardiopulmonary syndrome in the New world with fatality rates as high as 12% and 60%, respectively. The development of antiviral drugs and vaccines against these agents is hindered by a poor understanding of the hantavirus entry pathway. To better understand the entry process and the roles of cellular host factors, we developed a live cell imaging system to observe hantavirus entry in real time. Fusion kinetics of fluorescently-labeled recombinant VSV particles bearing glycoproteins of Andes or Hantaan virus, virulent New world and Old world hantaviruses, respectively, in primary human endothelial cells matched those of other late-fusing viruses, though Andes virus requires more time to fuse than Hantaan virus. Furthermore, we defined the compartments where Andes and Hantaan virus GP-dependent membrane fusion occurs. Together, these experiments support the model that hantavirus fusion occurs late in the endocytic pathway. In addition, we are also characterizing the roles of known hantavirus receptors during entry.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P018

STRUCTURAL CLASSES OF OLD WORLD ARENAVIRUS GP1 ATTACHMENT GLYCOPROTEIN Weng M. Ng* 1, Rhys Pryce1, Antra Zeltina1, Kamel El Omari2, Armin Wagner2, Thomas A. Bowden1 1Division of Structural Biology, University of Oxford, Oxford, 2Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, United Kingdom

Abstract: Arenaviruses categorize into two serogroups: Old World and the New World, which differ genetically and by geographical distribution. The arenaviral surface attachment glycoprotein, GP1, is responsible for host cell receptor recognition and is a primary determinant of zoonosis that enables the transmission of hemorrhagic fever arenaviruses from rodent hosts to humans. Previous crystallographic analyses of arenaviral GP1 glycoproteins have revealed two distinct pH- dependent conformational states. Here, our high-resolution crystal structures of the GP1 glycoproteins of Old World Loei River virus (LORV) and New World Whitewater Arroyo virus (WWAV), obtained at both neutral and acidic pH, suggest that these rearrangements are limited to Old World arenaviruses. We believe that Old World arenaviral GP1 glycoproteins presented on the mature virions adopt a structural class discrete to that formed upon detachment from the GP2 fusion protein during host cell entry. This detached conformation may constitute an immunological decoy and contribute to an ineffective humoral immune response during infection. Furthermore, the ability of detached New World arenaviral GP1 glycoproteins to bind receptors and neutralizing antibodies suggests that they are unlikely to undergo such conformational rearrangements. These data have implications for the development of detection diagnostics and immunogens specific to Old World arenaviruses.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P019

ARCHITECTURE OF THE METASTABLE HANTAVIRUS ENVELOPE

Robert Stass* 1, Ilona Rissanen1, Sai Li1, Thomas A. Bowden1, Juha T. Huiskonen1 1Department of Structural Biology (Strubi), UNIVERSITY OF OXFORD, Oxford, United Kingdom

Abstract: Members of the genus Hantaviridae are ssRNA viruses present in rodent, bat, shrew, and mole reservoirs worldwide that cause hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome upon zoonosis into humans. Hantaviruses are pleomorphic, enveloped viruses that exhibit a tetragonal lattice of glycoproteins on their surface comprised of Gn and the class-II fusion protein, Gc. Using a non-pathogenic hantavirus, Tula virus, we utilize cryo- electron tomography, combined with previously reported crystal structures of Gn and Gc glycoproteins, to provide the best currently available model of the Gn-Gc glycoprotein spike assembly. We also demonstrate the collapse of this lattice upon exposure to low pH, mimicking its entry into the endocytic pathway. Comparison of our hantaviral Gn-Gc spike model with other viruses highlights the diverse modes of assembly available to class-II fusion protein displaying viruses.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P020

CHARACTERIZATION OF DETERMINANTS FOR HUMAN RESPIRATORY SYNCYTIAL VIRUS SPECIES SPECIFIC CELL ENTRY Svenja M. Wiechert* 1, Dortothea Bankwitz1, Christine Krempl2, Sibylle Haid1, Sebastian Blockus1, Luisa Stroeh3, Thomas Krey3, Thomas Pietschmann1 1Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, 2Institute of Virology and Immunobiology, Julius-Maximilian-University, Wuerzburg, 3Institute of Virology, Hannover Medical School, Hannover, Germany

Abstract: Human Respiratory syncytial virus (HRSV) and Pneumonia virus of mice (PVM) belong to the genus of Orthopneumoviruses within the family of Pneumoviridae. Both viruses display a pronounced species and tissue tropism as they infect the respiratory tract of humans and mice, respectively. The determinants that govern tropism of these viruses are poorly defined. HRSV associated lower respiratory tract disease is a leading cause of hospitalizations and in-hospital deaths in young children. Existing mouse models of RSV infection do not precisely mirror the pathophysiology of human disease. We hypothesize that species-specific HRSV cell entry limits susceptibility of mice to HRSV infection. To dissect mechanisms and determinants of species-specific HRSV cell entry, we recently developed infectious lentiviral pseudo particles decorated with HRSV G, F and SH proteins. To complement this system, we cloned codon optimized genes of PVM F, G and SH. PVM G protein expression was confirmed by immunofluorescence microscopy using sera from PVM G immunized rabbits. For detection of PVM F, F-specific monoclonal antibodies were created and used to detect F protein expression in transfected cells and incorporation of F protein onto secreted lentiviral particles. Surprisingly, PVM F protein carrying lentiviral pseudo particles were non-infectious in various human and rodent cell lines. Notably, mobility of F protein species in transfected cells differed from the one of F protein detected in PVM-infected BHK cells suggesting that aberrant post-translational modification (e.g. proteolytic processing) may be responsible for production of non-infectious particles. Mechanistic and functional assays to pinpoint the relevance of post-translational modifications for infectivity of PVM pseudo particles and to dissect the species tropism of HRSV are ongoing and will be presented.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P021

THE NOVEL CEDAR HENIPAVIRUS ATTACHMENT GLYCOPROTEIN DISPLAYS IDIOSYNCRATIC USAGE OF THE HUMAN EPHRIN CELL SURFACE RECEPTORS Kristopher Azarm* 1, Rhys Pryce2, Thomas A. Bowden2, Benhur Lee1 1Icahn School of Medicine at Mount Sinai, New York, United States, 2Division of Structural Biology, University of Oxford, Oxford, United Kingdom

Abstract: The prototypical henipaviruses (HNVs), Hendra virus (HeV) and Nipah virus (NiV), are responsible for outbreaks of severe disease in domestic animals and humans. Zoonotic transmission of these pathogens is dependent upon the interaction of an attachment glycoprotein (HNV-G) with highly conserved ephrin receptors (EFNs). All extant HNVs whose receptors have been functionally defined use EFNB2. In 2012, a novel HNV, Cedar henipavirus (CedV), was isolated from a pteropid bat in Australia. Although overall sequence conservation of CedV-G with the extant HNV-Gs is very low (24- 31%), we see a substantial increase in conservation at the putative receptor binding site (47-54%). Thus, we examined whether CedV-G exhibited a conserved mode of binding to EFNB2. Moreover, given the low overall sequence conservation with extant HNV-Gs, we also sought to determine whether any of the other EFNs might serve as alternative receptors. Through functional analyses, we show that CedV-G uses not only EFNB2 as an entry receptor, but also EFNB1. Soluble EFNB2 and EFNB1 inhibit entry of a vesicular stomatitis virus pseudotyped with the CedV envelope glycoproteins (CedVpp) into susceptible cells, and stable expression of EFNB2 or EFNB1 in an EFN-negative cell line is sufficient to confer entry. Moreover, soluble EFNB2 and EFNB1 were able to compete CedVpp entry on both EFNB2- and EFNB1-expressing cells, suggesting that CedV-G interacts with EFNB2 and EFNB1 via an overlapping site. These results indicate CedV-G displays an idiosyncratic usage of EFN host receptors. Given that EFN receptor usage dictates viral tissue tropism and neurovirulence, this alternative receptor usage could partially explain why CedV is non-pathogenic in small animal models.

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BREAKING AND ENTERING - viral entry Abstract final identifier: P022

POTASSIUM ACTIVATES THE FUSION MACHINERY OF HAZARA VIRUS THROUGH SPIKE CONFORMATIONAL CHANGE Emma Punch* 1, Samantha Hover2, Jack Fuller1, Henry Blest1, Roger Hewson3, Juan Fontana4, Jamel Mankouri1, John Barr1 1SMCB, University of Leeds, 2SMCB, Uniersity of Leeds, 3Tropical Medicine and Health, PHE, 4ABSL, University of Leeds, Leeds, United Kingdom

Abstract: All enveloped viruses enter cells via fusion of viral and host membranes. Fusion is mediated by virus-encoded spikes that respond to the changing endosomal environment, which triggers spike conformational changes that drive the fusion process. Several fusion triggers have been identified including pH, membrane composition and endosome-resident proteins and these cues dictate when and where viral fusion occurs. We recently showed infection of enveloped bunyaviruses required elevated potassium ion concentration [K+] encountered during transit through maturing endosomes, resulting from cellular K+ channel influx. Here, we reveal the molecular basis for K+ requirement using cryo-electron tomography to compare the ultrastructure of model bunyavirus Hazara virus, exposed to low and high [K+]. We show that exposure to K+ alone results in spike extension and induces spike-membrane interactions, thus identifying K+ as a newly- defined fusogenic trigger. We further show that preventing K+ influx through channel blockade prevents virus replication, thus identifying cellular K+ channels as new antiviral targets.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P023

COMPETING LYSINE MODIFICATIONS IN INFLUENZA VIRUS PROTEINS. Edward Hutchinson* 1, Ervin Fodor2 1MRC-University of Glasgow Centre for Virus Research, Glasgow, 2Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Abstract: Ubiquitination is a common post-translational modification (PTM) of lysine, regulating protein stability and function. Using mass spectrometry, we found that influenza virions contained tens of ubiquitin molecules, linked through both the K48 and K63 positions. We also found that ubiquitin was conjugated to viral proteins.

As only a limited number of ubiquitination sites had been mapped in influenza virus proteins, we used our data to map ubiquitinated lysine residues in the proteins of influenza A and B viruses. In doing so, we discovered that viral lysines are subject to a variety of PTMs including formylation, methylation and acetylation. In the context of cellular proteins, the effects of ubiquitin are known to be modulated by other lysine PTMs. These competing PTMs therefore suggest a potential regulatory ‘lysine code’ for influenza proteins.

The modified lysines in viral proteins are typically surface exposed, as would be expected from regulatory PTMs, and highly conserved, underlining their importance to the virus. We found that KR mutations, which prevent PTMs, often have little effect on viral growth in tissue culture. However, KR mutations to any of a cluster of modified lysines in the RNA-binding groove of the viral nucleoprotein (K184, K229 and K273) substantially reduced viral replication. Evidence for multiple competing PTMs at these and other sites suggests that a balance of modifications may fine-tune the activity of viral proteins.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P024

ROLE OF THE NUCLEOCAPSID IN REGULATION OF VIRAL RNA SYNTHESIS Ming Luo* 1, Ryan H. Gumpper1, Weike Li1 1GEORGIA STATE UNIVERSITY, Atlanta, United States

Abstract: The nucleocapsid of vesicular stomatitis virus (VSV) serves as the template for viral RNA synthesis. Codon usage bias (CUB) of VSV shows that 50% of the codons have the opposite usage frequency compared to the host. Based on the interaction of the genomic RNA with the nucleocapsid N protein, we hypothesize that its CUB plays a role in regulation of viral RNA synthesis. A minigenome assay has been set up in which the high or low frequency codon usage in the VSV N gene was changed to match those of the host. The results showed that change to either high or low frequency usage dramatically reduced the level of vRNA and mRNA. In addition, the ratios among the three RNA species were also changed, indicating imbalance between transcription and replication. Study of the smallest number of codons that have a visible effect on viral RNA synthesis and the underlining mechanism for reduction of viral RNA synthesis is ongoing. One of the reasons may be the local stability of the nucleocapsid. Using a unique melting curve assay, we showed that the stability of the nucleocapsid is directly related to the release of the sequestered RNA. Nucleocapsid-like particles (NLP) are more stable when poly(rA) is incorporated or less stable when poly(rU) is incorporated than wtNLP. Stabilization of the nucleocapsid by a RNA binding polyamide as shown by a co-crystal structure could inhibit viral RNA synthesis. Mutagenesis studies also showed that mutations in a helix-loop motif, located next to the sequestered genomic RNA, in the N protein resulted in lower viral RNA synthesis. Compensatory mutations in the L protein are required to restore the activity and to rescue the recombinant VSV. All data suggest that the interactions of the N protein with the sequestered genomic RNA in the nucleocapsid play major roles in regulation of viral RNA synthesis.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P026

NUCLEAR IMPORT AND ASSEMBLY OF INFLUENZA VIRUS REPLICATION MACHINERY Amélie Donchet1, Jean-Marie Bourhis1, Rob W. Ruigrok1, Bernard Delmas2, Mariette Ducatez3, Thibaut Crepin* 1 1IBS, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, 2Unité de Virologie et Immunologie moléculaires, INRA, Jouy-en-Josas, 3IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France

Abstract: The specific nuclear replication of influenza viruses implies an efficient nucleocytoplasmic transport of viral components during the viral cycle. The nuclear import of neosynthesized viral proteins hijacks the cellular importins-a/β pathway, using several strategies. Whereas both PB2 and the nucleoprotein (NP) widely target the importins-a1-3, the nuclear transport of the two others polymerase subunits (PA and PB1) is performed through a ternary complex with the importin-β RanBP54,5. We have covered many aspects of the interaction between influenza virus replication machinery and the cellular importins- a/β pathway partners, in order to detail the assembly of this intricate viral architecture. Following our data on the complex between influenza B NP (B/NP) and the human importin-a76, we have detailed the inherent properties associated to the NP of the newly identified influenza D virus using several methods of the structural biology. The comparison of the X-ray structures of B/NP and D/NP reveals specific issues for the interaction with the cellular importins-a, that will be discussed7. In parallel, our work cover also the aspect of the interaction between RanBP5 and the viral PA-PB1 subcomplex prior its assembly with PB2. We have recently shown that RanBP5 impairs the vRNA recognition by the viral subcomplex8. We will present our recent results on the specific interaction between the human importin-β and its viral cargo9.

1. Tarendeau et al., (2007) NSMB; 2. Gabriel et al., (2011) Nat Com; 3. Nakada et al., (2015) Sci Rep; 4. Deng et al., (2006) J Virol; Hutchinson et al., (2011) J Gen Virol; 6. Labaronne et al., (2017) Sci Rep; 7. Donchet et al., in prep; 8. Swale et al., (2016) Sci Rep; 9. Swale et al., in prep

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P027

DEPHOSPHORYLATION OF RESPIRATORY SYNCYTIAL M2-1 PROTEIN BY THE CELLULAR PHOSPHATASE PP1 IS REQUIRED FOR ITS MRNA BINDING ABILITY Jean-Francois Eleouet* 1, Charles-Adrien Richard2, Vincent Rincheval3, Christophe Cardone4, Camille Esneau1, Sergei Nekhai5, Marie Galloux1, Marie-Anne Rameix-Welti3, Christina Sizun4 1Unite de Virologie et Immunologie Moleculaires, INRA Universite Paris-Saclay, 2Unite de Virologie et Immunologie Moleculaires, INRA, Jouy en Josas, 3UMR1173, INSERM-Universite de Versailles St Quentin, Montigny le retonneux, 4ICSN, CNRS-Universite Paris-Saclay, Gif-sur-Yvette, France, 5Center for Sickle Cell Disease, Howard University, Washington D.C., United States

Abstract: The M2-1 protein of respiratory syncytial virus (RSV) is essential for viral transcription. Previous reports suggested that dynamic regulation of M2-1 phosphorylation is critical for its function. M2-1 phosphorylation depends on the presence of the RSV phosphoprotein P, which is a multifunctional protein and the main cofactor of the large RNA polymerase L protein; formation of the P-M2-1 complex is required for viral transcription. However mechanisms involved in M2-1 phosphorylation and dephosphorylation in vivo have not been clarified. Using site-directed mutagenesis and NMR, we identified an RVxF-like motif located upstream of the M2-1 binding domain and involved in the capture of the host cell protein phosphatase-1 (PP1) by P and its recruitment to cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. We further show that the P-PP1 complex regulates M2-1 dephosphorylation and RSV transcription. In the absence of PP1 from inclusion bodies, M2-1 was excluded from IBs associated granules (IBAGs) formed by viral mRNA. These results suggest that M2-1 functions not only as a transcription antiterminator but plays also a critical role at late transcription steps. Graphical Abstract:

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P028

THE ISOLATION AND CHARACTERIZATION OF A N°-P COMPLEX OF THE RESPIRATORY SYNCYTIAL VIRUS OPENS THE WAY TO THE RATIONAL DESIGN OF NEW ANTIVIRALS Jean-Francois Eleouet* 1, Ronan Le Goffic1, Vanessa Gaillard2, Origène Nyanguile2, Marie Galloux1 1Unite de Virologie et Immunologie Moleculaires, INRA, Jouy en Josas, France, 2peptide and Protein Technologies, HSE- SO Valais, Sion, Switzerland

Abstract: The RNA genome of respiratory syncytial virus is encapsidated by the viral nucleoprotein N at all steps. This RNA-N complex is a template for the viral RNA-dependent RNA polymerase. Polymerization of N along the neo-synthesized genomic and anti-genomic RNAs requires the supply of neo-synthesized N protein. This N protein is maintained monomeric and RNA-free through the interaction with the N-terminal residues of RSV phosphoprotein P that plays the role of a chaperone, forming a soluble N0-P complex. In order to isolate a stable N0-P complex suitable for structural and functional studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that deletion of the 30 first N-terminal residues of N (NΔ30) impairs N oligomerization, and that the C-terminal arm of N is involved in the inhibition of N-RNA interaction in solution. Biochemical and functional analysis of RSV N mutants revealed the critical role of C-terminal acidic and hydrophobic residues of N in the folding of the C-arm close to the RNA groove, and the similarity of the binding surface of P40 on RSV N compared to hMPV N0-P complex. Altogether, these data led us to propose new antiviral approaches targeting the RSV N0-P complex. More specifically, we rationally designed peptides that mimic the P peptide and that were shown to be active in cellula and in vivo in a mouse model. Finally, an RNA-free, chimeric construct composed of the full-length N and the N-terminal region of P was purified and used for in vitro encapsidation studies. This protein represents a new tool to validate the specificity and efficacy of our compounds.

Graphical Abstract:

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P029

MECHANISM OF RESPIRATORY SYNCYTIAL VIRUS RNA SYNTHESIS Dongdong Cao1, Yunrong Gao1, Bo Liang* 1 1Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

Abstract: Cryo-electron microscopy (cryo-EM) is especially well suited for molecular systems traditionally challenging for structural characterization, including membrane proteins and large and heterogeneous assemblies. One such challenging system is the RNA synthesis machinery of a class of pathogenic and sometimes deadly non-segmented negative-sense (NNS) RNA viruses, including rabies, measles, Ebola, Marburg, and respiratory syncytial virus (RSV). Central to the life of these viruses is RNA synthesis, which is carried out by the RNA polymerase (the multifunctional enzyme). However, the structural and functional basis of the RNA synthesis machinery of these viruses remains largely unclear. The Liang laboratory is dedicated to understanding the mechanism of the RNA synthesis machinery of RSV, the top leading cause of severe pediatric respiratory tract diseases worldwide. The Liang laboratory focuses on establishing an RNA synthesis platform for RSV, elucidating how this RNA synthesis machine functions, and identifying potential antiviral therapeutic targets for more effective treatment. Our immediate research goal is to decipher the molecular architecture of the RSV RNA synthesis machine using single particle cryo-EM and x-ray crystallography. This could lead to the development of effective antiviral drugs to block the RSV activity.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P030

STRUCTURAL BASIS FOR REGULATION OF FILOVIRAL NUCLEOPROTEIN (NP) BY VP35 DURING VIRAL RNA SYNTHESIS Gaya Amarasinghe* 1 1Pathology and Immunology, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, St. Louis, United States

Abstract: Ebola virus and Marburg virus are two members of the zoonotic filovirus family likely maintained in nature in bats. These viruses cause highly lethal hemorrhagic fever in humans (filoviral hemorrhagic fever (FHF)), with case fatality rates approaching 90 percent during some outbreaks. Fatal outcomes correlate with increasing viremia over time and are associated with shock, convulsions and disseminated intravascular coagulation. Viral antagonism of host innate immunity is important for infection and a key protein called viral protein 35 (VP35) performs multiple immune evasion functions. Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis and its activity is controlled, in part by VP35. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We will describe significant structural differences between Ebola viral NP and Marburg viral NP proteins, including differences in how the VP35 binding site in NP is modulated. Importantly, our in vitro studies are consistent with viral life cycle models, such as minigenome and trVLP assays, supporting physiological relevance and biological significance. We will also provide data to support how these basic biochemical insights provide a framework for much needed small molecule therapeutic development.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P031

FIRST IN HUMAN STUDIES OF ONCOLYTIC VESICULAR STOMATITIS VIRUSES ENCODING INTERFERON FOR CANCER THERAPY Nanda Packiriswamy1, Bethany Brunton1, Jamie Bakkum-Gamez2, Stephen Russell1, Martha Lacy3, Kah-Whye Peng* 1 1Molecular Medicine, 2Gynecologic Surgery, 3Hematology, Mayo Clinic, Rochester, United States

Abstract: The Indiana strain of Vesicular Stomatitis Viruses (VSV) are being developed as anticancer drugs for the treatment of a variety of malignancies. To ensure tumor selective replication and spread, we have designed the VSV to encode interferon beta. Expression of IFNb also serves as a STING angonist to activate host immunity against the cancer. The sodium iodide symporter (NIS) is inserted as a reporter gene into the viral genome to enable noninvasive monitoring of viral spread using PET/CT imaging. Two Phase I clniical trials testing VSV-IFN-NIS are ongoing after intravenous administration of the virus in hematological malignancies and endometrial cancer. It is a classical 3+3 phase I trial, starting 9 11 at 5x10 TCID50 through 5x10 TCID50, given as a single IV dose. The primary objective is safety and tolerability; secondary objectives include monitoring the PK of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding, preliminary efficacy, changes in the immune profile of peripheral blood leukocytes, and immunohistochemistry for immune cell infiltrates in tumors. To date, nine patients have received IV VSV-IFNβ-NIS; three with EC and six with hematologic 10 malignancies. The highest dose administered to date is 1.7x10 TCID50 and dose escalation is ongoing. Multiple cytokines increased at 4h post infusion of virus, but most returned to baseline levels by 24h. Viremia was detectable in all patients at the end of infusion, and to varying levels at 30 mins, 1, 2, 4, 24, 48h or 72 hours post virus infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-VSV antibodies were present by day 29. Extensive immune phenotyping for immune cells showed a trend towards increased PD-1 expression on CD8+ cells. Dose escalation is ongoing and updated results will be reported.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P032

START-SNATCHING: A POTENTIAL NOVEL MECHANISM FOR TRANSLATION INITIATION IN SEGMENTED NEGATIVE-SENSE RNA VIRUSES Elizabeth Sloan* 1, Veronica Valentina Rezelj1, Ingeborg Van Knippenberg1, Quan Gu1, Edward Hutchinson1 1CVR, MRC-University of Glasgow, Glasgow, United Kingdom

Abstract: A number of segmented negative sense RNA viruses use cap-snatching to initiate transcription. As start codons have been found close to the cap in host mRNAs, we asked whether cap-snatching might append additional start codons to viral mRNAs. We analysed published deep sequencing data of influenza cap-snatched sequences and found that >6% contained start codons. To determine if these could initiate translation we used transfection-based minireplicon systems, in which viral polymerases transcribe a luciferase gene flanked by viral UTRs. We assessed three distantly related cap- snatching viruses, influenza A virus (IAV), Oropouche virus and Heartland virus (HRTV). Even after mutating the luciferase start codon we detected luciferase expression from all three minireplicon systems, providing evidence of functional start sites within cap-snatched sequences. Focusing on IAV, we next analysed mass spectra from viral proteins and identified peptides that mapped to the UTR of segment 5, upstream of any encoded start codons, consistent with translation initiation in the cap sequence, or ‘start-snatching’. Upon further investigation of published IAV sequences, we discovered out-of- frame ORFs which lack an encoded start codon at the 5’ end of a number of viral genome segments. These are highly conserved, suggesting they may be of biological importance. Overall, this study has found evidence for a novel model for viral translation initiation, which may be used by at least three negative stranded RNA viruses.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P033

STRUCTURAL INSIGHTS INTO REPTARENAVIRUS CAP-SNATCHING MACHINERY Maria Rosenthal* 1, Nadja Gogrefe1, Dominik Vogel1, Juan Reguera2, Bianka Rauschenberger1, Stephen Cusack3, Stephan Guenther1, Sophia Reindl1 1Virology, BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE, Hamburg, Germany, 2AFMB UMR 7257 Aix- Marseille Univ/CNRS, Marseille, 3Grenoble Outstation, European Molecular Biology Laboratory, Grenoble, France

Abstract: Arenaviruses occur worldwide and can cause severe, often fatal hemorrhagic fever in humans. Vaccines and effective treatments are not available. Arenaviruses replicate in the cytoplasm of infected cells and since they cannot synthesize cap-structures they use a mechanism called cap-snatching to steal cap structures from host mRNAs for viral transcription. This mechanism is an attractive drug target, as it is essential for virus replication and virus specific. However, the arenaviral components of this mechanism are poorly defined compared to influenza virus, the prototypic cap-snatching virus. We present the first crystal structures of two putative components of the California Academy of Sciences arenavirus cap-snatching machinery, namely the isolated N- and C-termini of the viral RNA polymerase (L protein). The N-terminus harbors what looks like a functional cap-snatching endonuclease. The L protein C-terminus, despite complete sequence divergence, shows overall structural similarity to the C-terminal region of influenza virus polymerase PB2 subunit, suggesting a common evolutionary origin. A domain clearly related to the PB2 cap-binding domain is present, although cap- binding could not be biochemically demonstrated. The determined structures provide the basis for future research to unravel the details of the arenavirus cap-snatching mechanism and its potential as a target for drug development.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P034

A MECHANISM FOR THE ACTIVATION OF THE INFLUENZA VIRUS TRANSCRIPTASE Itziar Serna Martin* 1, Narin Hengrung2, Max Renner3, Jane Sharps1, Ervin Fodor1, Jonathan Grimes3 1Dunn School of Pathology, UNIVERSITY OF OXFORD, Oxford, 2Francis Crick Institute, London, 3Structural Biology Division, University of Oxford, Oxford, United Kingdom

Abstract: The influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB1, PB2 and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. Transcription depends on capped RNA primers, generated from nascent cellular transcripts by the cap-snatching activity of the polymerase. Access to nascent capped transcripts for cap-snatching is ensured by the interaction between FluPol with the C-terminal domain (CTD) of RNA polymerase II. We have determined the co-crystal structure of influenza C virus polymerase (FluPolC) bound to a Ser5- phosphorylated CTD (pS5-CTD) peptide, identifying novel CTD binding sites distinct from those characterised in FluPolA and FluPolB. The position of the CTD-binding site at the interface of PB1, P3 and the flexible PB2 C-terminal domains suggests that CTD-binding stabilizes the transcription-competent conformation of FluPolC. In agreement with this, FluPolC in vitro activity assays show that both the cleavage of capped RNA and capped primer-dependent transcription initiation are greatly enhanced when pS5-CTD peptide is present. Mutagenesis of amino acid residues in the regions identified to be involved in CTD binding resulted in the specific inhibition of viral mRNA synthesis, confirming their importance. These data, in combination with cryo-EM analysis of the vRNA promoter bound FluPolC, have allowed us to propose a model where presence of promoter RNA and an interaction with the pS5-CTD of Pol II are key for the activation of the influenza virus transcriptase.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P035

N-TERMINAL ACETYLATION BY NATB IS REQUIRED FOR THE SHUTOFF ACTIVITY OF INFLUENZA A VIRUS PA-X Kohei Oishi* 1, Seiya Yamayoshi1, Hiroko Kozuka-Hata2, Masaaki Oyama2, Yoshihiro Kawaoka1 1Virology, 2Medical Proteomics Laboratory, University of Tokyo, Minato-ku, Japan

Abstract: N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. Influenza virus PA-X possesses endonuclease activity that suppresses (i.e., shuts off) host protein expression; however, the host proteins involved in this shutoff activity remain unknown. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of PA-X. The shutoff activity of wild-type PA-X was suppressed in NatB-deficient cells, and a PA-X mutant that was not acetylated by NatB showed reduced shutoff activity compared with wild-type PA-X. These data show that N-terminal acetylation by NatB is required for the shutoff activity of PA-X. To examine whether NatB is involved in the N-terminal acetylation of PA-X, PA-X expressed in wild-type or NatB- deficient cells, was analyzed for N-terminal modification. All detected N-terminal peptides of PA-X expressed in wild-type cells were N-terminally acetylated, whereas N-terminal acetylation was detected in approximately 50% of N-terminal peptides of PA-X derived from NatB-deficient cells. These data suggest that NatB is involved in the N-terminal acetylation of PA-X. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal amino acid sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells, whereas mutant PA that was not acetylated by NatB lost its functions in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation plays roles in both virus protein function and replication.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P036

IDENTIFICATION OF AN EBOLA VIRUS VP30-SPECIFIC KINASE THAT REGULATES VIRAL TRANSCRIPTION Yuki Takamatsu* 1, Nadine Biedenkopf1, Verena Krähling1, Larissa Kolesnikova1, Sandro Halwe1, Stefan Baumeister2, Stephan Becker1 1Institute of Virology, Philipps University Marburg, 2Department of Parasitology, Faculty of Biology, Philipps University Marburg, Marburg, Germany

Abstract: Ebola virus (EBOV) protein VP30 is phosphorylated predominantly at six N-proximal serine residues (S29-S31, S42, S44, and S46). While the EBOV polymerase complex composed of the polymerase L and VP35 executes genome replication, viral transcription requires VP30, an EBOV-specific transcriptional activator. Reversible phosphorylation of VP30 regulates EBOV polymerase function. Nonphosphorylated VP30 promotes genome transcription, whereas phosphorylated VP30 favors genome replication. Although phosphatases which dephosphorylate VP30 are identified as PP1 and PP2A, VP30- specific kinases remained elusive. Here we identified a VP30-specific kinase that is able to phosphorylate VP30 in vitro and in cellulo at the important position S29, to interact with VP30 in immunoprecipitation, colocalized with VP30 in viral inclusion bodies and regulate viral transcription. Our findings imply a novel therapeutic approach which focuses on VP30 phosphorylation.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P037

FUNCTIONAL CHARACTERIZATION OF THE HANTAVIRUS S SEGMENT UNTRANSLATED REGION Janne Tynell* 1, Jonas Klingström1 1MedH, KAROLINSKA INSTITUTET, Stockholm, Sweden

Abstract: Functional characterization of the hantavirus S segment untranslated region

Janne Tynell & Jonas Klingström

Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Hantaviruses are rodent- and insectivore-borne viruses estimated to cause over 100 000 human cases annually around the world. The trisegmented hantavirus RNA genome contains 3-4 open reading frames (ORFs) coding for the N, Gn, Gc and RNA-dependent RNA polymerase (RdRp) proteins as well as the non-structural NSs protein found in some hantavirus species. In addition to the ORFs, the hantavirus S segment contains a large untranslated region (UTR) comprising more than 20% of the entire segment. In an effort to assign functional significance to the S segment UTR, we have performed in vitro RNA pulldown assays and mass spectrometry analysis on human umbilical vein endothelial cell (HUVEC) lysates to identify possible interactions between host cell proteins and the S segment UTR. We present data obtained with various different pulldown techniques using S UTRs from the Puumala and Andes hantaviruses and discuss the significance of our results for hantavirus pathogenesis.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P038

DETAILED MAPPING OF THE VARIOUS POLYMERASE COFACTOR FUNCTIONS WITHIN THE PHOSPHOPROTEIN OF MEASLES VIRUS Louis-Marie Bloyet1, Antoine SCHRAMM 2, Carine Lazert3, Sonia Longhi2, Denis Gerlier* 1 1CIRI INSERM U1111, CNRS UMR5307, UNIVERSITY LYON 1, ENS LYON, UNIV LYON, Lyon, 2Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, CNRS, Aix Marseille University Marseille, Marseille, 3CIRI, International Center for Infectiology Research, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France

Abstract: The phosphoprotein (P) of Mononegavirales plays a central role in viral expression and multiplication. In Measles virus, P associates with nascent nucleoprotein (N) via an α-MoRE located at its N-terminus, to form the monomeric N0P complex. N0P is used as substrate for genome encapsidation by the polymerase. Via its C-terminal X domain, P dynamically tethers the L protein endowed with polymerase enzymatic activities on the helicoidal nucleocapsid (NC). NC is made of a continuous homopolymer of N protein wrapping the genomic RNA. The binding strength of XD-NC interaction tightly regulates the efficiency of polymerase scanning and/or transcription re-initiation at each intergenic junction. P associates with L to yield a functional polymerase complex. Its polymerase cofactor activity can be decomposed in at least three components: P provides HSP90-mediated chaperone and proper assistance to the folding of L into a fully functional polymerase; P allows the polymerase to dynamically bind to its NC template; and P enables the polymerase to be processive all along the genome. A protein complementation assay between a large panel (> one hundred) of P variants (e.g. truncated, chimeric and mutated forms) and L protein fragments indicated a major binding site located at the C terminus of P. L folding assays revealed the requirement of the C-ter of P multimerisation domain (PMD). Minigenome assays and recombinant biGbiS viruses coupled to biochemical, biophysical and structural studies unveiled a critical contribution of protein dynamics to the polymerase cofactor activity. The conserved modular organisation of P within the Paramyxoviridae family argues for a common functional organization.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P039

VIRAL TRANSCRIPTION DRIVES THE FORMATION OF VIRAL RNA GRANULES WITHIN RESPIRATORY SYNCYTIAL VIRUS INCLUSION BODIES V Rincheval1, Mickael Lelek2, Charles-Adrien Richard3, Jing Jing Cao4, Camille Bouillier1, Delphine Sitterlin1, Sabine Blouquit-Laye1, Marie Galloux3, Christophe Zimmer2, Ralf Altmeyer4, Jean-Francois Eleouet3, Marie-Anne Rameix-Welti* 1 1UMR 1173, INSERM - Université de Versailles St Quentin, Montigny-le-Bretonneux, 2UMR 3691, Institut Pasteur - CNRS, Paris, 3UR892 VIM, INRA, Jouy-en-Josas, France, 4Shandong University-Helmholtz Institute of Biotechnology , Qingdao, China

Abstract: We investigated functional organization of respiratory syncytial virus (RSV) cytoplasmic inclusion bodies (IBs). We found that viral RNA synthesis mainly occur in IBs and that newly synthetized viral mRNA concentrate in IBs sub compartments called IB associated granules (IBAG). Interestingly confocal microscopy and super-resolution microscopy revealed that M2-1, a RSV transcription antiterminator, accumulated in IBAGs when the other components of the viral polymerase (N, P, L) are excluded therefrom. Using a dicistronic minigenome system we found that IBAG formation is strictly dependent on viral RNA synthesis, but may occur in the absence of M2-1. Indeed, when omitting M2-1, FISH experiments revealed some IBAGs when using probes against the first gene (that can be transcribed without M2-1), but not probes against polyA or the second gene. IBAGs are highly dynamic structures, which seem to release their content periodically into the cytosol. Noteworthy, we found that RSV IBs exhibit properties of liquid organelles: spherical shape, fusion to form larger spherical structures, disappearance upon osmotic shock. Thus, IBAG formation could be regarded as a liquid-liquid phase separation resulting from accumulation of viral mRNA in IBs. The strict dependence of IBAG formation on viral RNA synthesis but not on M2-1 suggests that viral mRNA are the driving force of IBAG formation. Surprisingly, however, Cyclopamine, described as an RSV inhibitor targeting M2-1, was found capable of quickly disrupting IBAGs in RSV infected cells.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P040

INFLUENZA VIRUS RNA-POLYMERASE: TEMPERATURE-SENSITIVE MUTANTS IN PB1 DISPLAY A DEFECT IN NUCLEAR TARGETING OF THE PA-PB1 DIMER

Bruno Da Costa1, Laura Sedano1, Nathalie Lejal1, Ronan Le Goffic1, Rob Ruigrok2, Thibaut Crépin2, Bernard Delmas* 1 1Unité VIM, INRA, Jouy-en-Josas, 2IBS, Université de Grenoble, CEA, CNRS, Grenoble, France

Abstract: The influenza virus RNA-dependent RNA polymerase catalyses genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits, PB1, PB2, and PA are critical steps in replication and secondary transcription. We previously found that the PA linker plays a key role in nuclear targeting of the PA-PB1 dimer. Temperature-sensitive mutants in the linker are defective in the transport of the PA-PB1 dimer into the nucleus at restrictive temperature (39.5°C), resulting in a poor replication/transcription activity and suggesting an alteration of folding kinetic parameters. In this study, we generated numerous PB1 mutants engineered at the PA linker interface, the PB1 nuclear location signal, at positions known to promote a ts-phenotype to FluMist vaccine strains or at buried positions in the PB1 subunit or in the PA-PB1 dimer. Several PB1 mutants exhibited temperature-sensitivity with a reduced growth at 39.5°C versus 37°C/33°C. The ts-phenotype was also associated with a reduced efficiency of replication/transcription as measured in a minireplicon assay and to a defect in the transport of the PA-PB1 dimer into the nucleus at restrictive temperature. Complementation assays using PB1 mutants, wt-PA and importin-beta IPO5 revealed PB1 residues associated to the formation of a stable complex and involved in the efficient transport of PA-PB1 dimer into the nucleus. Taking our results as a whole, we propose that ts-sensitivity marks a local misfolding of the PB1 subunit that results in a defect of the stability of the PA-PB1 complex and consequently an inability of the PA-PB1-IPO5 complex to assemble and reach the nucleus.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P041

OPTIMISING INFLUENZA A REPORTER VIRUSES BY IN VIVO PASSAGING Monique Spronken* 1, Altagracia Russel1, Ron Fouchier1, Kirsty Short1, 2 1Viroscience, Erasmus Medical Centre, Rotterdam, Netherlands, 2School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Abstract: Influenza A reporter viruses are a valuable tool to study fundamental research questions regarding virus tropism and replication. To date, several influenza A reporter virus systems have been described that differ in attenuation, stability and expression levels. Additionally, it was shown that these systems are either dysfunctional or inferior when incorporated in human and avian circulating virus strains. In order to use reporter viruses to answer key research questions it’s essential that they can be detected by live in vivo imaging. Luminescent influenza A reporter viruses have been successfully used in live in vivo imaging experiments. However, this requires the administration of expensive substrates and it can’t be used for single cell detection and analysis. Therefore, the ideal reporter virus is fluorescently labelled so it can be used for both in vivo imaging and single cell analysis. With this in mind, we sought to improve the replication and signal strength of our previously described fluorescently labelled influenza viruses. We inserted reporters in the red spectrum (mCardinal and Katushka_2S) into the A/PR/8 PA gene segment containing promotor up mutations and a duplicated packaging region. The resultant A/PR/8 2UP_PA_mCardinal reporter virus was then passaged four times in C57BL/6 mice. This resulted in enhanced virus replication and increased percentages of reporter expression in the lungs of infected mice. Full sequencing of viral genomes from infected lungs showed a double peak at amino acid position 640 of the PB1 gene segment. This mutation was introduced in PB1 and the 2UP_PA_mCardinal virus was further studied in several in vitro assays to assess reporter expression levels, minireplicon activity and virus replication. As this mutation also enhanced reporter expression in vitro, it was further assessed in mice and preliminary experiments show enhanced replication and reporter expression.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P042

RIFT VALLEY FEVER PHLEBOVIRUS NUCLEOPROTEIN PROTEOMIC STUDIES IDENTIFY IMPORTANT WNT PATHWAY INTERACTIONS Timothy J. Mottram* 1, Margus Varjak1, Benjamin Brennan1, Alain Kohl1 1MRC-University of Glasgow Centre For Virus Research, Glasgow, United Kingdom

Abstract: Rift Valley fever phlebovirus (RVFV, Phenuviridae, Bunyavirales) is an important pathogen of both humans and livestock. RVFV transmission by mosquitoes across sub-Saharan Africa and the Arabian Peninsula has a significant impact on the socio-economics of these areas. Research interests have primarily focused on identifying interacting partners of the non-structural protein (NSs), encoded within the viral S RNA segment. However, the interaction partners of the nucleocapsid protein (N), remain largely unknown. Using a proteomics-based approach, we identified 24 potential mammalian host- derived N protein interaction partners. Following an siRNA screen utilising a viral minigenome system, the cellular proteins: β-Catenin, Polyadenylate binding protein 4, Scaffold attachment factor B and Annexin A2 appeared to be important for the formation of functional ribonucleoprotein (RNP) complexes. As β-Catenin is a known effector molecule of the WNT pathway, analysis following RVFV infection and minigenome transfection on the WNT pathway indicated a cell specific inhibition of the pathway. Additionally, β-Catenin knockout resulted in reduced viral replication indicating an important host-viral interaction. This was evidenced by a direct interaction of β-Catenin with RVFV N protein and evidence of relocalisation of β-Catenin from the plasma membrane. Understanding the fundamental biology, followed by further characterisation of these interactions will aid future development of new intervention strategies

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P043

IDENTIFICATION OF MUMPS VIRUS NUCLEOPROTEIN RESIDUE ENHANCES PRODUCTION OF DEFECTIVE INTERFERING PARTICLES Jacquline C. Risalvato* 1, James R. Zengel2, Ming Luo3, Biao He1 1Department of Infectious Disease, University of Georgia College of Veterinary Medicine, Athens, 2Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, 3Department of Chemistry, Georgia State University Center for Diagnostics and Therapeutics, Atlanta, United States

Abstract: Mumps virus (MuV) is a negative-sense single-stranded RNA virus belonging to the family Paramyxoviridae. A human pathogen, MuV is responsible for acute infection of the parotid glands, and can cause severe cases of encephalitis, meningitis, and deafness. The nonsegmented RNA genome of MuV is encapsidated by the nucleocapsid protein (NP), which forms the ribonucleoprotein (RNP) complex – which serves as a template for RNA synthesis. To make RNA accessible to the viral polymerase, a conformational change within NP must occur. Crystal structure analysis of the NP of parainfluenza virus 5 (PIV5), a paramyxovirus closely related to MuV, indicates that an α-helix close to the RNA genome becomes flexible when RNA is removed. This region of the NP is likely responsible for the conformational change which allows the polymerase to access RNA for transcription and replication. To examine the functionality of MuV’s NP, point mutations were made in MuV NP protein corresponding to PIV5 at sites G185P, A197Q, Q200R, and regions denoted as Top (N63G, P139D, A197Q), Tip (P109E, N121G, A124R), and Bottom (G21S, S29T, P43N, R93Q, R304Q). The “Top” MuV mutant exhibited normal growth kinetics at low multiplicity of infections (MOIs); however, at high MOI’s the virus could not efficiently replicate. Further analysis indicates that production of defective interfering (DI) particles was enhanced in the mutant virus. Understanding the production of DI particles, which can lead to increased interferon production, will invariably lead to a better understanding of MuV pathogenesis as well as its replication/transcription process.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P044

ALTERNATIVE SPLICING UNMASKS AN ENDOPLASMIC RETICULUM TARGETING SIGNAL OF BORNA DISEASE VIRUS NUCLEOPROTEIN Shohei Kojima* 1, 2, Ryo Sato3, Mako Yanai1, 2, Yumiko Komatsu2, 4, Masayuki Horie2, 5, Keizo Tomonaga1, 2, 6 1Graduate School of Biostudies, 2Lab. of RNA viruses, 3Faculty of Medicine, KYOTO UNIVERSITY, 4K-CONNEX, 5Hakubi Center, 6Graduate School of Medicine, KYOTO UNIVERSITY, Kyoto, Japan

Abstract: Borna disease virus (BoDV) is a non-segmented negative strand RNA virus which establishes intranuclear infection in mammals. BoDV exploits host pre-mRNA splicing machinery to express polycistronic transcripts of M, G and L. However, the detailed analysis of splice sites in other transcripts was not performed. Therefore, we set out to comprehensively analyze the splice junctions of BoDV mRNA using deep sequencing. We discovered that the transcripts encoding nucleoprotein (N) undergo mRNA-splicing. There were two introns within the N ORF, and spliced transcripts retained the protein reading frame, suggesting that the N gene expresses at least two truncation isoforms of N, named N2 and N3, by splicing. While full length N and N2 mainly localized in the nucleus, N3 translocated to the endoplasmic reticulum (ER) and was cleaved into a shorter form by host protease. We also found that the N protein intrinsically harbors a signal peptide and the truncation of full-length N into N3 works as a molecular switch for ER targeting. Lastly, we unveiled both N2 and N3 inhibited the BoDV minigenome activity, indicating that the N isoforms can regulate BoDV replication. Some reports previously showed that BoDV generates a N-terminally truncated isoform of N, which is important for elaborate control of BoDV polymerase activity. Our study adds novel insights into the molecular framework for how BoDV generates N isoforms of different cellular localization.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P045

THE OLIGOMERIC STATE OF MARBURG VIRUS VP40 INFLUENCES ITS ROLE IN MODULATING VIRAL TRANSCRIPTION AND REPLICATION Alexander Koehler* 1, Sebastian Pfeiffer1, Larissa Kolesnikova1, Stephan Becker1 1Institute of Virology, Philipps University Marburg, Marburg, Germany

Abstract: Marburg virus matrix protein, VP40, is crucial for viral assembly, budding and release; it counteracts the innate immune response and inhibits viral replication and transcription. VP40 is a peripheral membrane protein, which is partially membrane-associated forming an ordered VP40 lattice beneath the plasma membrane and the viral envelope, and partially soluble being diffusely distributed throughout the cell and detected also in viral inclusion bodies. Structural studies have suggested that the transition from VP40 monomers to oligomers is accompanied by the switch from a soluble to a membrane-associated form, the latter is essential for viral assembly and budding. It remains unclear whether the oligomeric state or membrane-binding of VP40 or both are important for the regulation of the innate immune response and/or viral replication and transcription. In the present study several mutations have been introduced into VP40 resulting in the formation of monomeric or dimeric forms of VP40; or oligomeric forms of VP40 with a reduced interaction with membranes. Monomeric or dimeric VP40 mutants co-localized partially with the viral inclusions and inhibited viral transcription and replication only weakly. VP40 mutants that formed higher oligomers but lack membrane association induced significant rearrangements of perinuclear inclusion bodies and strongly inhibited viral replication. Intriguingly, these VP40 mutants did not co-localize with the inclusion bodies, but were arranged along ER-bound ribosomes. Our results indicate that the oligomeric state of VP40 and lack of plasma membrane transport of VP40 oligomers, regulates VP40’s capacity to inhibit viral transcription and replication.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P046

TOWARDS THE DEVELOPMENT OF REVERSE GENETICS SYSTEMS FOR TOMATO SPOTTED WILT VIRUS Richard Kormelink1, Andre Bertran* 1, Magdalena Mazur1 1Laboratory of Virology, WAGENINGEN UNIVERSITY, Wageningen, Netherlands

Abstract: TOWARDS THE DEVELOPMENT OF REVERSE GENETICS SYSTEMS FOR TOMATO SPOTTED WILT VIRUS André Bertran, Magdalena J. Mazur and Richard Kormelink Laboratory of Virology, Wageningen University and Research, the Netherlands The order of Bunyavirales comprises a large group of emerging arthropod-borne viruses that primarily infect animals and of which some even pose a worldwide biological threat. During the last two decades various reverse genetics systems have been established to generate (mutant) viruses from cDNA clones for many different non-segmented and segmented (- )ssRNA viruses including bunyaviruses. Infectious clones are now available for almost all type species of the bunyaviruses, with the exception of the hantaviridae and tospoviridae. Here, we present our strategies towards the development of minireplicon systems for Tomato spotted wilt (TSWV), the prototype of the plant-infecting bunyaviruses, in mammalian and insect cell lines. To generate bona fide viral RNA templates, two distinct expression systems are being tested, namely T7 RNA polymerase and RNA polymerase I/II (pol I/ pol II). For both systems, we developed templates that allow qualitative (fluorescent reporter proteins) and quantitative (firefly and Renilla luciferase) measurements of minireplicon activity. As the replication /transcription activity is dependent on the formation of viral ribonucleoproteins (RNPs), the viral RNA templates are co-transfected with helper plasmids coding for the viral nucleocapsid (N) and polymerase (L) proteins. Using immunostaining we have successfully detected the N and L proteins in both mammalian and insect cells, and showed that they localize to cytoplasmic bodies. Current activities aim to establish RNP-dependent expression of the reporter genes and will be discussed.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P047

ROLE OF HOST PHOSPHATASE PP2A IN FILOVIRUS REPLICATION CYCLE Nadine Biedenkopf* 1, Cornelius Rohde1, Thomas Kruse2, Emil P. T. Hertz2, Jakob Nilsson2, Stephan Becker1 1Institute of Virology, PHILIPPS-UNIVERSITÄT MARBURG, Marburg, Germany, 2University of Copenhagen, Copenhagen, Denmark

Abstract: Ebolavirus and Marburg virus, both belonging to the family of Filoviridae, are the causative agents of a severe fever with high fatality rates among humans. The only nonpathogenic member within the Filoviridae is Ebolavirus subtype Reston Ebolavirus. Little is known regarding the molecular mechanisms these viruses utilize in order to transcribe and replicate their genome. Ebolavirus transcription is strongly dependent on dephosphorylation of the essential viral transcription factor VP30. Recently, we could show that the nucleoprotein NP recruits the host phosphatase PP2A-B56 via a B56 binding motif (LxxIxE) in order to dephosphorylate and thereby activate VP30. A peptide inhibitor of the LxxIxE motif resulted in inhibition of Ebolavirus transcription. Interestingly, the B56 binding motif LxxIxE is also found in other members of the family Filoviridae, including Marburg and Reston Ebolavirus. The role of VP30 dephosphorylation for transcription of these viruses is currently not understood. Here, we present data indicating that dephosphorylation of VP30 by PP2A B56 plays an important role for transcription of Reston Ebolavirus and Marburgvirus, as well.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P048

STRUCTURAL CHARACTERISATION OF THE METAPNEUMOVIRUS TRANSCRIPTASE COMPONENTS P AND M2-1 BY MODELLING, CRYSTALLOGRAPHY, AND SAXS Max Renner* 1, Jonathan Grimes1, Cédric Leyrat2 1Division of Structural Biology, UNIVERSITY OF OXFORD, Oxford, United Kingdom, 2Institut de Génomique Fonctionnelle, Montpellier, France

Abstract: The phosphoprotein (P) is the essential cofactor of the RNA-dependent RNA polymerase (L) of unsegmented, (- )RNA viruses. P serves as a bridge between the viral polymerase and its nucleoprotein-RNA template and prevents unproductive encapsidation of host nucleic acids by the nucleoprotein (N). In addition, P recruits the viral transcription antiterminator M2-1, which is crucial for the synthesis of full-length mRNAs in pneumoviruses. P proteins of negative strand viruses form multifunctional homomultimers with large intrinsically disordered regions (IDRs). In human metapneumovirus (HMPV) P forms homotetramers via a stable central coiled-coil domain (Pcore) flanked by IDRs. Here we combined x-ray crystallography with small angle x-ray scattering (SAXS) and molecular modelling to characterise the P protein and its interaction with the transcription antiterminator M2-1. Our structural description of P and its complex with M2-1 captures the dynamic and flexible character of pneumoviral transcriptase components and highlights the presence of transiently stable elements within the IDRs.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P049

CHARACTERIZING DEFECTIVE VIRAL GENOMES OF CANINE DISTEMPER VIRUS Natasha L. Tilston-Lunel* 1, Linda J. Rennick1, Stephen R. Welch2, Sham Nambulli1, Christina F. Spiropoulou2, Stuart T. Nichol2, W. Paul Duprex1 1Microbiology, NEIDL, Boston University School of Medicine, Boston, 2VSPB, Centers for Disease Control and Prevention, Atlanta, United States

Abstract: The error-prone nature of RNA-dependent RNA polymerases (RdRp) drives the diversity we observe in RNA viral populations. A sub-species of this diversity are defective viral genomes (DVGs) which arise due to premature termination of genome synthesis by the RdRp and its reinitiation either onto the same template or onto the newly synthesized strand. Replication competent DVGs alter the dynamics of a viral population by their ability to interfere with standard virus replication and/or by stimulating type-I IFN induction. Such DVGs are known as defective interfering (DI) genomes and these have been studied for many RNA viruses. Dissecting molecular mechanisms that contribute to DI generation and inhibition could potentially allow us to harness the interfering traits for use as vaccine adjuvants or broad-spectrum antivirals. An idea that has been proposed and studied for influenza virus. Here, we present work carried out using canine distemper virus (CDV). CDV is a tractable biosafety level-2 (BSL-2) paramyxovirus. Using recombinant (r) CDVRI we cultured various independently generated rCDVRI stocks across 10 passages in Vero-cCD150 cells. Passages were subjected to a combination of Sanger sequencing and next-generation sequencing to identify DVGs arising naturally in vitro. Isolated DVGs were cloned and assessed for their ability to interfere with rCDVRI replication in vitro. Further, using a CDV minigenome expressing a fluorescent reporter we demonstrate that a synthetic DVG can be maintained along with the full-length virus over several passages in an IFN competent canine B-cell line (CLBL-1). We have an established pipeline to engineer natural and synthetic DIs which is transferrable to other paramyxoviruses. Understanding the common mechanisms of inhibition by natural DIs could potentially be powerful against the continuously evolving nature of RNA viruses.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P050

RABIES VIRUS L-PROTEIN C-TERMINAL DOMAINS ARE STRUCTURALLY COORDINATED BY THE N-TERMINUS OF THE VIRAL PHOSPHOPROTEIN Joshua Horwitz* 1, Stephen Harrison2, Sean Whelan1 1MBIB, 2BCMP, Harvard Medical School, Boston, United States

Abstract: The 250 kDa rabies virus large protein (L) contains all of the enzymatic activities necessary for mRNA transcription and genome replication. The template for RNA synthesis is the viral genomic RNA completely coated by a sheath of the viral nucleocapsid protein (N), and L must bind the viral phosphoprotein (P) to engage the N-RNA template. Using an in vitro assay comprising purified rabies L protein and chemically synthesized short template RNA, we previously defined a minimal region of P that stimulates the RNA dependent RNA polymerase activity of rabies virus L. Using electron cryo-microscopy we previously provided an atomic model of L for the related vesicular stomatitis virus. L comprises five distinct domains, including three enzymatic domains: an N-terminal RNA-dependent RNA polymerase (RdRP) and RNA capping enzyme (Cap), and three globular C-terminal domains, including a connector domain (CD), methyltransferase domain (MT), and C-terminal domain (CTD). Using negative-stain electron microscopy (nsEM) of rabies virus L we show that the globular C-terminal domains of L are highly flexible relative to the N-terminal RdRP and Cap domains in the absence of P. We identify a minimal region of P that coordinates these C-terminal domains atop the RdRP and Cap domains. Additionally, by fusing GFP to the N-terminus of P, we can approximate the location of the N-terminus of P with respect to the C-terminal domains of L.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P051

EFFECT OF MUTATIONS IN THE GENE-END SEQUENCE ON RSV TRANSCRIPTION Jean-Francois Eleouet1, Charles-Adrien Richard* 1 1Unité de Virologie et Immunologie Moléculaires, INRA - Université Paris-Saclay, Jouy en Josas, France

Abstract: The respiratory syncytial virus (RSV) genome is a single strand, negative sense RNA of about 15 kb that is packaged by the nucleoprotein (N) and maintained as a left-handed helical N-RNA ribonucleoprotein complex (RNP). This RNP is the template for two distinct activities: RNA replication and RNA transcription that generates 10 capped and poly- adenylated mRNAs. RNA transcription is carried out by the viral RNA-dependent RNA polymerase complex (RdRp), composed of the viral N, P, L and M2-1 proteins. RSV transcription proceeds through sequential stop-and-restart events, in which the RdRp recognizes gene start (GS) and gene end (GE) sequences that flank each gene and direct initiation and termination of transcription, respectively. By increasing the processivity of the RdRp, RSV transcription antiterminator protein M2-1 prevents premature transcription termination. M2-1 is an RNA binding protein that binds preferentially to GE and A-rich sequences present on RSV mRNAs. The exact mechanism of how M2-1 improves transcription and its relation with its RNA binding abilities remain to be clarified. In this work, the effect of GE sequence variation was analyzed by using a bicistronic RSV minigenome coding for Gaussia and Firefly luciferases. Relative expression of the Gaussia and Firefly luciferase reporters were compared between wild type or mutated GE sequences in the absence or in the presence of M2-1. The results highlighted the critical role of some nucleotides in the GE sequence for efficient transcription termination of the first gene and reinitiation at the second GS signal.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P052

THE C-TERMINAL FRAGMENT OF THE RESPIRATORY SYNCYTIAL VIRUS PHOSPHOPROTEIN INHIBITS THE VIRAL POLYMERASE ACTIVITY Koyu Hara* 1, Kenichiro Yaita1, Takahito Kashiwagi1, Hiroshi Watanabe1 1Department of Infection Control and Prevention, KURUME UNIVERSITY SCHOOL OF MEDICINE, Kurume, Japan

Abstract: 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 demonstrate that a fragment of RSV phosphoprotein (P) has an ability to inhibit the viral polymerase activity and suggest a significant potential to treat RSV infection. Viral ribonucleoprotein (RNP) was reconstituted by transfecting plasmids expressing P, RNA polymerase (L), nucleoprotein (N), M2-1 and vRNA-like luciferase RNA into BHK-21 derived BSR-T7/5 cells, stably expressing T7 RNA polymerase (gifted from Dr. Jean-François Eléouët). Concurrently, a plasmid expressing serial P fragments was transfected as a competitor and the luciferase activity was measured at 24h post transfection to evaluate the polymerase activity. We found that C-terminal half fragment of P severely inhibited the polymerase activity. A precise deletion analysis identified the three domains essential for the inhibitory activity: the oligomerization domain (aa 130-150), N-binding domain (aa 160-180) and L binding domain (aa 212-241). When the TAP- tagged P fragment was co-expressed with each component of RNP and purified by TAP-tag method, the P fragment was able to pull-down P, L and M2-1. Specifically, a strong interaction with the full-length P was observed. The P protein forms homotetramers and acts as an essential cofactor of the viral polymerase L by recruiting L to the nucleocapsid. Our results suggest that the P fragment primarily targets the full-length P, possibly interfering the tetramer formation, thereby inhibiting the polymerase activity.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P053

INVESTIGATION OF A PROTEIN KINASE RESPONSIBLE FOR THE PHOSPHORYLATION OF MEASLES VIRUS NUCLEOPROTEIN Akihiro Sugai* 1, Hiroki Sato1, Misako Yoneda1, Chieko Kai1 1The Institute of Medical Science, Laboratory Animal Research Center, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Measles virus (MeV) N and P proteins are known to be phosphorylated to modify viral gene expression and viral genomic stability. It has been reported that Casein kinase II phosphorylates the P protein, while a responsible protein kinase for the phosphorylation of the N protein has not been identified. The N protein is typically phosphorylated in its tail-domain at S479 and S510. The mechanism phosphorylating these sites of the N protein, however, has not been clarified. In this study, we conducted in vitro kinase assay in the presence or absence of various protein kinase inhibitors to identify a responsible protein kinase for the phosphorylation of the N protein. We first purified non-phosphorylated form of the GST- tagged N-tail domain (GST-Nt) as a substrate and conducted in vitro kinase assay using 293 or COS7 cell lysate. We demonstrated that these cell lysate phosphorylated the GST-Nt successfully. This phosphorylation signal was not derived from GST-tag. Conversely, these cell lysate failed to phosphorylate the GST-Nt in the presence of c-Jun N-terminal kinase (JNK) inhibitor (SP600125). We also showed that the JNK inhibitor suppressed the phosphorylation of transiently expressed full-length N protein in COS7 cells. Using the specific antibodies against phosphorylated S479 or S510, we demonstrated that JNK inhibitor was able to block both of the phosphorylation modification, suggesting that S479 and S510 sites were phosphorylated by the same protein kinase. These findings are helpful in identifying a responsible kinase that phosphorylates S479 and S510 of N protein and also in understanding the mechanism regulating phosphorylation of the N protein.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P054

DISCOVERY AND DETECTION OF EBOV-ENCODED MICRORNA-LIKE MOLECULES IN INFECTED CELLS AND NON-HUMAN PRIMATES Ashley L. Silvia* 1, 2, Anthony Griffiths1, 2 1Texas Biomedical Research Institute, 2UT Health, San Antonio, United States

Abstract: MicroRNAs (miRNAs) are important regulators of various cellular processes and can be used as biomarkers for disease. Some viruses have been shown to encode miRNAs that may act using similar mechanisms as host miRNAs to affect viral or host transcripts. Several reports have predicted Ebola virus (EBOV) encoded miRNA-like molecules using computer-based algorithms, however, studies to detect EBOV-encoded miRNAs during infection have been limited to one study using human serum. Using small RNA deep sequencing, we detected both novel and previously predicted EBOV- encoded miRNA-like molecules in various infected cell lines 26 hours post infection. Using the sequencing data, we designed custom qPCR TaqMan miRNA assays to quantify expression in various cell lines at 1, 6, and 26 hours post infection and in sera from EBOV-infected non-human primates (NHPs). Future experiments will aim to address the biogenesis and potential role of these molecules during EBOV infection.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P055

STRUCTURAL CHARACTERIZATION OF RSV PHOSPHOPROTEIN Christophe Cardone* 1, Nelson Pereira1, Safa Lassoued1, Charles-Adrien Richard2, Jenna Fix2, Marie Galloux2, Jean- François Eléouët2, Christina Sizun1 1CNRS, ICSN, Gif-sur-Yvette Cedex, 2INRA, VIM, Jouy-en-Josas, France

Abstract: In Mononegavirales, the phosphoprotein (P) is the main co-factor of the viral RNA polymerase. It positions the polymerase complex onto its template, a ribonucleoprotein complex formed of genomic RNA and the associated nucleoprotein. P proteins recruit viral and cellular proteins to the viral RNA dependent RNA polymerase (RdRp) complex. The mechanisms of action of these proteins are still not fully understood. In particular they often elude structural characterization, owing to extensive structural disorder. Among Mononegavirales, human Respiratory Syncytial Virus (hRSV), of the Pneumoviridae family, is the main viral cause of lower respiratory tract illness worldwide. We have undertaken a structural investigation of hRSV P and of its interactions by using nuclear magnetic resonance spectroscopy, an atomic scale tool well adapted to study highly dynamic proteins. hRSV P is a 241-residue protein with a short ~40 residue oligomerization domain (OD), flanked by large intrinsically disordered regions (IDRs) at its N-and C-termini. On closer inspection, NMR shows that these IDRs are very heterogeneous. Almost stable C-terminal helices are formed downstream of the OD. Very weak helical propensity is observed in two N-terminal regions. All transient helices as well as the OD mediate transient internal long-range contacts. These likely contribute to overall compaction of P in the absence of a defined tertiary structure, protection against proteolysis and/or unspecific contacts. Indeed, these transient secondary structure elements in hRSV P provide protein binding sites that are specifically recognized by RdRp proteins, like the hRSV transcription antitermination factor M2-1. Very recently we found that a transient extended region next to the M2-1 binding site was recognized by the cellular phosphatase PP1. The proximity of both sites in P tetramers explains how P-dependent dephosphorylation of M2-1 by PP1 can take place.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P056

DETECTION AND CHARACTERISATION OF INFLUENZA VIRUS RNA POLYMERASE DIMERS USING BIMOLECULAR FLUORESCENCE COMPLEMENTATION (BIFC) Alex Walker* 1, Haitian Fan1, David Bauer1, Ervin Fodor1 1Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Abstract: Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA. The RdRP carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins (RNPs). Replication of negative-sense viral RNA is a two-step process, progressing via a positive-sense complementary RNA intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP-RdRP interactions may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography and SAXS analysis our group has identified the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We establish a bimolecular fluorescence complementation (BiFC) assay to monitor intermolecular interactions between RdRPs in cells expressing viral RNPs. Using this system we confirm the existence of RdRP dimers in the context of actively replicating RNPs. Mutating amino acid residues at the identified dimer interface causes loss dimerization and inhibition of RNA replication in minigenome assays. These data suggest that dimerisation of RdRP via the PA C-terminal domain is important for replication of the viral RNA genome.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P057

THE CONNECTOR DOMAIN OF VESICULAR STOMATITIS VIRUS LARGE PROTEIN IS AN ACCEPTOR SITE FOR PHOSPHOPROTEIN BINDING Joseph Gould* 1, Shihong Qiu1, Qiao Shang1, Peter Prevelige1, Chad Petit2, Todd Green1 1Microbiology, 2Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, United States

Abstract: Vesicular stomatitis virus (VSV) is an archetypical negative strand virus with a simple molecular biology. In addition to its importance as a model system for the study of Mononegavirales, VSV has been explored as a potential oncolytic agent and as a vaccine platform. VSV encodes for a large (L) protein, which supplies all catalytic activities for viral RNA synthesis and transcript modification, and phospho- (P) protein, a multifunctional molecular chaperone and cofactor. In order for viral mRNA and genomic RNA synthesis to take place, an interaction between the L and P proteins must occur. Despite the importance of L-P interaction(s), the interface or interfaces at which they occur have not been described. Using structural and biophysical methods, we report here that the connector domain of VSV L, which previously had no assigned functional significance, is an acceptor site for P-protein binding. We offer a model of the connector-phosphoprotein interface, supplemented by functional information from targeted disruption of the interaction in a minigenome reporter system. Given the extensive conservation of large and phosphoproteins in Mononegavirales, we assert this discovery has implications for studies of negative strand viral RNA synthesis as a whole.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P058

MEASLES VIRUS PHOSPHOPROTEIN MULTIMERIZATION DOMAIN: COILED-COIL FEATURES AND FUNCTION Antoine SCHRAMM* 1, Louis-Marie BLOYET2, Carine LAZERT2, Maggy HOLOGNE3, Olivier WALKER3, Denis GERLIER2, Sonia LONGHI1 1CNRS UMR 7257, Aix Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, 2CIRI INSERM U1111, CNRS UMR5307, University Lyon 1, ENS Lyon, 3CIRI INSERM U1111, CNRS UMR5308, University Lyon 1, ENS Lyon, Univ Lyon, Lyon, France

Abstract: Measles virus (MeV) phosphoprotein (P) plays a central role in viral transcription and replication. Understanding the relationships between sequence, structure, dynamics and function of P is crucial to draw a consistent mechanistic model for viral transcription and replication. This 54 kDa protein is prevalently intrinsically disordered with the notable exception of a small 3-helix bundle (termed X domain, XD) and a coiled-coil multimerization domain (PMD). Coiled-coils consist in multiple a-helices packed together in a fiber shape that results from a repeated seven residues motif (heptad). MeV PMD is tetrameric and features a stammer, i.e. a three-residues insertion between two heptad repeats. This induces the formation of a 310 helix and hence to a local distortion referred to as “kink”. To achieve further insights onto the roles played by PMD, PMD variants were characterized in cellula, in vitro and in silico in order to understand how PMD may influence the polymerase cofactor activity of P. Biochemical and biophysical characterization made use of circular dichroism, X-ray crystallography and molecular dynamic simulations. The introduction of a proline causes the 310 helix to shift with no concurrent functional impact on transcription and replication. In parallel, we investigated the coiled-coil cohesion by substituting buried residues with residues of varying hydrophobicity. The variants possess the same oligomeric state as that of the wild-type P protein but exhibit different stabilities and activities in a minigenome assay.

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EXPRESSING AND MULTIPLYING – viral gene expression Abstract final identifier: P059

COMBINED PROTEOMICS AND TRANSCRIPTOMICS SUGGESTS THAT M1 MRNA SPLICING INFLUENCES IAV HOST SPECIFICITY Boris Bogdanow* 1, 2, Anne Sadewasser1, Gudrun Heins1, Immanuel Husic2, Katharina Paki1, Barbara Vetter3, Xi Wang4, Jingyi Hou4, Wei Chen4, Lüder Wiebusch3, Thorsten Wolff1, Matthias Selbach2 1Unit 17, Robert-Koch-Institute, 2Proteome Dynamics, Max-Delbrück-Center, 3Laboratory for Pediatric Molecular Biology, Charité University Medicine, 4BIMSB, Max-Delbrück-Center, Berlin, Germany

Abstract: A successful Influenza A virus (IAV) infection requires that viral proteins are synthesized at the right time in the right order and correct proportions. Many avian IAV strains lack adaptation to the human host, which leads to abortive non- productive infections in human cell lines. Here we used transcriptomic and proteomic methods to assess the regulation of viral and host mRNA and protein production comparatively for an avian (H3N2/A/Mallard) and a human (H3N2/A/Panama) influenza isolate. First, we monitored protein synthesis profiles in human A549 cells in 4 h intervals over a 16 h period. We observed the expected overall shut-down in host protein synthesis for both strains, which was more pronounced at later intervals. Cluster analyses revealed subsets of host proteins that escaped the shut-down, including proteins involved in antiviral response, immune response and translation. Concomitantly, synthesis of viral proteins was potently induced and showed striking strain differences for the M1, HA and NA proteins. mRNA levels obtained via RNA-seq indicate that the differences in protein production between the two strains could largely be explained by differences at the mRNA level. The impairment of the avian strain to produce high amounts of M1 was linked to an increased intensity of M1 mRNA splicing. Experiments using eukaryotic expression vectors containing the coding sequence of the M segment indicate that the 3' splice site is responsible for strain-specific control of M1 mRNA splicing. We then integrated the avian segment 7 3’ splice site into the Panama strain. This mutant virus replicated to lower titers and produced lower levels of M1 mRNA and protein than the wildtype. This suggests that control of M1 mRNA splicing by the 3’ splice site influences IAV host specificity.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P060

MXA-DEPENDENT INFLAMMASOME RESTRICTS INFLUENZA A VIRUS INFECTION IN RESPIRATORY EPITHELIAL CELLS SangJoon Lee1, Peter Staeheli2, Kyosuke Nagata3, Atsushi Kawaguchi* 3 1PhD Program in Human Biology, University of Tsukuba, Tsukuba, Japan, 2Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 3Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

Abstract: Inflammasomes are cytosolic molecular complexes that typically consist of ASC, caspase-1, and cytoplasmic pathogen recognition receptors (PRRs) such as NOD-like receptor family proteins. Previous studies demonstrated that influenza A virus (IAV) infection triggers NLRP3-mediated inflammasome in immune cells. However, NLRP3 is not expressed in respiratory epithelial cells, although an inflammasome is formed upon IAV infection. To identify the novel respiratory epithelium-specific inflammasome receptor, we performed the high-content shRNA library screening and LC-MS proteomics analysis with human respiratory epithelial cells. Using two different screening systems, we identified human myxovirus resistance protein 1 (MxA) as a novel inflammasome receptor in respiratory epithelial cells that specifically forms inflammasome complexes with ASC and caspase-1 upon viral infection. The secretion of IL-1β upon IAV infection was reduced by MxA knockdown. The reduction of IL-1β secretion by MxA knockdown was complemented by NLRP3 expression in respiratory epithelial cells. In vivo analysis using hMxA-transgenic mice revealed that the rapid activation of the MxA inflammasome in bronchiolar epithelial cells represses virus spread from the bronchioles to distal alveolar regions. Our study highlights a novel function of MxA that is required for inflammasome activation in the respiratory epithelium after IAV infection which enhances virus resistance.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P061

CHARACTERIZATION OF VIRUS - HOST INTERACTION DYNAMICS WITHIN THE RESPIRATORY EPITHELIUM Ronald Dijkman* 1, 2, Melle Holwerda1, 2, Hulda Jonsdottir1, 2, Volker Thiel1, 2 1Federal Food Safety and Veterinary Office, Institute of Virology & Immunology, Bern and Mittelhäusern, 2Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty of the University of Bern, Bern, Switzerland

Abstract: The respiratory epithelium is the main entry port of respiratory pathogens and serves as an important barrier to infection. Within the respiratory epithelium the innate immune system plays a major protective role as the first line of defence. However, the dynamics of the innate immune response towards respiratory pathogens at the main entry port is not well understood. To characterize these complex dynamics,we established transgenic primary human Airway Epithelial Cell (hAEC) cultures harbouring a reporter cassette with either fluorescent or enzymatic reporter proteins under the control of a promoter element known to be induced during the intermediate (e.g. IFNB1) or late (e.g. MxA) stage of the interferon (IFN) response. Besides phenotypical comparison, the functional comparison of these reporter hAECs with naïve hAECs using exogenous stimuli revealed similar characteristics. Furthermore, infection experiments with Influenza A virus H1N1 (pdm2009) wildtype, or a NS1 mutant, demonstrated no significant differences of viral kinetics between reporter or naïve hAECs. We confirmed that pdmH1N1 does not evoke a pronounced IFN response during infection, in contrast to the pdmH1N1 NS1 mutant, suggesting that pdmH1N1 is well equipped to evade the first line of defence at the main entry port. Most importantly, the reporter hAECs allow for active monitoring of the innate immune response, providing a robust method to gain detailed knowledge on virus – host interaction dynamics within the respiratory epithelium.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P062

NUCLEAR RESIDENT RIG-I SENSES INFLUENZA A VIRUS REPLICATION MOUNTING AN ANTIVIRAL RESPONSE Guanqun Liu1, Yao Lu1, Qiang Liu1, Yan Zhou* 1 1University of Saskatchewan, Saskatoon, Canada

Abstract: The spatiotemporal detection of influenza A virus (IAV) by RIG-I is puzzling; as a rare nuclear-replicating RNA virus, IAV conceals its genome replication in the nucleus potentially limiting the access of cytoplasmic RNA sensors. Here, we report the existence of nuclear RIG-I under homeostatic conditions sensing IAV nuclear replication. We demonstrate an intimate association of RIG-I activation with nuclear viral RNA accumulation, and specific nuclear RIG-I staining unless circumvented by the introduction of a nuclear export signal. A nuclear-localized RIG-I efficiently mediated a type I interferon response via the canonical signaling axis, and exhibited greater signaling capacity following the recognition of viral replication. Reconstitution of cells with nuclear RIG-I exerted exclusive sensing and restriction of IAV, but not of cytoplasmic- replicating Sendai virus. These results demonstrate for the first time, the sensing of a RNA virus by RIG-I within the nucleus and implicate a previously unrecognized subcellular milieu for RLR sensing.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P063

SPECIFICITY AND FUNCTIONAL INTERPLAY BETWEEN INFLUENZA VIRUS PA-X AND NS1 SHUTOFF ACTIVITY Toru Takimoto* 1, Chutikarn Chaimayo1, Megan Dunagan1 1Microbiology & Immunology, University of Rochester Medical Center, Rochester, United States

Abstract: Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS1 proteins, the virus is capable of suppressing host protein synthesis, termed “host shutoff.” Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (pH1N1) viruses containing mutations affecting the expression of active PA-X and NS1. We then analyzed viral growth, the kinetics of viral RNA and protein synthesis, host shutoff function, and specific mRNA targets of these mutants. Our results show that PA-X is the major protein that affects general host protein expression. Intriguingly, our RNA-Seq data from infected human airway A549 cells indicate that active NS1 specifically targets host mRNAs related to interferon (IFN) and cytokine signaling pathways. Specificity of target mRNAs was less evident in PA-X, although it preferentially degraded transcripts from genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of active NS1, PA-X also degraded viral mRNAs, especially NS segments. The virus expressing active NS1 with reduced amount of PA-X most efficiently suppressed antiviral and innate immune responses in human cells, indicating the need for influenza virus to optimize the contribution of these two shutoff proteins to circumvent the host environment for optimum growth.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P064

THE AIRWAY EPITHELIUM MAINTAINS THE BARRIER FUNCTION AFTER INFLUENZA VIRUS INFECTION DESPITE THE EXTENSIVE LOSS OF CILIATED CELLS Georg Herrler* 1, Nai-Huei Wu1, Wei Yang1, Peter Valentin-Weigand2 1Virology, 2Microbiology, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany

Abstract: We established an air-liquid interface (ALI) culture system to analyze the infection of differentiated airway epithelial cells by influenza viruses. Porcine ALI-cultures were sensitive to infection by swine and human influenza viruses. Release of virus at a high level was observed for up to eight dpi. Infection was characterized by a dramatic change of the epithelium. A large number of ciliated cells were lost due to virus-induced apoptosis. As a consequence, the thickness of the epithelial layer was reduced. However, the epithelial cell layer remained intact and there was no decrease of the transepithelial electrical resistance (TEER) and the tight junction (TJ) network was not destroyed. Our findings are explained by the regeneration of epithelial cells to compensate for the loss of ciliated cells. Basal cells had started to differentiate into specialized cells. The early differentiation process was sufficient to maintain the barrier function as indicated by the TJ network and the TEER. However, the differentiation process had not yet proceeded to the generation of ciliated cells. During differentiation, the epithelial cells showed different surface properties as compared to well-differentiated cells. The latter cells were characterized by the presence of a2,6-linked sialic acid, whereas basal cells mainly contained a2,3-linked sialic acid. Lectin staining indicated that both linkage types are present on the surface of regenerating cells. Because of the different surface markers, regenerating and well-differentiated airway epithelial cells may have different susceptibilities to infection by viral and bacterial pathogens Taken together, the ALI culture system allows to analyze the regeneration of airway epithelial cells after influenza virus infection. These cells will be valuable to study viral-viral and viral-bacterial co-infections.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P065

INSIGHTS INTO TETHERIN COUNTERACTION BY THE EBOLA VIRUS GLYCOPROTEIN Markus Hoffmann1, Mariana González-Hernández1, Inga Nehlmeier1, Constantin Brinkmann1, Verena Krähling2, Laura Behner2, Anna-Sophie Moldenhauer1, Nadine Krüger3, Julia Nehls4, Michael Schindler4, Andrea Maisner2, Stephan Becker2, Stefan Pöhlmann* 1 1Infection Biology Unit, German Primate Center, Göttingen, 2Institute of Virology, Philipps-University Marburg, Marburg, 3Institute of Virology, University of Veterinary Medicine Hannover, Hannover, 4Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany

Abstract: Tetherin is an interferon (IFN) induced host cell factor that can inhibit release of progeny virions from infected cells. The Ebola virus glycoprotein (EBOV-GP) counteracts tetherin in transfected cells but the determinants in GP that control tetherin antagonism are incompletely understood. Moreover, it is unclear whether tetherin counteraction occurs in infected cells and contributes to viral spread. Here, we show that residues in the receptor binding domain of GP and a GXXXA motif in the transmembrane domain (TMD) of GP are required for counteraction of human tetherin. In addition, we provide the first evidence that GP can antagonize human tetherin in the context of an infectious, replication-competent EBOV surrogate – chimeric VSV encoding EBOV-GP. Finally, we found that tetherin of fruit bats, the EBOV reservoir, is largely resistant to counteraction by EBOV-GP, at least when expressed at high levels, and that tetherin expression is IFN- inducible in fruit bat cells. More importantly, we discovered that tetherin expression is critical for efficient IFN-mediated inhibition of EBOV and Nipah virus (NiV) infection of fruit bat cells. Collectively, our results identify determinants in GP that are required for counteraction of human tetherin and provide evidence that counteraction may promote viral spread in infected cells. Moreover, our findings indicate that tetherin might be important for IFN-dependent control of EBOV and NiV infection of fruit bats, the natural reservoir.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P066

INFLUENZA VIRUS MOUNTS A TWO-PRONGED ATTACK ON RNA POLYMERASE II TRANSCRIPTION David L. V. Bauer* 1, Michael Tellier1, Mónica Martínez-Alonso1, Takayuki Nojima1, Nick J. Proudfoot1, Shona Murphy1, Ervin Fodor1 1Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Abstract: Influenza virus intimately associates with host RNA Polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian nascent elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, cleavage of nascent host transcripts by the viral polymerase causes Pol II to terminate prematurely at the start of genes. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously-reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions, but also fundamental aspects of mammalian transcription. Graphical Abstract:

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P067

FLYING UNDER THE (R)ADAR: ADAR1-P150 EDITING COUNTERACTS INTRINSIC IMMUNITY ACTIVATION BY SELF AND VIRAL DOUBLE STRANDED RNA Christian K. Pfaller* 1, Ryan C. Donohue1, 2, Stepan Nersisyan3, 4, Leonid Brodsky4, Roberto Cattaneo1, 2 1Molecular Medicine, 2Mayo Clinic Graduate School of Biomedical Sciences, MAYO CLINIC, Rochester, United States, 3Lomonosov Moscow State University, Moscow, Russian Federation, 4Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel

Abstract: Adenosine deaminase acting on RNA 1 (ADAR1) edits and destabilizes double-strand RNA (dsRNA). This prevents translational shutdown through PKR, but favors viral replication. To understand how, we generated ADAR1- knockout cells, and cells expressing either nuclear ADAR1p110, or interferon-inducible cytoplasmic ADAR1p150. We show here that ADAR1 primarily edits Alu elements embedded in opposite polarity in 3’ untranslated regions (UTRs) of more than hundred polymerase II transcripts. Through fine mapping of A-to-I editing events in RNA deep sequencing analyses, we determined editing scores for each position in these long UTRs, and validated the editing patterns in primary human samples. Edited elements form extensive dsRNA structures conserved in primates, and even in rodents, confirming their broad biological relevance. They activate intrinsic immune responses in ADAR1-deficient cells, inhibiting viral replication. Reconstitution with catalytically active ADAR1p150 suppresses autoimmunity and rescues replication of measles virus (MeV), but not that of a mutant generating excess dsRNA, which activates PKR. In contrast, catalytically inactive ADAR1p150 has modest effects. We also analyzed editing of dsRNA structures in MeV genomes, concluding that these structures underlie the same type of editing as cellular dsRNA. Thus, our data indicate that intrinsic immunity recognizes self and foreign dsRNA through the same ADAR1-dependent mechanism. By interfering with the detection of low amounts of dsRNA, ADAR1 increases tolerance to autoimmunity but weakens the antiviral response.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P068

GLOBAL SIRNA SCREEN IN HUMAN MACROPHAGES IDENTIFY TBC1D5 AS A NOVEL CELLULAR RESTRICTION FACTOR FOR INFLUENZA A VIRUS REPLICATION Laura Martin-Sancho* 1, Ariel Rodriguez-Frandsen1, Shashank Tripathi2, Maite Sanchez-Aparicio2, Judd Hultquist3, David Jimenez-Morales3, Paul De Jesus1, Max Chang4, Hong Moulton5, David Stein5, Chris Benner4, Megan Shaw2, Nevan Krogan3, Adolfo Garcia-Sastre2, Sumit Chanda1 1Infectious and Inflammatory Center, SBP Medical Discovery Institute, La Jolla, 2Microbiology, Icahn School of Medicine at Mount Sinai, New York, 3Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, 4School of Medicine, University of California San Diego , La Jolla, 5Department of Biomedical Sciences, Oregon State University, Corvallis, United States

Abstract: Influenza A virus (IAV) is an important respiratory pathogen. Current treatments to combat influenza infection are suboptimal and new strategies are required. Targeting cellular factors that naturally prevent influenza infection represents a powerful alternative approach, since these will benefit from broad spectrum activities and are likely to confer a higher barrier to viral resistance. Using genome-wide siRNA screening in human macrophages, we successfully identified 104 novel restriction factors that affect the replication of various IAV strains differing in pathogenesis. This data was integrated with global transcriptomic and proteomic datasets generated using the same experimental settings, to identify those host factors that are regulated upon IAV infection and that modulate their responses through physical interactions. Among the identified host factors, we focused on the GTPase-activating protein TBC1D5. TBC1D5 negative effect on viral replication was validated using CRISPR KO cells and in vivo models. Additionally, we detected co-localization of TBC1D5 and M2 in the perinuclear region of IAV-infected cells. Critically, these meta-analyses provide comprehensive maps of cellular factors and pathways that regulate IAV infection, and that have the potential to serve as the basis to develop new strategies to combat IAV infection.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P069

INTERFERENCE BEFORE INTERFERON: CHARACTERIZATION OF INTERFERON SIGNALING INDEPENDENT ANTIVIRAL INNATE IMMUNITY AGAINST INFLUENZA A VIRUSES Shashank Tripathi* 1, 2, Sumit Chanda3, Adolfo Garcia-Sastre1, 2 1Microbiology, 2Global Health and Emerging Pathogen Institute, Icahn School of Medicine at Mount Sinai, New York, 3Immunity and Pathogens Program, Sanford Burnham Prebys Medical Discovery Institute, San Diego, United States

Abstract: The mammalian antiviral innate immune response begins with the detection of viral pathogen associated molecular patterns by host pattern recognition receptors, which leads to activation of Interferon Regulatory Factors (IRFs), which transcribe Interferons (IFN), which in turn activate the JAK-STAT pathway and leads to expression of a battery of antiviral genes collectively called as Interferon Inducible Genes (ISGs). Among IRFs, IRF3 is the key driver of IFN induction. In this study we examined the cellular transcriptome induced by IRF3 in absence of downstream IFN signaling. For this we overexpressed constitutively active form of IRF3 in STAT1 -/- mouse fibroblasts and performed expression profiling using Illumina microarray. Results indicated that IRF3 induces a large set of ISGs, even in the absence of downstream IFN signaling. Importantly, this also resulted in the antiviral response demonstrated by reduced Influenza A virus (IAV) replication in STAT1-/- cells overexpressing IRF3. We compiled additional similar gene lists from published studies, which included genes induced by IRF1, IRF3 and IRF7 in STAT1-/- or IFNAR-/- background, and combined them with IRF3 overexpression dataset. From the resulting combined list, we tested the anti-IAV activity of 158 genes, on a lentiviral overexpression setup with a Luciferase reporter-IAV. This resulted in the identification of a set of anti-IAV genes which manifest antiviral activity in absence of IFN signaling. Further characterization of the underlying mechanisms of novel anti-IAV factors identified in this study and validation of their independence from IFN signaling is being carried out presently.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P070

COMPARATIVE TRANSCRIPTOMICS REVEAL THAT ANTIVIRAL GENE EXPRESSION IN THE EGYPTIAN ROUSETTE BAT IS ANTAGONIZED IN VITRO BY MARBURG VIRUS INFECTION Jonathan C. Guito* 1, Catherine E. Arnold2, Louis A. Altamura3, Elyse Nagle2, Sean Lovett2, Mariano Sanchez-Lockhart2, Jonathan S. Towner1, Gustavo Palacios2 1Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, 2Center for Genome Sciences, 3Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, United States

Abstract: Marburg virus (MARV) causes severe human disease and is responsible for several large, sporadic outbreaks in sub-Saharan Africa. Recently, the Egyptian rousette bat, Rousettus aegyptiacus (ERB), was identified as a MARV reservoir host. When infected by MARV, these bats develop a productive infection with viremia and shedding but without overt disease, which current thinking hypothesizes is due to unique ERB antiviral responses. In humans, an insufficient interferon (IFN) response early in infection may be linked to fatal outcomes, likely due to MARV IFN antagonist activity. Unfortunately, investigative efforts into ERB immune responses in vitro or in vivo have been hampered by a lack of species-specific reagents. Helpfully, the newly-annotated ERB genome and transcriptome can now be used to study immune gene function on a transcriptional level. Employing two platforms, RNA-seq and NanoString nCounter, we assessed antiviral responses in a MARV-infected ERB cell line. Strikingly, both platforms showed that MARV suppressed the IFN response in ERB cells, while an IFN antagonist-impaired MARV mutant significantly stimulated responses, as previously reported for human cells. Interestingly, despite evolutionary expansion of IFN loci in ERBs, we saw almost no induction of IFNs by either virus, but did observe high basal expression for several key response genes. Our findings suggest that, in vitro, antiviral gene upregulation unlikely determines ERB resistance to MARV infection, but that a putative role may exist for an IFN- independent, constitutively-transcribed antiviral system.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P071

TRANSPOSON-MEDIATED ACTIVATION SCREENING IN HUMAN CELLS IDENTIFIES THE CLASS II TRANSACTIVATOR AS A RESTRICTION FACTOR FOR EBOLA Anna Bruchez* 1, Ky Sha1, Joshua Johnson2, Li Chen3, Gene Olinger2, Lynda Stuart1, 4, Adam Lacy-Hulbert1, 5 1Immunology Research Program, BENAROYA RESEARCH INSTITUTE, Seattle, 2Integrated Research Facility, NIAID, Fort Detrick, 3Center for Cancer Research, Massachusetts General Hospital, Boston, 4Discovery and Translational Sciences, Bill and Melinda Gates Foundation, 5Immunology, University of Washington, Seattle, United States

Abstract: Ebola virus causes sporadic outbreaks of severe hemorrhagic fever. Recent outbreaks have exposed our limited understanding of cellular mechanisms to counteract this virus. Viral infections can be controlled by restriction factors, which although poorly defined, represent potential targets for host-directed anti-viral therapies.

We employed a forward genetic approach to identify host genes conferring resistance to viral infection. In this system, cells are mutagenized by widespread, near-random transposon insertion, which is capable of either up- or downregulating genes depending on orientation and location. Candidate resistance genes are identified by sequencing of transposon insertion sites in cells that survive challenge with cytotoxic virus.

We used this approach to identify genes involved in resistance to recombinant vesicular stomatitis virus bearing the Ebola virus glycoprotein. Validating our screen, we found that transposon-mediated inactivation of NPC1, the Ebolavirus entry receptor, conferred resistance. We also identified genes that conferred resistance when overexpressed. These included the MHC class II transactivator (CIITA), which was a potent host restriction factor, increasing cellular resistance over 1,000 fold. CIITA induces resistance through transcription of host target genes, including CD74. These in turn trigger reorganization of the endo-lysosomal pathway, directing Ebola glycoprotein containing virions into the intraluminal vesicles of multi-vesicular bodies. These data implicate CIITA and CD74 in an intrinsic host defense mechanism against infection, independent from their roles in antigen presentation. Furthermore, these findings provide evidence for the power of transposon-mediated gene-activation as a screening strategy able to reveal roles for genes and pathways that elude conventional screening approaches.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P072

PARAINFLUENZA VIRUS 5 (PIV5) P PROTEIN IS MODIFIED IN RESPONSE TO IFNΑ, AND THIS INHIBITS PIV5 INFECTION Jelena Andrejeva* 1, Steve Goodbourn2, Richard Randall3, David Hughes3 1School of Biology, UNIVERSITY OF ST. ANDREWS, SCOTLAND, UK, St. Andrews, 2St. George's University of London, Institute of Infection and Immunity, London, 3School of Biology, University of St. Andrews, St. Andrews, United Kingdom

Abstract: The ubiquitin-like protein (Ubl) ISG15 is strongly induced by type I interferon (IFNα/β) and is critical for regulating how cells respond to infections. Like other Ubls, it can be covalently attached to target proteins in a process known as ISGylation, and in many cases, modification of viral proteins forms part of the antiviral response. Here we show that the parainfluenza virus 5 (PIV5) P protein (a component of the viral polymerase complex) is modified in response to IFNα, and this inhibits PIV5 infection. ISGylation of P requires the E3 ligase HERC5, and a lack of HERC5 alleviates inhibition of PIV5 infection. We also show that P is the only PIV5 protein that is modified, suggesting that ISGylation specifically inhibits the PIV5 polymerase complex. ISG15 only modifies a minority of the P ‘proteome’; as paramyxovirus P proteins function as tetramers, we hypothesise that only a small fraction of modified P is sufficient for inhibition. This aligns well with the ‘dominant-negative’ function that has recently emerged for the antiviral function of ISGylation. Consequently, ISGylation blocks the formation of a competent ribonucleoprotein complex, leading to an inhibition of virus transcription and replication.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P073

STRUCTURAL BASIS OF IMPORTIN ALPHA SPECIFICITY FOR HENIPAVIRUS W PROTEINS Megan R. Edwards* 1, Kate M. Smith2, Sofiya Tsimbalyuk2, David Aragão3, Jade K. Forwood2, Christopher F. Basler1 1Center for Microbial Pathogenesis, Georgia State University, Atlanta, United States, 2School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, 3Australian Synchrotron, Australian Synchrotron, Melbourne, Australia

Abstract: Nucleocytoplasmic trafficking of proteins is important for a number of cellular processes, including viral infection. However, how cargo bearing classical nuclear localization signals (NLSs) can selectively utilize a subset of importin alpha nuclear transport proteins present in human cells remains unclear. To address this question, we studied the Hendra (HeV) and Nipah (NiV) virus W proteins. HeV and NiV, members of the family Paramyxoviridae, are recently emerged, highly lethal zoonotic pathogens. The non-segmented, negative-sense RNA genome encodes for nine-proteins, including the W protein, expressed from the P gene through mRNA editing. W plays a role in controlling the inflammatory response to NiV infection in ferret models. It also inhibits responses to interferon by STAT1 tyrosine phosphorylation and inhibits signalling through TLR3, IKKε and TBK1, likely through inhibition of IRF3. Notably, W localizes to the cell nucleus, through the specific interaction of its classical NLS with the Qip-1 subfamily of importin alpha nuclear transporters, importin α3 and importin α4. To gain insight into the basis of this specificity, we combined structural, biophysical and molecular approaches. X-ray crystal structures between HeV/NiV W and importin α3 or importin α1 (Rch-1 subfamily) identified differential positioning of the armadillo (ARM)-repeat domains 7 and 8 in the importins that allows for a more extensive interface between W and importin α3. Co-immunoprecipitation assays using mutagenesis and chimeras of importin α3/importin α1 confirmed the basis of specificity is present in the C-terminal ARM-repeat domains. These findings explain how W selectively uses Qip-1 transporters and, together with a previously defined mechanism for isoform specificity, suggest that differential importin alpha usage may depend on how the NLS is positioned in the cargo.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P074

PUUMALA HANTAVIRUS INFECTION MODULATES THE CIRCADIAN CLOCK Agnieszka Szemiel* 1, Brian Willett1 1MRC - University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom

Abstract: The circadian clock synchronises host physiology in approx. 24 h oscillations. These processes are controlled by the central oscillator located in the hypothalamus, and peripheral oscillators present in organs, tissues, and most cell types. At the molecular level, circadian timekeeping system is regulated by transcriptional feedback loops involving two activators, CLOCK and BMAL transcription factors; which promote expression of their repressors PER and CRY proteins. Circadian disruption has been associated with multiple diseases like cancer, neurodegeneration, metabolic syndrome, cardiovascular diseases and recently with susceptibility to viral infections. Puumala hantavirus (PUUV), from the family Hantaviridae (order Bunyavirales) causes haemorrhagic fever with renal syndrome, and it is the most common rodent-borne pathogen in Europe. As a hantavirus that causes relatively mild symptoms, it is an excellent model to study infections caused by highly pathogenic hantaviruses, like Hantaan or Seoul. Our study investigated the interplay between PUUV infection and the circadian clock machinery in cell culture. We demonstrate that PUUV infection in BHK21 cells is enhanced when the host circadian rhythm is activated. This enhancement is abolished when the clock machinery is inhibited. Our investigation showed that PUUV infection slows down the oscillation of PER1 mRNA, therefore lengthening the circadian cycle in BHK21 cells. In addition, this function can be attributed to PUUV N protein, as transient expression of PUUV N protein alters the oscillation pattern of PER1 protein. Our results suggest that PUUV infection modulates the molecular circadian clock to facilitate its own replication.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P075

HUR RESTRICTS EBOLA VIRUS REPLICATION Kristina L. Schierhorn* 1, Rui P. Galao1, Harry Wilson1, Stuart Neil1, Chad Swanson1 1Infectious Diseases, King's College London, London, United Kingdom

Abstract: Ebola virus (EBOV) is a nonsegmented, negative-strand RNA virus that replicates in the cytoplasm of infected cells. The presence of viral RNA can trigger antiviral mechanisms, mostly involving RNA-binding proteins (RBPs) which can lead to the formation of stress granules (SGs). These are non-membrane-bound cytosolic organelles that assemble due to different stress conditions, such as viral infection, and have been shown to directly or indirectly inhibit viral replication. To identify cellular RBPs that regulate EBOV replication, we performed an overexpression screen with candidate RBPs that localise to SGs. Using a transcription- and replication-competent virus-like particle (trVLP) system, we identified five proteins which inhibit EBOV trVLP propagation at least 10-fold. All these proteins presented a dose-dependent inhibition which cannot be overcome by high trVLP input. To determine if the endogenous proteins inhibit trVLP replication, we generated single cell clone knockout cell lines using CRISPR genome editing. Using these knockout cell lines as producer as well as target cells for trVLPs, we identified HuR as a potential antiviral factor targeting EBOV propagation. To map the antiviral effect to the viral life cycle, we examined the impact of overexpression on viral transcription and replication. Strand-specific quantitative RT-PCR revealed a block of transcription as well as replication of the viral genome in the target cells. This suggests HuR inhibits EBOV trVLP propagation at an early step in the viral life cycle. We are currently investigating the exact mechanism how HuR inhibits EBOV in the trVLP system.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P076

TERMINAL DEPTH SINGLE-MOLECULE SEQUENCING OF CAPPED TRANSCRIPTS REVEALS HOST-PATHOGEN DYNAMICS IN HUMAN MACROPHAGES. Sara Clohisey* 1, Nicolas Bertin2, Helen Wise3, Andru Tomiou4, Piero Carninci5, Yoshihide Hayashizaki5, David Hume 6, Paul Digard3, J. Kenneth Baillie1 1Genetics and Genomics, The Roslin Institute, Edinburgh, United Kingdom, 2Division of Genomic Technologies, RIKEN, Edinburgh, Japan, 3Infection and Immunity, 4Developmental Biology, The Roslin Institute, Edinburgh, United Kingdom, 5Division of Genomic Technologies, RIKEN, Yokohama, Japan, 6Developemental Biology, The Roslin Institute, Edinburgh, United Kingdom

Abstract: Immune-regulatory actions of macrophages in the lung determine the severity of pathology in influenza. In order to obtain parallel observations of both host and influenza mRNA production, we used cap analysis of gene expression (CAGE) to sequence all capped RNA species, at terminal depth, from influenza-infected primary human macrophages from multiple donors at multiple timepoints. Exploiting both the 5’ and 3’ non-coding regions in viral RNA, we directly observe the dynamics of viral mRNA production over 24 hours.

Influenza viruses replicate by “snatching” 5’ capped mRNA sequences from host mRNAs from abundant transcripts. By sequencing these transcripts from the 5’ end, we have identified the genomic origin of these snatched sequences, revealing that non-codingRNAs are significantly over-represented compared to their abundance. Additionally, pathway analysis on identified sources of cap-snatched transcripts allows us to speculate on the targets of this process. Finally, by comprehensively polling host gene expression, we not only recapitulate canonical antiviral signalling pathways, but also identify numerous host transcripts with highly-specific expression in influenza-treated macrophages.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P077

IS THE PATHOGENICITY OF SFTSV DETERMINED BY VIRAL NSS PROTEIN? Rokusuke Yoshikawa* 1, 2, Jiro Yasuda1, 2 1Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 2National Research Center for Control and Prevention of Infectious Diseases (CCPID), Nagasaki University, Nagasaki, Japan

Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS phlebovirus (SFTSV), which is a novel phebovirus of the Phenuiviridae. SFTSV was first isolated in China and subsequently identified in South Korea and Japan. SFTSV is pathogenic to human, while immunocompetent adult mice infected with SFTSV never show apparent symptom. However, mice lacking interferon (IFN)-a/b receptor are highly susceptible to SFTSV infection and result in death. We found that mice deficient for the signal transducer and activator of transcription 2 (STAT2) which is the key factor of type I IFN signaling pathway are also highly susceptible to SFTSV infection. It suggests that innate immunity depending on STAT2 inhibits the efficient replication of SFTSV in mice. Recently, NSs of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT2 protein in viral cytoplasmic inclusion bodies. In this study, we examined anti-STAT2 activity of NSs in human and mice and proposed a hypothesis that the inhibitory activity of NSs to STA2 is associated with the difference in susceptibility to SFTSV between human and mice. Reporter assay revealed that type I IFN signaling was antagonized by NSs in human cells, while NSs did not inhibit the signaling in mouse cells. Moreover, NSs suppressed the phosphorylation of human STAT2 protein. In contrast, phosphorylation of murine STAT2 was not inhibited by NSs. In addition, co-immunoprecipitation assay revealed that NSs bound to human STAT2, but not murine STAT2. Our results imply that the activity of NSs to antagonize STAT2 may be attributed to the pathogenesis of SFTSV in human.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P078

REGULATION OF CELL INTRINSIC IMMUNITY BY THE TRANSCRIPTIONAL REPRESSOR CAPICUA Julianna Han* 1, Jasmine Perez1, Cindy Chen1, Yan Li2, Asiel Benitez3, Matheswaran Kandasamy1, Yoontae Lee4, Jorge Andrade2, Benjamin tenOever3, Balaji Manicassamy1 1Microbiology, 2Center for Research Informatics, The University of Chicago, Chicago, 3Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States, 4Life Sciences, Pohang University of Science and Technology, Pohang, Korea, Republic Of

Abstract: Host innate immune pathways are important for sensing viruses and mounting robust responses against viral infections. However, negative regulation is critical to prevent erroneous antiviral responses and to ramp down these pathways after clearance of viral infection. Through a genome-wide CRISPR/Cas9 screen to discover host factors critical for influenza virus replication, we have identified capicua (CIC) as a novel host factor critical for regulating intracellular antiviral responses. CIC is a highly conserved DNA binding repressor and mutations in CIC lead to neuropathogenesis and cancer progression. However, the role of CIC in viral infection remains unknown. In CIC knockout cells (KO), we observed higher expression of interferon and interferon stimulated genes. Additionally, replication of viruses from diverse families were highly attenuated in CIC KO cells due to the heightened antiviral status in these cells. In contrast, ectopic expression of CIC repressed antiviral gene promoter reporter constructs containing CIC binding sites. Interestingly, CIC protein and mRNA levels were downregulated during influenza virus infection. Thus, our studies have identified a novel function of CIC in modulating cell intrinsic immunity. We hypothesize that under steady-state conditions CIC represses the transcription of antiviral genes, and upon viral infection, CIC is downregulated to facilitate robust expression of antiviral genes. Our future investigations will reveal a mechanism by which CIC regulates the intracellular antiviral state and may have significant implications in our understanding of host-pathogen evolution. Graphical Abstract:

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P079

ACUTE PLASMODIUM INFECTION PROMOTES RESISTANCE TO EBOLA VIRUS VIA TYPE 1 IMMUNITY IN MACROPHAGES Kai Rogers* 1, Rahul Vijay1, Chester J. Joyner2, Mary R. Galinski2, Noah S. Butler1, Wendy J. Maury1 1Microbiology and Immunology, University of Iowa, Iowa City, 2Emory Vaccine Center, Emory University, Atlanta, United States

Abstract: Ebola virus (EBOV) outbreaks occur sporadically in Africa with enormous fatality rates. Individuals are often infected with other endemic pathogens, but consequences of co-infections are understudied. During the 2014-2016 epidemic, a significant number of EBOV patients were co-infected with P. falciparum when admitted to Ebola treatment units. Currently, there is no consensus regarding how malaria affects EBOV infection, with studies suggesting better or worse outcomes in co-infection patients. Here, we investigated the effect of pre-existing malaria on EBOV challenge using a model virus, recombinant VSV bearing EBOV glycoprotein (EBOV/rVSV). Interferon a/b receptor knock out mice were infected with Plasmodium yoelii (Py) and subsequently challenged with EBOV/rVSV. Acute infection with Py protected against lethal virus challenge. Further, mice were protected against EBOV challenge for weeks after resolution of malarial disease, indicating that innate host responses to Py rendered mice resistant to EBOV. Protection against EBOV was linked to IFNγ-mediated M1 polarization of peritoneal macrophages (pmacs) in Py-infected mice. Serum from acutely Py infected mice also reduced EBOV/rVSV infection of pmacs in an IFNγ-dependent manner. Similarly, human macrophages treated with serum from rhesus macaques acutely infected with P. cynomolgiwere protected against EBOV/rVSV. Protection was abolished by neutralization of IFNg. Finally, Py-infected mice lacking the IFNg receptor were not protected from EBOV/rVSV, yet their serum, containing IFNg, protected wild-type pmacs. These experiments support the hypothesis that acute malaria infection protects against EBOV by production of IFNγ, which in turn elicits an M1 state in macrophages.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P080

RECOGNITION OF INFLUENZA A VIRUS BY DNA SENSORS Miyu Moriyama* 1, Takeshi Ichinohe1 1Division of Viral Infection, UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Influenza A virus is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. The recognition of influenza virus plays a key role not only in limiting virus replication and inflammatory responses at early stages of infection but also in initiating and orchestrating virus-specific adaptive immune response. It has become increasingly evident that influenza viral infection is recognized by at least three classes of pattern-recognition receptors, including TLR-7, the retinoic acid inducible gene-I (RIG-I) and nucleotide-binding domain and leucine-rich-repeat-containing protein 3 (NLRP3), a member of the Nod-like receptor family. In contrast, the role of DNA sensing pathway in recognition of influenza virus remains to be defined. Here we show that recombinant influenza virus lacking the NS1 gene (ΔNS1 virus) stimulates translocation of mitochondrial DNA (mtDNA) into the cytosol and elicits stimulator of interferon genes (STING)-dependent innate antiviral immunity. Release of mtDNA into the cytosol dependent on MAVS and mitochondrial permeability transition. The STING-dependent antiviral signaling was amplified by bystander cells via gap junction. Our results show a mechanism by which influenza virus activates STING pathway and provide insight into the role of DNA sensing pathway in recognition of influenza virus.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P081

REGULATION OF CELL DEATH BY THE EBOLAVIRUS VP24 PROTEIN Diego Cantoni* 1, Jeremy S. Rossman1 1School of Biosciences, University of Kent, Canterbury, United Kingdom

Abstract: Ebolavirus (EBOV), a member of the filoviridae family, is a highly pathogenic virus which causes cell death through a variety of mechanisms, including as as-yet-undefined necrosis pathway. Here we have investigated the pathway and mechanisms of EBOV necrotic cell death and define a crucial role of VP24 in regulating cell death signalling pathways. EBOV VP24 is a 251 amino acid protein that has been shown previously to counteract antiviral defences and increase stability of the viral nucleocapsid. Using a combination of biochemical and microscopy-based assays, we have investigated the role of VP24 in the regulation of cell death following the induction of different signalling pathways, including stimulation by tumour necrosis factor alpha. We find that VP24 interacts with several mitochondrial proteins, including VDAC1 and plays a crucial role in the upregulation of necroptosis and the downregulation of apoptosis, thus regulating the pathway of cell death following EBOV infection.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P083

SANDFLY FEVER SICILIAN VIRUS NSS SPECIFICALLY TARGETS IRF3 TO INHIBIT TYPE I INTERFERON INDUCTION Jennifer Wuerth* 1, Matthias Habjan2, Andreas Pichlmair3, Giulio Superti-Furga4, Friedemann Weber1 1Justus-Liebig University, Giessen, 2Max Planck Institute of Biochemistry, Martinsried, 3Technical University of Munich, Munich, Germany, 4Center for Molecular Medicine, Vienna, Austria

Abstract: Different members of the Phlebovirus genus exhibit different degrees of virulence. Rift Valley fever virus (RVFV), for example, causes severe symptoms, whereas Sandfly fever Sicilian virus (SFSV) is much milder. The major virulence factor of phleboviruses is the non-structural protein NSs, an antagonist of the type I interferon (IFN) system. Whereas RVFV NSs is known to entirely shut off cellular transcription by targeted sequestration and destruction of host factors, the function of SFSV NSs has remained elusive. Here, we show that SFSV NSs inhibits the transcriptional induction of type I IFN. Applying tandem affinity purification and mass spectrometry (Pichlmair et al. Nature 2012), we identified IFN regulatory factor 3 (IRF3) as host interactor. Co- immunoprecipitation and reporter assays confirmed IRF3 as interactor and functional target of SFSV NSs: SFSV NSs alone abrogated IRF3-dependent promoter activity induced via MAVS, TRIF, TBK1, or constitutively active IRF3. However, neither phosphorylation, dimerization nor nuclear accumulation of IRF3 were affected. Instead, SFSV NSs was found to act by obstructing the DNA-binding domain of IRF3, thereby hindering enhanceosome formation. To our knowledge, this is the first report of an RNA virus factor that directly disrupts the binding of activated IRF3 to the IFN promoter. Thus, SFSV NSs is different from the NSs of highly virulent RVFV, as it does not induce a general host transcription block but instead specifically targets IRF3-driven gene expression. Although these viruses are highly related, their NSs proteins display remarkably diverse strategies of counteracting the IFN system, probably contributing to their respective virulence levels.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P084

IDENTIFICATION OF TWO NEW POLYPEPTIDES FROM SEGMENT 2 OF IAV THAT MODULATE THE TYPE I INTERFERON RESPONSE Rute M. Pinto* 1, Helen Wise1, Liliane M. W. Chung1, Marlynne Quigg-Nicol1, Samantha Lycett1, Gregory Heikel2, Gracjan Michlewski2, Bernadette Dutia1, Paul Digard1 1The Roslin Institute, 2Wellcome Centre for Cell Biology, The University of Edinburgh, Edinburgh, United Kingdom

Abstract: Alternative translation events that produce additional viral polypeptides have been shown to be significant in influenza A virus (IAV) biology. IAV segment 2 is a pathogenicity determinant, known to encode PB1, PB1-F2 and PB1-N40 proteins, starting from AUG codons 1, 4 and 5 respectively. We investigate the expression of two additional polypeptides, arising from translation initiation at AUGs 10 and 11 of segment 2. These codons are highly conserved in IAV and direct the translation of two N-terminally truncated versions of the primary PB1 product (PB1-N92 and -N111 respectively). Mutation of AUGs 10 or 11 (M92V, M111V) in the background of PR8 had minor effects on virus replication kinetics although the viruses displayed elevated levels of IRF3 phosphorylation and type-I IFN secretion compared to WT virus. However, simultaneous mutation of AUGs 10 and 11 severely decreased viral fitness, despite the full-length mutant PB1 possessing normal genome transcription and replication activity. Similar patterns of defective replication and elevated innate signalling were seen when mutating AUGs 10 and/or 11 in other mammalian virus isolates. The propagation deficit of the ΔAUG10,11 mutant recovered in IFN-deficient models, including in ovo in pre-day-8 embryonated eggs, in vitro in IFN a/β receptor (IFNAR) knockout bone marrow-derived macrophages and in vivo in IFNAR-/- mouse lungs. Moreover, expression of PB1- N92 or –N111 polypeptides blocked poly I:C-induced activation of IFN-β and ISRE promoters in transfected cells. In vitro, knockout of TRIM25 did not increase growth of the mutant viruses, but the addition of a TBK1/IKKε inhibitor did. In conclusion, segment 2 expresses previously undescribed N-terminally truncated versions of PB1 which play a role in antagonising the host IFN response, most likely independently of RIG-I/TRIM25, but upstream of IRF3 phosphorylation.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P085

EARLY HOST RESPONSES OF HUMAN PRIMARY LUNG MICROVASCULAR ENDOTHELIAL CELLS AND RESTING MONOCYTES TO BLACK CREEK CANAL ORTHOHANTAVIRUS (BCCV) Evan P. Williams1, Elizabeth A. Fitzpatrick1, Colleen B. Jonsson* 1 1Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, United States

Abstract: Hantaviruses are single-stranded, negative-sense, trisegmented RNA viruses that persistently infect species within the Rodentia, Eulipotyphla and Chiroptera. Of these, only those viruses harbored by rodents cause disease in humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (L-MVEC). Studies suggest that monocytes may also become infected at some point during infection. Since monocytes are one of the first responders to infection, it is possible that these immune cells may become infected upon arrival; however, this is not known. We hypothesized that the addition of monocytes to infected-LMVEC would elevate the standing proinflammatory response. We asked how host gene expression would be modulated following BCCV infection of L-MVEC or monocytes or L-MVEC and monocytes following BCCV infection from 24 to 96 hours with time-points every 12 hours. QuantiGene probes were used to measure the kinetics of genes representing antiviral, proinflammatory, anti-inflammatory, metabolic pathways. As expected in L-MVEC, anti-inflammatory genes were not upregulated in any experimental group while proinflammatory genes (e.g., IL8, NFKB IFNA1/B1) were upregulated. Surprisingly, upon addition of monocytes, the proinflammatory genes of the L-MVEC were suppressed. Notably, ITGAM was highly upregulated in L-MVEC-monocytes (presumably in the monocytes); however, a number of genes (e.g., TNF, TLR7, and IL1RN) were not affected. In experiments with monocytes alone, IFNG was upregulated along with IDO1, IL15, PPARG and TNF. In conclusion, in contrast to our original hypothesis, monocytes plus L-MVEC did not amplify the proinflammatory response suggesting that monocytes may contribute to evasion of the innate immune response.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P086

TWO CONSERVED AMINO ACIDS WITHIN THE NSS PROTEIN OF SFTS VIRUS ARE ESSENTIAL FOR ANTI- INTERFERON ACTIVITY Takeshi Ichinohe* 1, Miyu Moriyama1 1Department of Virology, UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infec tious disease caused by the SFTS virus (SFTSV). The nonstructural protein (NSs) of SFTSV sequesters TANK-binding kinase 1 (TBK1) into NSs- induced cytopl asmic structures to inhibit the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-β) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-β responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Herein, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV NSs protein are essential for suppression of IRF3 phosphorylation and IFN-β mRNA expression following infection with recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, association between SFTSV NSs and TBK1 is required for suppression of mitochondrial antiviral signalling protein (MAVS)-mediated activation of IFN-β promoter activity. Our findings strongly implicate the N-terminal region of SFTSV NSs in the inhibition of host antiviral immunity, and will aid the development of novel therapeutic strategies to treat SFTSV infection and associated diseases.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P088

ANATOMY OF CYTOTOXIC T LYMPHOCYTE RESPONSE DURING THE 2013-2016 EBOLA OUTBREAK IN WEST AFRICA Saori Sakabe* 1, Kristian Andersen1, Michael B. Oldstone1, Brian M. Sullivan1 1of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, United States

Abstract: Ebola virus (EBOV) causes severe febrile disease in humans and large scale outbreak occurred in West Africa in 2013-2016, resulting in 28,616 cases and 11,310 deaths. EBOV-vaccinated non-human primates are protected from lethal challenge of EBOV if their CD8+ T cell response is operational (Sullivan et al., Nat Med, 2011). Passive transfer of high titered anti EBOV antibodies failed to protect in the absence of CD8 T cells. This data and observations that T cell responses are observed in humans surviving acute viral hemorrhagic fevers indicate that T cells play a key role in the disease severity. We defined the Ebola viruses-specific cytotoxic T lymphocyte (CTL) response in 31 Ebola survivors living in Sierra Leone to seven of the eight Ebola full-length proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40), and 36-67 aa truncations of these proteins by using recombinant single cycle VSVs library encoding EBOV proteins. Our results indicated a minor of (only 10) of the 27 individuals (37%) showed CD8+ CTL response to GP or sGP. By contrast 26/27 (96%) showed response to NP, 81% to VP24, 74% to VP40, 44% to VP35, and no response to VP30. Further, we mapped various novel CD8+ CTL epitopes derived from EBOV proteins and their .HLA restriction. Our data suggest that an EBOV vaccine designed to include not only the Ebola GP but also NP would be more effective to elicit both cellular and humoral responses in most individuals than a vaccine designed just include the Ebola GP which may elicit good humoral but limited CTL responses. This research is funded by NIH Contract HHSN272201400048C under BAA-NIAID-DAIT-NIHAI2013167.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P089

THE OVARIAN TUMOR (OTU) DOMAIN OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS (CCHFV) INFLUENCES THE POLYMERASE RDRP ACTIVITY, BUT DOES NOT COUNTERACT INNATE IMMUNITY Stéphanie Devignot* 1, Ali Mirazimi2, Friedemann Weber1 1Institute for Virology, FB10 Veterinary medicine, Giessen, Germany, 2Public Health Agency, Solna, Sweden

Abstract: The Crimean-Congo Hemorrhagic fever virus (CCHFV) polymerase L encodes an Ovarian Tumor (OTU) domain, with deubiquitination and deISGylation activities. Studies based on the ectopic expression of the OTU domain showed an inhibition of both NFκB and type I interferon (IFN) induction. Hence, the OTU domain has been suggested to take part in immune evasion.

Since the OTU domain is small compared to the full-length polymerase (169 versus 3549 amino acids), we asked whether the OTU domain retains the same activities in the context of the entire, RdRp active L.

Using reporter assays, we confirmed that the ectopic OTU domain inhibits both NFκB and IFN induction, but this effect was lost in the context of the full-length L. Then, we used the minireplicon (Bergeron et al., JVI, 2010) and the transcriptionally- competent virus-like particle (tc-VLP) (Devignot et al., JVI, 2015) systems to assess the role of the OTU domain on the RdRp activity. While the OTU domain is not required in the minireplicon system, only few tc-VLPs could be produced with an OTU-inactive L. However, both wild-type and OTU-deficient polymerases are equally sensitive to IFN. Moreover, neither trans-complementation with wild-type OTU domain, nor blocking IFN signalling were able to rescue OTU-inactive mutant tc-VLP production. This suggests distinct trans- and cis- activities of the OTU domain. Lastly, and very surprisingly, we could rescue tc-VLP production over the wild-type polymerase levels, by over-expressing a conjugable Interferon Stimulated Gene 15 (ISG15).

To conclude, the role of the CCHFV OTU domain regarding innate immune response needs further careful investigation, which should be made in the context of the full-length polymerase.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P090

NIPAH VIRUS C PROTEIN INHIBITS INFLAMMATORY CYTOKINE INDUCTION BY INTERACTION WITH PHOSPHATASE 2A INHIBITOR. Ryo Horie1, Misako Yoneda1, Shotaro Uchida1, Asuka Yoshida* 1, Hiroki Sato1, Chieko Kai1 1Laboratory Animal Research Center, THE INSTITUTE OF MEDICAL SCIENCE, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. 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. In present study, we isolated human proteins binding to the NiV-C using an affinity purification method. 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. In the cells stably expressing C protein, total cellular PP2A activity was significantly increased and induction of proinflammatory cytokines, IL-8 and CXCL2, by poly(I:C) treatment were suppressed. 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.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P091

MX1 REQUIREMENTS FOR INFLUENZA A VIRUS RESTRICTION Olivier Moncorgé* 1, Joe McKellar1, Wendy Barclay2, Caroline Goujon1 1IRIM, CNRS, MONTPELLIER, France, 2Department of Medicine, Imperial College London, LONDON, United Kingdom

Abstract: Type 1 interferons (IFNs) are produced by infected cells upon detection of pathogenic agents and are the first line of defence against viral infections. IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs), both in infected and neighbouring cells. The products of these ISGs in turn induce in cells a potent antiviral state, capable of limiting viral replication. The dynamin-like, high-molecular weight GTPases MX1 and MX2 play a significant role in the IFN-induced inhibition of viral replication. Human MX1 is a restriction factor of broad antiviral activity, able to inhibit influenza A virus and a great diversity of RNA and DNA viruses at different stages of their life cycles. Human MX2 is notably able to inhibit HIV-1 and some primate lentiviruses. Although the antiviral activity of human MX1 has been studied extensively, the molecular mechanism of action remains largely unsolved. MX1 and MX2 are 63% identical at the amino acid level, share a similar domain organization and their crystal structures are almost practically superimposable. Taking advantage of chimeras between MX1 and MX2 in which their different domains have been swapped as well as point mutants, we have notably identified a new motif required for influenza A restriction by MX1. Importantly, some MX1/MX2 chimeric proteins are highly active against influenza A viruses but not in the context of minireplicon assays. Additional ongoing efforts to better characterize MX1’s requirement for influenza A restriction and mechanism of action will be presented.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P092

UBIQUITINATION OF TOSCANA VIRUS NSS UNDERMINES ITS STABILITY AND HAS A ROLE IN RIG-I DEGRADATION Claudia Gandolfo* 1, Gianni Gori Savellini1, Shibily Prathyumnan1, Maria G. Cusi1 1Medical Biotechnologies, University of Siena, Siena, Italy

Abstract: Toscana Virus (TOSV) is a Phlebovirus (Phenuiviridae family) responsible for central nervous system (CNS) injury in humans. Although TOSV pathogenicity is largely unclear, its non structural protein (NSs), when over-expressed, inhibits host innate immunity, silencing the IFN-β pathway. This antagonistic effect is due to the interaction with RIG-I, by addressing the latter to proteasome degradation upon NSs ubiquitination. Some lysine residues of NSs targeted for ubiquitination were identified by theoretical prediction and mutated to arginine. K150R and K154R point mutations restored RIG-I activation. Indeed, besides the accumulation of RIG-I in cells co-transfected with NSs mutants, an activation of IFN promoter was observed by luciferase assay. Unexpectedly, K104R and K109R mutations, although not determining RIG-I degradation, showed an activation of the luciferase expression. These results suggested a dual function mediated by NSs protein on both RIG-I degradation and interferon activation. In parallel, the effects on NSs stability was evaluated for all of these mutants, determining a considerable increase of protein accumulation in the cytoplasm. Mass-spectrometry analysis performed on wt-NSs confirmed that lysine at 104, 109, and 154 position are linked to K48-polyubiquitin chains. Unfortunately, K150 ubiquitination was not confirmed, due to the undetectable size of the peptide generated during sample preparation. Taken together, these results provide, for the first time, evidence that Toscana virus NSs is ubiquitinated, opening a new insight on NSs function. Moreover, it is probable that TOSV NSs could exert an E3 ubiquitin ligase activity either on RIG-I and NSs itself, suggesting an important role of this protein among Phleboviruses.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P093

THE GLYCOPROTEIN OF VESICULAR STOMATITIS VIRUS IS A TETHERIN ANTAGONIST IN TRANSFECTED BUT NOT INFECTED CELLS Markus Hoffmann* 1, Constantin Brinkmann1, Anastasia Lübke1, Inga Nehlmeier1, Annika Krämer-Kühl2, Michael Winkler1, Stefan Pöhlmann1 1Infection Biology Unit, Deutsches Primatenzentrum GmbH Leibniz-Institut für Primatenforschung, Göttingen, 2Poultry Viral Vaccine R&D, Boehringer Ingelheim Veterinary Research Center GmbH & Co. KG, Hannover, Germany

Abstract: Vesicular stomatitis virus (VSV) is a prototype member of the Rhabdoviridae family and used as a model virus for various scientific endeavors. VSV release from infected cells is inhibited by the interferon-inducible antiviral host cell factor tetherin (BST-2, CD317). Since several viruses encode tetherin antagonists the present study investigated whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the VSV glycoprotein (VSV-G) acts as a tetherin antagonist in transfected cells, but does so with reduced efficiency as compared to the Vpu protein of human immunodeficiency virus 1. Tetherin antagonism by VSV-G did not rely on alteration of tetherin expression and could further be linked to a glycine-X-X-X-glycine (GXXXG) motif within the transmembrane domain of VSV-G. However, when the GXXXG motif was investigated in the context of replication- competent VSV, disruption of this motif had no impact on tetherin sensitivity of VSV spread and thus, no evidence for a tetherin-antagonizing activity of VSV-G in infected cells could be detected. In sum, we identified VSV-G as a tetherin antagonist in transfected cells but could not provide evidence for a contribution of tetherin antagonism to spread of authentic VSV.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P094

HUMAN PARAINFLUENZA VIRUS TYPE 2 V PROTEIN INDUCES FILAMENTOUS ACTIN FORMATION Keisuke Ohta* 1, Yusuke Matsumoto1, Natsuko Yumine1, Masato Tsurudome2, Machiko Nishio1 1Department of Microbiology, Wakayama Medical University, Wakayama, 2Department of Microbiology and Molecular Genetics, Mie University Graduate School of Medicine, Tsu, Japan

Abstract: The growth of human parainfluenza virus type 2 (hPIV-2) is promoted by RhoA activation, through the binding of its V and P proteins with Graf1 (Ohta et al., 2016, J. Virol.). However, it remains unknown how RhoA activation affects hPIV-2 growth. Here, we focused on actin reorganization that is regulated by RhoA activation. We have found that hPIV-2 infection induced filamentous actin (F-actin) formation, and that cytochalasin D, an actin polymerization inhibitor, reduced hPIV-2 growth. To investigate whether RhoA-induced F-actin formation affects hPIV-2 growth, wild type (wt) RhoA- or dominant negative (DN) RhoA-expressing HEK293 cells were established. Overexpression of wt RhoA caused an apparent F-actin formation even in mock-infected cells, and promoted hPIV-2 growth. In contrast, hPIV-2 growth was inhibited in DN RhoA-expressing cells where F-actin formation was not observed. Immunoprecipitation studies revealed that hPIV-2 V protein bound to inactive DN RhoA but not to wt and active RhoA, while P protein bound to none of these RhoAs. We also found that mutation of the Trp residues within the C-terminal region of V protein lost the capacity to bind DN RhoA. Infection with recombinant hPIV-2 carrying this Trp-mutated V did not cause F-actin formation. Furthermore, F-actin formation was observed in HEK293 cells constitutively expressing wt V, but not Trp-mutated V. The interaction between V protein and inactive RhoA is important for F-actin formation. This interaction might enhance the exchange of GDP for GTP, thereby converting inactive to active. The analysis of this possibility is now in progress.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P095

REGULATION OF HAZARA VIRUS GROWTH THROUGH APOPTOSIS INHIBITION BY VIRAL NUCLEOPROTEIN Takashi Nouchi1, Yusuke Matsumoto* 1, Keisuke Ohta1, Machiko Nishio1 1Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan

Abstract: Hazara virus (HAZV) is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) but is nonpathogenic to humans. Since HAZV was first isolated in 1954, the biological characteristics of this virus, particularly its behavior within cultured cells, have not been well studied, despite the importance of HAZV as a surrogate model for CCHFV. Nucleoprotein (N), the main component of viral ribonucleoprotein complex and the most abundant protein in the virion, is believed to play a pivotal role in the virus lifecycle. The generation of a series of anti-HAZV N monoclonal antibodies were used to directly examine the involvement of this protein in viral growth. HAZV infection was found to induce severe apoptosis, which was further characterized by DNA ladders and elevated caspase-3/7 activity. HAZV titers initially increased in culture cells, but rapidly turned to decline after reaching the peak titer. HAZV particles were found to be very unstable in the culture medium at 37 oC, suggesting that progeny virions tends to lose infectivity. We also found that HAZV N inhibits the apoptosis, which supports the maintenance of viral infectivity. The contrary effects of induction and inhibition of apoptosis are considered to be an important property of this virus lifecycle.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P096

MEASLES VIRUS INFECTION TRIGGERS CGAS-DEPENDENT ANTIVIRAL RESPONSES. Hiroki Sato* 1, Fusako Ikeda1, Miho Hoshi1, Misako Yoneda1, Chieko Kai1 1Laboratory Animal Research Center, INSTITUTE OF MEDICAL SCIENCE, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: In mammalian cells, intracellular RNA produced by RNA viruses are recognized by RIG-I or MDA5, and cytosolic DNA viruses are sensed by cGAS, in principal, and trigger a series of downstream host innate antiviral signaling. Interestingly, recent reports revealed that endogenous cytoplasmic DNA such as mitochondrial DNA (mtDNA) released from mitochondria by cellular stresses can also activates cGAS pathway. Our previous report indicated that measles virus (MeV) induces rapid antiviral responses, and proceeding of the infection causes comprehensive downregulation of host gene expressions including genes encoding mitochondrial proteins in an epithelial cells. In the present study, we found that increased amount of mtDNA in the cytosol was detected accompanied by proceeding of the infection. Furthermore, we revealed that the cytosolic mtDNA was captured by cGAS in the infected cells, and knockdown of cGAS suppressed IFN-β expression after MeV infection similarly to knockdown of MAVS which mediates RIG-I/MDA5 signaling. Our study proposes two-step induction of antiviral responses by MeV infection; intracellular RNA sensors are activated directly at early phase, and prolonged infection induces mtDNA-activated cGAS pathway for full innate control of MeV.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P097

EBOLA VIRUS INHIBITS CELLULAR NRF2-DEPENDENT ANTIOXIDANT SIGNALING PATHWAY VIA EXPRESSION OF STRUCTURAL PROTEIN VP24. Valentina Volchkova* 1, Kirill Nemirov1, Mathieu Mateo1, St Patrick Reid1, Audrey Page1, Nicolas Danet1, Viktor Volchkov1 1BMPV, CIRI 1111, Lyon, France

Abstract: Virus replication in cells is often associated with release of reactive oxygen species and oxidative stress. Transcription factor Nrf2 (Nuclear factor erythroid-related factor-2) plays a central role in cellular defence against oxidative stress conditions. During stress, Nrf2 is translocated into the nucleus and activates a panoply of genes implicated in cell protection but also in the regulation of oxidative stress responses. Deregulated inflammatory response is a hallmark of Ebola virus (EBOV) pathogenesis. Inflammation and oxidative stress are known to be interrelated. In this study we investigate whether EBOV infection affects Nrf2 dependent anti-oxidative cell responses. We demonstrate that: (i) expression of Nrf2-dependent genes is impaired in EBOV infected cells, (ii) this is mediated by VP24 and that (iii) VP24 prevents Nrf2 transport into the nucleus, which is essential for Nrf2 function. Moreover, we show that a VP24 mutant defective in binding with karyopherin alpha (KPNA) lacks the ability to prevent Nrf2 nuclear transport, suggesting that these two VP24 functions are some way interdependent. Using reverse genetics for EBOV we demonstrate that an EBOV mutant defective in binding of VP24 and KPNA is not capable to antagonize IFN signaling. Remarkably, this EBOV mutant is severely attenuated in Vero E6 cells albeit that this cell line is known to be defective for IFN production. The data obtained support the notion that impairment of Nrf2-dependent anti-oxidative pathway may play a role in productive virus replication but may also facilitate virus induced pro-inflammatory responses, inflicting additional damage to the host and thus contributing to high pathogenicity of infection.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P098

SMALL RNAS DERIVED FROM AN ENDOGENOUS BORNAVIRUS ELEMENT IN MICE SUPPRESS EXPRESSION OF A REPORTER WITH BORNA DISEASE VIRUS SEQUENCES IN GC2 CELLS Tomoyuki Honda* 1, 2, Xiaoshu Liu1, Bea C. Garcia1, Nicholas F. Parrish1, Keizo Tomonaga1 1Kyoto University, Kyoto, 2Osaka Univeristy, Osaka, Japan

Abstract: Endogenous bornavirus-like nucleoprotein (EBLN) is a fossil record of an ancient bornavirus infection in vertebrate genomes and homologous to the nucleoprotein (N) gene of current bornaviruses, such as Borna disease virus (BoDV). Recently, we have found that EBLNs give rise to small RNAs, which appear to be PIWI-interacting RNAs (piRNAs), in the adult mouse testis. piRNAs associate with their partner PIWI proteins and silence their target sequences. Species with EBLNs seem to have resistance to the current bornavirus infection, leading to reasonable doubt about whether EBLN- derived small RNAs might target the viral N sequences. In this study, therefore, we evaluated this possibility using a mouse spermatocyte cell line GC-2spd (GC2), which reportedly has functional piRNA pathway. We first confirmed that EBLN- derived small RNAs were loaded onto the MIWI protein. When we transduced a mouse EBLN-derived synthesized small RNA of 26 nt into GC2 cells, the small RNA significantly suppressed expression of GFP reporter containing its target sequence of N gene, suggesting that EBLN-derived small RNAs have a potential to silence the target N sequence. We also found that BoDV transcription was upregulated by downregulation of Myb, a transcription factor for the piRNA loci. Collectively, EBLN-derived small RNAs may function as piRNAs to silence bornavirus transcripts in the mouse germline cells.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P100

THE NSS PROTEIN FROM THE ANDES VIRUS INHIBITS THE TYPE I IFN RESPONSE PATHWAY Jorge Vera Otarola* 1, Loretto Solis1, Karla Pino1, Marcelo Lopez Lastra1 1Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica , Escuela de Medicina, Pontificia Universidad Católica De Chile, Santiago, Chile

Abstract: Andes virus (ANDV) is a rodent-borne hantavirus member of the Bunyaviridae family of viruses. ANDV is endemic in Argentina and Chile and is the major etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in south America. ANDV features a tripartite genome consisting of three negative polarity single-stranded RNA segments designated large (L), medium(M) and small (S), packed into helical nucleocapsids.The L and M messenger RNAs (mRNAs) encode the RNA polymerase and a glycoproteinprecursor that is co-translationally processed to yield two envelopeglycoproteins (Gc and Gn), respectively. The SmRNA encodes the nucleocapsid (N) protein and the NSs protein from an overlapping (+1) open reading frame. At early stages of infection ANDV inhibits the cellular type I IFN response, through a yet not fully understood mechanism. The ANDV Gn and N proteins have been shown to inhibit the type I IFN pathway by blocking its signaling at level of TANK-binding kinase 1 (TBK1). TBK1 is involved in the activation of NFkB and IRF-3 which in turns triggers transcription of the IFN genes. Here, we use an assay in which the luciferase reporter is under the control of IFN-beta promoter to demonstrate that the ANDV-NSs protein blocks the IFN-beta activation pathway. Thus, findings reveal a role for the ANDV-NSs protein in the evasion of the cellular innate immunity response. Work supported by FONDECYT Iniciación 11150611, CONICYT-PIA ACT1408 and P09/016-F, Iniciativa Científica Milenio del Ministerio de Economía, Fomento y Turismo de Chile.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P101

INFLUENZA A VIRUS INFECTION TRIGGERS PYROPTOSIS AND APOPTOSIS OF RESPIRATORY EPITHELIAL CELLS THROUGH TYPE I IFN SIGNALING PATHWAY IN A MUTUALLY EXCLUSIVE MANNER Sangjoon Lee* 1, Kyosuke Nagata2, Atsushi Kawaguchi1, 2 1Ph.D. Program in Human Biology, School of Integrative and Global Majors, 2Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

Abstract: Respiratory epithelial cell death by influenza A virus (IAV) infection is responsible for induction of inflammatory responses. Previous studies suggest that apoptosis is a major cell death pathway triggered by IAV infection in cultured epithelial cells isolated from malignant tumors. However, apoptotic cells are rapidly phagocytized without inflammatory responses. Thus, the exact cell death mechanism responsible for inflammatory responses by IAV infection is still unclear. Here we found that IAV infection induced apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced at early phases of infection, but the cell death pathway was shifted to pyroptosis at late phases of infection. We also found that the type I IFN-mediated JAK-STAT signaling pathway promotes the switch from apoptosis to pyroptosis by inhibiting apoptosis possibly through the induced expression of Bcl-xL anti-apoptotic gene. Further, the inhibition of JAK- STAT signaling repressed pyroptosis, but enhanced apoptosis in infected respiratory epithelial cells. Taken together, type I IFN signaling pathway plays an important role to induce pyroptosis but represses apoptosis in the respiratory epithelial cells to initiate pro-inflammatory responses against IAV infection.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P102

DEVELOPMENT OF AN EBOLA VIRUS INFECTION MODEL USING IPSC-DERIVED HEPATOCYTES Whitney Manhart* 1, 2, 3, Steve Mazur4, Ignacio Caballero2, Liliana Mancio Silva5, John G. Bernbaum4, Sangeeta Bhatia5, Reed F. Johnson6, Elke Mühlberger1, 3, Gustavo Mostoslavsky2, 7 1National Emerging Infectious Disease Laboratory (NEIDL), Boston University, 2Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, 3Microbiology, Boston University School of Medicine, Boston, 4Division of Clinical Research, Integrated Research Facility, National Institute for Allergy and Infectious Disease, National Institute of Health, Frederick, 5Institute for Medical Engineering and Science, Massachusetts Institute of Technology , Cambridge, 6Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute for Allergy and Infectious Disease, National Institute of Health, Frederick, 7Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, United States

Abstract: Background Ebola virus (EBOV) infection causes a severe disease in humans which causes liver damage. It is thought that hepatocytes are a key site of viral replication in vivo and potentially a promising target for therapeutic interventions. Primary liver cells are difficult to grow in vitro, and not all animal models recapitulate liver pathology. The goal is to use induced pluripotent stem cell (iPSC)-derived hepatocytes to develop a disease-relevant in-vitro infection platform for modeling EBOV pathogenesis. Methods Hepatocytes were differentiated from human iPSCs using a published, directed differentiation protocol. iPSC-derived hepatocytes were characterized using flow cytometry, intracellular staining, qRT-PCR, and functional assays. iPSC-derived hepatocytes, primary human hepatocytes (PHH), and immortalized hepatic carcinoma cells (Huh7) were infected with EBOV at an MOI of 3 and harvested for analysis at 1 and 4 dpi. Infected cells were analyzed by immunofluorescence, electron microscopy, and 3’ RNA-seq (DGE, Broad Institute). Results iPSC-derived hepatocytes express mature hepatic markers and key hepatic enzymes are active. All three cell platforms supported EBOV infection as indicated by the presence of cytoplasmic viral inclusions. iPSC-derived hepatocytes contained significant amounts of replicating and nascent virions as shown by electron microscopy. iPSC-derived hepatocytes and PHHs were less susceptible to EBOV infection compared to Huh7s. Using RNA-seq data, iPSC-derived hepatocytes and PHHs respond more similarly to infection compared to Huh7 cells, and iPSC-derived hepatocytes express an interferon signature upon infection that is not observed in Huh7 cells. Conclusions iPSC-derived hepatocytes are a suitable in vitro model for EBOV infection of hepatocytes. The model can be used to better understand the molecular mechanisms leading to liver damage during human EBOV infection.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P103

KNOCKING ON THE NUCLEAR DOOR: RIFT VALLEY FEVER VIRUS VIRULENCE FACTOR NSS TARGETS NUCLEAR-CYTOPLASMIC TRANSPORT Simone Lau* 1, Matthias Habjan2, Giulio Superti-Furga3, Andreas Pichlmair2, Friedemann Weber1 1Justus-Liebig University, Giessen, 2Max-Planck Institute of Biochemistry, Martinsried/Munich, Germany, 3CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

Abstract: Rift Valley fever virus (RVFV, genus Phlebovirus, family Phenuiviridae) causes large epizootics and epidemics among livestock and humans in Africa. Its main virulence factor NSs is known to inhibit the transcriptional upregulation of interferons (IFNs) by attacking the general transcription factor IIH (TFIIH) via subunit sequestration and proteasomal degradation. However, being such a strong IFN antagonist, NSs may engage additional strategies to manipulate the host cell. Accordingly, we observed that NSs induces a complete retention of poly(A) RNAs in the nucleus, as previously reported by Copeland et al. (Copeland et al., J.Virol 2013 Nov, 87(21):11659-69). Moreover, our mass spectrometry analysis of NSs- interacting host cell factors suggested a direct interaction with the mRNA export factors Nup98 and RaeI, albeit verification by less sensitive methods have failed so far. However, using immunofluorescence analysis, we detected a disruption of the nuclear pore complex and an NSs-dependent displacement of specific nucleoporins. Our current investigations focus on the question whether RVFV also impairs protein transport. Overall, our results suggest that RVFV, which replicates in the cytoplasm and has mRNAs lacking the prototypical poly(A) tail, constrains the nuclear export of host cell mRNAs and possibly the transport of proteins by directly targeting nucleoporins.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P104

SINGLE DOMAIN ANTIBODIES TO PROBE THE ANTIVIRAL FUNCTION OF INTERFERON-INDUCED MX1 PROTEINS Eline Soetens* 1, 2, Jan Spitaels1, 2, Xavier Saelens1, 2 1Center for Medical Biotechnology, VIB, 2Department for Biomedical Molecular Biology, Ghent University, Ghent, Belgium

Abstract: For centuries, our immune system has been trapped in an evolutionary arms race with viruses. This relationship has shaped our immune defenses and led to the evolution of several specialized anti-viral proteins. An example of these are the Mx proteins; large GTPases that are part of the antiviral response induced by type I and type III interferons, and thus belong to the first line of defense against viral infections. Their importance is emphasized by their presence in almost all vertebrates and their ability to counteract a wide range of viruses. The human MxA and mouse Mx1 proteins confer resistance against influenza viruses, by blocking entry into the nucleus and transcription and replication, respectively. Despite the importance of these proteins, little is known about their anti- influenza mode of action. Therefore, we aim to elucidate the interactions between human and mouse Mx proteins and influenza vRNPs by using single domain antibodies (VHHs) as tools. We have isolated and sequenced 20 VHHs directed against human MxA and 20 against mouse Mx1. These VHHs have been purified from the culture medium of Pichia pastoris transformants and are currently being characterized functionally for their capacity to interfere with the in vitro GTPase activity of MxA and Mx1. We have also purified the vRNP complexes from an H7N7 influenza virus that is highly sensitive to Mx1. In the near future, we also aim to visualize the interaction between Mx proteins and IAV by high resolution microscopy techniques, using directly labeled VHHs as imaging tools. Overall, our findings will lead to novel insights in the mechanism of IAV restriction of human and murine Mx proteins.

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SUPPRESSING AND CONQUERING – antiviral defenses and their suppression Abstract final identifier: P105

INVOLVEMENT OF ADAR2 IN BORNA DISEASE VIRUS INFECTION Mako Yanai* 1, 2, Shohei Kojima1, 2, Nadine Gillich3, Masayuki Horie1, 4, Akiko Makino1, 2, Keizo Tomonaga1, 2 1Lab. of RNA Viruses, 2Graduate School of Biostudies, KYOTO UNIVERSITY, Kyoto, Japan, 3Institute of Virology, University Medical Center, Freiburg, Germany, 4Hakubi Center, Kyoto, Japan

Abstract: Borna disease virus (BoDV) is, a non-segmented, negative-strand RNA virus, establishes a persistent infection in the cellular nucleus without cytopathic effect. Our previous study revealed that recombinant BoDV harboring certain foreign gene in the genome underwent A-to-G substitution only in the foreign gene, suggesting that the A-to-I editing catalyzed by adenosine deaminase acting on RNA (ADAR) may be involved in BoDV replication. Here, therefore, we evaluated the involvement of ADARs, ADAR1 and ADAR2, in BoDV life cycle. To assess the significance of ADARs on BoDV infection, we infected ADAR1- or ADAR2- knockdown cells with BoDV, and the viral replication was monitored. The ADAR1- or ADAR2-knockdown was shown to significantly decrease the infection efficiency at the early phase of BoDV infection, compared to the control cells. Next, we evaluated the number of days required to reach almost 100% infection within the cultured cells. While the control and ADAR1-knockdown cells took 20 and 16 days to expand the infection in the cultures, respectively, the ADAR2-knockdown cells required 40 days to establish the persistent infection. These observations suggested that ADAR2 plays important role both in the initial infection and expansion of BoDV. To clarify the mechanisms of how ADAR2 affects BoDV infection, we performed microarray analysis and identified 65 differentially expressed genes between control and ADAR2-knockdown cells. To verify the result of microarray analysis, siRNA screening was conducted with 50 genes, whose expression levels were elevated in ADAR2- knockdown cells and identified a number of genes associated with the deficiency of BoDV infection by knockdown of ADAR2.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P107

CHARACTERIZATION OF THE H1 SUBTYPE-SPECIFIC NONCODING REGIONS OF INFLUENZA A VIRUSES IN REGULATING HA SEGMENT VIRION INCORPORATION Tao Deng* 1, Yue Xiao1 1Institute of Pathogen Biology,Chinese Academy of Medical Sciences, Beijing, China

Abstract: We previously reported that the noncoding region at both the 3′ and 5′ ends of the HA segments of influenza A viruses, are subtype-specific and vary significantly in sequence and length. Our study of the substitutions of the H1 subtype- specific NCR (ssNCR) with H1-H7 and H9 ssNCRs indicated that both the length and particular nucleotide(s) of the HA ssNCR might be involved in regulating HA segment virion incorporation. In this report, we further characterized the length and sequence requirements of the H1 ssNCR in determining H1 segment packaging in the WSN (H1N1) rescue system. In terms of the length requirement, we used serial 3′ and 5′ end H1 ssNCR truncation mutants with progressively reduced length. We described the differential effects of the length at the two ends of the H1 ssNCR. In terms of the sequence requirement, we first used serial H1 ssNCR mutants in which we replaced 3′ and 5′ H1 ssNCRs by random sequences with the wildtype length. We found that big sequence variations at both ends showed distinct HA packaging efficiencies. We then further performed Packaging Sequence Selection Assay (PSSA) with H1 ssNCR libraries containing random nucleotides in the H1 ssNCR. The results suggested that both the high A/U content and the specific locations of the A/U in the H1 ssNCR play important roles in regulating the H1 segment packaging efficiencies. We concluded that the H1 segment packaging efficiency may not be determined by a single nucleotide or specific successive nucleotides but by discrete nucleotides with low G/C content in the H1 ssNCR. Therefore, our experiments further illustrated detailed characteristics of the H1 ssNCR in regulating the H1 segment incorporation efficiency.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P108

THE COMPLEXITY OF THE N-GLYCOSYLATIONS NEAR SITE SA ON THE HA OF THE INFLUENZA A MODULATE ITS ANTIGENIC PROPERTY AND THE AVIDITY FOR ITS RECEPTOR Rafael A. Medina* 1, 2, Gabriel Guajardo-Contreras2, Richard Cadagan1, Adolfo Garcia-Sastre1 1Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States, 2Pediatric Infectious Diseases and Immunology, PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE, Santiago, Chile

Abstract: Antigenic drift is a key process that incorporates mutations that become fixed in the surface of the Hemagglutinin (HA) immunodominant glycoprotein of Influenza A Virus (IAV). HA also recognizes the sialic acid receptors in the host cells. One strategy used by IAVs to evade pre-existing immunity is to add N-glycosylation sites, in the globular head of HA. Glycosylation at residue 144 within site Sa of the 2009 H1N1 can induce a polyclonal response capable of neutralizing other glycosylated H1N1 variants. This glycosylation is also very effective at shielding this antigenic site. We hypothesized that the complexity of glycosylation 144 modulates the antigenic properties of HA and it has an effect in viral fitness. We made recombinant viruses and soluble HA proteins harboring variants of the A/Netherlands/602/09 H1N1 virus HA containing glycosylations at sites 144, 142, 172, and 144-172. We also generated either high-mannose or complex glycosylations, by growing these viruses in the presence or absence of the mannosidase inhibitor, kifunensine. Analyzes indicated a higher molecular weight of the HAs containing the 144 glycosylation in both, the recombinant viruses and soluble proteins, implying that this position allows the assembly of longer glycans compared to glycosylations in other positions. Reducing the length of the glycans increased the avidity of HA for its receptor, suggesting that glycosylations near the receptor-binding site can have an impact on viral infection. Additionally, infection of mice with viruses with complex glycosylated HAs induced more broadly neutralizing and higher titers antibodies as compared to the high-mannose glycosylated HAs viruses. These results provide new insights of the biological relevance of the N-glycosylations near site Sa and their role in antigenicity and viral fitness.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P110

ASSEMBLY AND SPREAD OF NIPAH VIRUS ENCODING A MATRIX PROTEIN WITH A DEFECTIVE NUCLEAR EXPORT SIGNAL Marc Ringel* 1, Laura Behner1, Anja Heiner1, Lucie Sauerhering1, Larissa Kolesnikova1, Andrea Maisner1 1PHILIPPS-UNIVERSITY MARBURG, INSTITUTE OF VIROLOGY, Marburg, Germany

Abstract: The NiV matrix protein (M) plays a major role in virus assembly by mediating the contact between viral RNPs and surface glycoproteins. As many paramyxoviral matrix proteins, M has been shown to undergo a nuclear transit before becoming assembly competent (Pentecost et al., 2015). So, aside of its role in cytoplasmic virus assembly, M might play a role in the nucleus. However, the influence of nuclear M expression on host cell functions or virus replication is mostly unclear yet. To determine if nuclear M expression affects the propagation of an assembly-defective NiV, we wanted to characterize NiV replication in the presence of an assembly-defective M protein that is readily expressed in the nucleus. For this, we generated a recombinant NiV encoding an M protein with a disrupted nuclear export signal (rNiV-MNESmut). Immunofluorescence analysis confirmed that mutant M was predominantly expressed in the nucleus. Though some MNESmut colocalized with cytoplasmic inclusions, it was not found in inclusion bodies associated with the plasma membrane. Corroborating the expected assembly defect, cell-free virus titers and the relative particle infectivity of rNiV-MNESmut were decreased by about 100 to 1000-fold compared to NiV wildtype. Furthermore, cells infected with rNiV-MNESmut showed an increased syncytia sizes suggesting that MNESmut is not only assembly defective but also fails to downregulate cell-cell fusion mediated by cell surface expressed NiV glycoproteins. Side by side analysis of rNiV-MNESmut with rNiV∆M did not reveal any differences in replication rates and particle infectivity suggesting that growth kinetics of an assembly-defective NiV is neither supported nor counteracted if M protein is functionally expressed in the nucleus.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P111

TYROSINE 132 OF INFLUENZA A VIRUS MATRIX PROTEIN 1 IS ESSENTIAL FOR EFFICIENT VIRAL GENOME PACKAGING AND PARTICLE ASSEMBLY Angeles Mecate-Zambrano1, Darisuren Anhlan1, André Schreiber1, Lilo Greune2, Stephanie Grothe3, Ludmilla Wixler1, Alexander Schmidt2, Klaus Langer3, Tianlai Shi4, Stephan Ludwig1, Yvonne Börgeling* 1 1Institute of Virology (IVM), 2Institute of Infectiology, 3Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Muenster, Germany, 4Immunology, Inflammation and Infectious Diseases (I3) DTA, Roche Pharma Research and Early Development, Roche Innovation Center, Basel, Switzerland

Abstract: Influenza is a zoonotic disease with pandemic potential. The limited protection provided by current antivirals emphasizes the urgent need for a deeper understanding of the virus life cycle to develop novel therapeutics with a broad spectrum. The highly conserved matrix protein 1 (M1) is a master regulator of the virus life cycle and essential for particle morphogenesis. Its multifunctionality qualifies M1 as a promising target for combating influenza, since it provides the opportunity to potentially block infections at various stages. M1 tyrosine 132 (Y132) was previously suggested to be essential for virus fitness as viruses carrying a mutation at this site could not be rescued. Based on overexpression data, it was hypothesized that this might be due to defective nuclear entry of M1. In the present study, we were able to rescue a virus mutant carrying an alanine at Y132 allowing for analysis of the role of this tyrosine during genuine infection. WSN M1 Y132A showed strongly decreased viral replication compared to wild type. While we did not detect any reduced nuclear import, coarse M1 protein clusters were observed at the plasma membrane in late stages of infection. Deeper characterization showed random defects in viral genome packaging, resulting in an increased production of non-infectious progeny. Furthermore, while general M1 Y132A association to membranes was not altered, resistance to detergent solubilization from membrane fractions was decreased. This suggests a potential defect in M1 recruitment to IAV assembly sites in lipid raft domains, which resulted in a diminished structural stability of viral progeny and the presence of filamentous particles. These findings indicate that M1 Y132 is crucial at late stages of IAV replication, and that efficient particle assembly including genome packaging is triggered by Y132 of the M1 protein.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P112

ARENAVIRUS EXIT, UBIQUITIN, AND THE ESCRT PATHWAY Christopher Ziegler* 1, Loan Dang1, Emily Bruce1, Bryan Ballif2, Jason Botten1 1Medicine, 2Biology, UNIVERSITY OF VERMONT, Burlington, United States

Abstract: Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPXL late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We recently showed that this domain in Z and the ESCRT pathway are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism driving this process, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of LCMV and Lassa virus. Nedd4 family E3 ubiquitin ligases were common host partners between the two Z proteins and this interaction with LCMV Z required an intact PPXY. We demonstrated that these ligases directly ubiquitinate LCMV Z and have mapped the specific lysine residues modified. We are currently testing recombinant LCMVs containing mutations at these particular lysine residues for their ability to produce both infectious virus and DI particles and these findings will be discussed.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P113

THE IQGAP FAMILY PROTEINS IN MARBURG VIRUS INFECTION Olga Dolnik* 1, Susanne Berghöfer1, Stephan Becker1 1Institut für Virologie, PHILIPPS-UNIVERSITY MARBURG, Marburg, Germany

Abstract: IQGAPs are evolutionary conserved proteins involved in the regulation of many cellular processes including cytokinesis, cell migration and proliferation, intracellular signaling, vesicle trafficking and cytoskeletal dynamics. Humans express three isoforms IQGAP1, IQGAP2 and IQGAP3. IQGAP1 is expressed ubiquitously, whereas IQGAP2 is mainly expressed in the liver and IQGAP3 in the brain. We could show earlier that IQGAP1 forms a comet-like structure at the rear end of Marburg virus (MARV) nucleocapsids during their transport in infected Huh-7 cells and siRNA down-regulation of IQGAP1 resulted in decreased MARV release. To further characterize the role of IQGAP1 in MARV infection we analyzed the release of infectious MARV particles from IQGAP1 knockout HAP1 cells. We observed almost no differences or even slightly increased titers in comparison to the parenteral HAP1 cells. Interestingly, an upregulation of IQGAP2 expression was detected in the IQGAP1 knockout cells in comparison to the HAP1 cell line, suggesting redundant usage of IQGAP2 for efficient virus release. Accordingly, MARV release from a IQGAP2 knockout cell line was slightly reduced and expression of IQGAP1 was elevated. We then analyzed the expression of IQGAP3 in the HAP1 cells and detected IQGAP3 being expressed and recruited into the MARV-induced inclusion bodies. Further analyses revealed elevated IQGAP3 expression levels in a IQGAP1 and IQGAP2 double knock-out cell line, suggesting that all the three human IQGAP proteins are involved in MARV release. To determine the importance of the IQGAP protein family in MARV infection we generate a triple-knock-out cell line for all three IQGAP proteins (IQGAPtriple-) using the CRISPR/Cas9 methodology. MARV infected IQGAPtriple- cells are currently analyzed for virus release and nucleocapsid transport.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P114

THE VRNA-VRNA INTERACTIONS IMPORTANT FOR HA VRNA PACKAGING OF THE INFLUENZA A VIRUS Sho Miyamoto* 1, Yukiko Muramoto1, Keiko Shindo1, Jamie L. Gilmore1, Masahiro Nakano1, Takeshi Noda1 1Laboratory of Ultrastructural Virology, Institute for frontier life and medical sciences, Kyoto University, Kyoto, Japan

Abstract: The influenza A virus genome is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight vRNAs are selectively packaged into progeny virions in a regular arrangement. For efficient vRNA packaging, each vRNA possesses the segment-specific packaging signal sequence located in the noncoding and terminal coding regions of both 3’ and 5’ ends. The incorporation of eight different vRNAs evokes specific interactions among the vRNAs via the packaging signals. However, vRNA-vRNA interactions involved in the selective genome packaging remains largely uncertain at the nucleotide level. Here, to identify vRNA(s) that potentially interact with HA vRNA during genome packaging, we generated several mutant influenza viruses (A/WSN/33) possessing silent mutations in the packaging signal of HA vRNA. Amongst them, one mutant virus, which showed a significant reduction in viral replication, was serially passaged in cultured cells until the replication efficiency was restored. RT-qPCR analysis showed that the mutant virus had a specific defect in HA vRNA incorporation. After the passages, the mutant virus obtained point mutations in the packaging signals of HA and PB2 vRNAs, both of which synergistically contribute to efficient virus replication and HA vRNA packaging. Furthermore, a direct RNA-RNA interaction between HA and PB2 vRNAs was confirmed in vitro. Overall, our data suggest that direct interactions of HA vRNA with PB2 vRNA are involved in the HA vRNA packaging into influenza virions.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P115

ANALYSIS OF HIGHER ORDER RNA STRUCTURES IN THE INFLUENZA A VIRUS GENOME Bernadeta Dadonaite* 1, Ervin Fodor1, Alain Laederach2, David Bauer1 1Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 2Department of Biology, University of North Carolina, Chapel Hill, United States

Abstract: Influenza A viruses (IAVs) are respiratory pathogens that constitute a major threat to human health. IAVs have negative sense, segmented, single stranded RNA genomes, which are maintained in viral ribonucleoprotein complexes (vRNPs). Each vRNP consists of the viral RNA (vRNA), viral polymerase and an oligomer of the viral nucleoprotein (NP). However, little is known about how the IAV RNA genome is structured inside virions. In this study we have used multiple high-throughput sequencing methods to analyse native secondary RNA structures in the context of vRNPs inside IAV virions. Using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) we show that secondary RNA structures form in the context of vRNPs. Different viral RNA segments have unique secondary RNA structures, suggesting an uneven distribution of NP along the vRNA. In addition, using sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), we show that IAV maintains a redundant network of inter-vRNP RNA interactions. Mutation of nucleotides involved in these interactions resulted in nonviable or attenuated influenza viruses with vRNP packaging defects. Further research into the vRNA interaction networks present in different influenza strains may lead to improved understanding of the molecular mechanisms governing the reassortment of influenza viruses.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P116

THE INFLUENZA VIRUS M2 PROTEIN CYTOPLASMIC TAIL INTERACTS WITH HOST PROTEINS TO FACILITATE VIRAL MORPHOGENESIS. Matthew Badham* 1, Jeremy S. Rossman1 1School of Biosciences, UNIVERSITY OF KENT, Canterbury, United Kingdom

Abstract: Influenza A virus (IAV) is an unusual pathogen in that it is pleomorphic, capable of forming virions ranging in shape from spherical to filamentous. Despite decades of research on IAV, much remains unknown about the formation of filamentous IAV and their role in the viral replication cycle. Hijacking of the host cell systems not only for replication but also for subversion of defence mechanisms is crucial for efficient proliferation of progeny virus. The 97 amino acid protein M2 possesses ion channel activity, necessary for acidification of the interior of the virus during initial infection and enabling uncoating of the viral RNP. However, the cytoplasmic tail of M2 (M2-CT) is known to also undergo interactions with multiple cellular proteins during virus assembly and budding, affecting the morphology of budding virions. It was recently found that the M2-CT is essential for the IAV evasion of autophagy and for filamentous virion formation. The M2-CT binds and sequesters LC3, a small ubiquitin-like protein and key autophagy regulator, thus attenuating the progression of autophagy and facilitating viral morphogenesis though an unknown mechanism. In this work we use mass spectrometry, confocal microscopy and biochemical assays to further characterise the interactions between the M2-CT and the small ubiquitin-like proteins (including LC3) and define their affect on virus morphology. We find that IAV does not form filamentous virions in the absence of the M2-CT and that interaction between the M2-CT and several small ubiquitin-like proteins are essential for viral morphogenesis, though many of these interactions are expendable for virus replication in vitro.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P117

EVIDENCE FOR A LINK BETWEEN BUDDING MORPHOLOGY AND TRANSMISSION OF AVIAN INFLUENZA A VIRUSES Carina Conceicao* 1, Helen Wise2, Nikki Smith1, Lita Murphy1, Seema Jasim1, Samantha Lycett1, Anamika Mishra3, Ashwin Raut3, Dave Burt1, Darrell Kapczynski4, Munir Iqbal5, Lonneke Vervelde1, Paul Digard1 1Infection and Immunity, The Roslin Institute, University of Edinburgh, Roslin, 2Institute of Genetics and Molecular Medicine, Heriot Watt University, Edinburgh, United Kingdom, 3ICAR-NIHSAD, NIH, Bhopal, India, 4Exotic & Emerging Avian Viral Diseases, USDA, Athens, United States, 5Avian influenza, The Pirbright Institute, Pirbright, United Kingdom

Abstract: Pleomorphic virus particles are a characteristic of influenza A virus (IAV), which can produce spherical virions of ˜100nm in diameter and filaments up to 30µm long. The majority of non-laboratory adapted mammalian IAV strains are thought to be filamentous however avian IAV budding morphology remains largely uncharacterised. To investigate this, 22 avian viruses representing the 11 major clades found from phylogenetic analysis of segment 7 were characterized for budding morphology, either as virus isolates and/or as 7:1 segment 7 reassortants in an PR8 backbone. The majority of viruses produced filaments up to 10µm but a sizeable minority were non-filamentous. Budding phenotype did not correlate with any particular subtype of virus or clade. However, the filamentous phenotype was common in duck viruses but less common in chicken viruses. Previous “rules” determined for M1 sequence polymorphisms affecting virion shape in mammalian strains were not confirmed, but mutagenesis of closely related strains with differing budding morphologies identified positions 59, 169 and 234 as IAV strain-dependent determinants. Furthermore, statistical analysis of the galliforme and anseriforme strains characterized here coupled with bioinformatic analysis of all avian segment 7s identified Ile at position 234 as significantly overrepresented in chicken IAVs and underrepresented in duck. Chicken viruses with Ile at this position produced spherical virions, while filamentous duck strains possessed Leu. Initial analyses of in vivo transmission experiments in chickens with the normally spherical A/chicken/Pakistan/UDL-01/08 and its filamentous mutant showed significantly raised buccal shedding of the latter from two sets of contact-infected birds. These results suggest that filamentous viruses are more common in ducks than chickens and the filamentous trait may be associated with enhanced shedding.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P118

CHARACTERIZATION OF THE MOLECULAR MECHANISMS UNDERLYING INTERACTION OF LASSA VIRUS Z PROTEIN WITH THE HOST FACTOR KIF13A Sarah Fehling* 1, Annika I. Meyer1, Birthe Ehlert1, Shuzo Urata2, Jiro Yasuda2, Thomas Strecker1 1Institute of Virology, PHILIPPS UNIVERSITY MARBURG, Marburg, Germany, 2Department of Emerging Infectious Diseases, Nagasaki University, Nagasaki-shi, Japan

Abstract: Lassa virus (LASV), a member of the Arenaviridae family, exits its host cell by budding from the plasma membrane, a process that is driven by the viral matrix protein Z. We have shown earlier that the microtubule-dependent motor protein KIF13A mediates the intracellular transport of Z protein. However, the molecular details of Z-KIF13A interaction remained elusive. KIF13A is characterized by an N-terminal motor domain, a forkhead-associated (FHA) domain, and five predicted coiled-coil domains. To identify domains within KIF13A that are important for interaction with Z protein, we generated a series of KIF13A deletion constructs. Biochemical and microscopic analyses pinpointed the Z-KIF13A binding interface to a C-terminally located coiled-coil motif and adjacent amino acids, while the FHA domain is dispensable for interaction. Our results describe a novel interaction domain for KIF13A-mediated cargo transport, which is distinct to previously defined cargo binding sites. To shed light on the interaction domain within Z protein, we also used a series of Z deletion constructs. Co-immunoprecipitation analyses and co-localization studies of Z protein mutants with KIF13A revealed a region spanning amino acids 31 to 50 within the highly conserved RING domain of Z that is critical for binding. The precise mapping of the binding sites within Z protein and KIF13A will allow the development of therapeutic strategies using inhibitors that target Z-KIF13A interaction.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P119

ACCESSIBILITY ASSESSMENT OF THE TERMINAL REGION OF INFLUENZA A VIRUS GENOME SEGMENT BY FLUORESCENCE IN SITU HYBRIDIZATION Fumitaka Momose* 1, Yuko Morikawa1 1Kitasato Institute for Life Sciences, KITASATO UNIVERSITY, Tokyo, Japan

Abstract: The genome of influenza A virus consists of eight vRNA segments and forms vRNP complexes. It is widely accepted that the segments selectively assemble, and one set of the segments is packaged into a virion. The packaging signals of each vRNA segment have been narrowed to both terminal regions by reverse genetic analyses using deletion and substitution mutants. However, the core nucleotide sequences essential for the recognition and interaction of the segments remain to be elucidated. It is possible that the core sequences would be covered if a segment assembled with others by base pairing or a mediating factor. In this study, fluorescence in situ hybridization (FISH) was applied to assess the accessibility of the 5' terminal region of the eighth segment (Seg. 8). Each cDNA probe binding within the 5' terminal region of Seg. 8 has a complementary sequence (ca 25 bases) overlapping each other, followed by a common detection sequence. MDCK cells were infected with A/Puerto Rico/8/34 strain and were subjected to FISH at six hours post infection. The maintenance of vRNP structure was confirmed using the anti-NP mAb61A5 preferentially binding to NP in an RNP complex. Fluorescent signals of cDNA probes bound to vRNA were intensified and detected with a fluorescence microscope. When hybridization was performed in the presence of 13% formamide, the hybridization efficiencies of three cDNA probes targeting 37-62, 62-86, and 100-127 bases of 5' terminal region were low. No correlation of the hybridization efficiency with the Tm values or the GC contents of complementary sequences suggested that the low efficiencies of the three probes were not due to poor probe characteristics but rather were due to the competition with other segments or factors. We supposed that the 5' terminal selective packaging signal of the Seg. 8 had been split into multiple regions.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P120

DETAILED FUNCTIONAL MAPPING OF THE PARAMYXOVIRUS ACCESSORY PROTEINS IN VIRAL INFECTION Ryoko Kawabata1, Asuka Yoshida2, Takemasa Sakaguchi1, Takashi Irie* 1 1Department of Virology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 2Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Abstract: Among Mononegavirales, paramyxoviruses are unique in producing more than one polypeptide species (C and/or V) from the P gene, in addition to the P protein. The C and V proteins are known as “accessory” proteins because they are not essential for minimal viral growth in cultured cells. However, they play a variety of important roles in viral growth and pathogenicity in vitro and in vivo. Indeed, a growing number of functions of the accessory proteins have been revealed, such as counteracting host innate immunity, avoiding host detection of infection, inhibiting apoptosis, induction of necroptosis, regulating polarized viral RNA synthesis, and promoting viral assembly and budding. These functions can be assessed by generating recombinant viruses in which the accessory proteins are knocked out. However, due to overlaps of their open reading frames, a more detailed evaluation of their functions during actual viral infection is severely limited by the difficulties in introducing amino acid substitutions only into the accessory proteins without any alterations within the P protein. To avoid this restriction, we generated recombinant Sendai viruses (rSeVs) in which the C or V protein was expressed from an independent cistron introduced between the HN and L genes, not from the P gene. Both of the viruses replicated as well as the parental wild-type virus. Based on this rSeV system, a series of rSeVs possessing five alanine substitutions throughout the Y2 (31 - 204 amino acid [aa]) region of the C protein, 204 amino acids long, were generated and examined in terms of their effect on viral replication as well as the other known functions of the C protein. These results show, for the first time, a detailed functional map of the SeV C protein in an actual viral infection.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P121

INFLUENCE OF NUCLEAR LOCALIZATION SITES IN HENDRA VIRUS MATRIX PROTEIN ON INTERACTION WITH ANP32B AND VIRUS LIKE PARTICLE FORMATION Maria Günther* 1, Stefan Finke1 1Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany

Abstract: In addition to the essential role of paramyxovirus matrix (M) proteins in assembly and budding of the cytoplasmically replicating viruses at the plasma membrane, many M proteins are also transported into the nucleus. For some paramyxoviruses, interactions with nuclear host factors have been described and host manipulatory functions are assumed. M protein of Hendra virus (HeV) comprises a bipartite nuclear localization signal (NLS) and interaction with acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) has been shown. Notably, nuclear import of henipaviral Ms is not only considered to be important for host manipulation but also for virus budding. The NLS contains a lysine residue that is mono-ubiquitinylated in the nucleus and nuclear mono-ubiquitinylation has been shown to be involved as a trigger for nuclear export of M. Since also budding activity at the plasma membrane was affected by mutations at that position, it has been suggested that nucleo-cytoplasmic trafficking and mono- ubiquitinylation are requirements for virus budding at the plasma membrane. In order to map interaction sites for ANP32B, we performed Ala scanning mutagenesis on HeV M and screened for effects of ANP32B on intracellular localization of M and co-purification with ANP32B. Here we focus on NLS-mutants with amino acid exchanges in the bipartite NLS of HeV M. Immunofluorescence analyses showed that mutations in one part of the NLS led to the loss of nuclear localization, whereas plasma membrane localization was not affected. First budding assays suggest the release of virus like particles (VLPs) after expression of the NLS mutant. These data indicate that nuclear shuttling and mono-ubiquitinylation in the nucleus are mechanistically not directly linked.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P122

SURFACE-EXPOSED LYSINE RESIDUES OF THE CDV MATRIX PROTEIN CONTRIBUTE TO MEMBRANE ASSOCIATION AND BUDDING ACTIVITY Nicole Kadzioch* 1, 2, Matthieu Gast1, 2, Philippe Plattet2 1Graduate School for Cellular and Biomedical Sciences, University of Bern, 2Division of Experimental Clinical Research, University of Bern, Vetsuisse Faculty, Bern, Switzerland

Abstract: The Matrix (M) protein of Morbilliviruses coordinates viral packaging, membrane budding and particle formation. Two monomers presumably assemble to form dimer and higher oligomers. The current model of cell-exit postulates that M associates with lipids to ultimately trigger membrane budding at the plasma membrane. Additionally, recent data revealed that ubiquitination of a lysine residue within Nipah Virus M, likely mediated within the nucleus, played a key role in viral particle production. Based on a homology model of Canine Distemper Virus (CDV) M, surface-exposed positively charged lysine (K) residues locating within M-CTD were selected for substitutions. Indeed, lysines are well-known to potentially interact with the negatively charged groups of the phospholipid heads. The selected lysines were mutated into either arginine (R) or glutamic acid (E), and the derivative M-mutants submitted to assays investigating Virus-Like-Particles (VLPs) production (biochemical analyses) and cellular localization (immunofluorescence). Preliminary data indicated that the conservative mutations K227R, K228R, K265R and K268R did not affect budding and the plasma membrane-targeting capacity of M. In contrast, the non-conservative M-variants K227E, K228E, K265E and K268E were deficient in budding and exhibited a clear cytosolic phenotype. Remarkably, substituting residue K240 of M led to opposite profiles: while mutant M-K240R was inefficient in VLPs production, M-K240E remained fully functional, although both M proteins were properly localized at the cell periphery. Since it was reported that Morbillivirus M-K240 undergoes ubiquitination, the functional impacts of several substitutions at this position is currently under investigation. In summary, our preliminary data already revealed a potential role of some lysine residues in membrane association and budding activity.

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BUILDING AND ESCAPING – viral assembly and budding Abstract final identifier: P123

INVESTIGATION OF Α-HELICES OF CANINE DISTEMPER VIRUS MATRIX PROTEIN IN OLIGOMERIZATION AND MEMBRANE BUDDING ACTIVITY Matthieu Gast* 1, 2, Nicole Kadzioch1, 2, Philippe Plattet2 1Graduate School for Cellular and Biomedical Sciences, University of Bern, 2Division of Experimental Clinical Research, University of Bern Vetuisse Faculty, Bern, Switzerland

Abstract: The Canine Distemper Virus (CDV), related to the human Measles virus, induces devastating epidemics in numerous wild species. It is supposed that specific antiviral compounds associated with vaccination campaigns may allow a better control of ongoing epidemics. CDV Matrix (M) protein orchestrates viral assembly and budding of progeny virions at the plasma membrane. Two M-protomers, composed of N- and C-terminal domains (NTD and CTD), likely assemble in a “head-to-tail” fashion, via a large dimeric binding interface, to form dimers. Interestingly, M-NTD and -CTD contain surface exposed α-helices (α2 and α9, respectively) that are proposed to be involved in dimer-dimer and higher oligomers formation. To investigate a potential role of high M-oligomers assembly in membrane budding and Virus-Like-Particles (VLPs) formation, we substituted multiple residues mapping within the critical α2 and α9 helices. Production of VLPs was assessed by biochemical analyses as well as a newly developed very sensitive NanoLuc Luciferase (NLuc)-based M-budding assay, which enables direct detection of VLPs in the cell supernatant. Surprisingly, among the various mutations introduced, two non-conservative mutants (M-E89R [α2] and M-L239D [α9]) exhibited strong impairments in VLPs formation using both assays, while displaying unaltered cell periphery accumulation. Other mutants were characterized by wild type-like profiles in localization and budding activity. Dimerization and ubiquitination propensities as well as membrane association capacity of the two identified budding-deficient M-mutants (E89R and L239D) are currently under investigation. Overall, these preliminary data suggest an important role of two specific amino acids locating within the α-helices of CDV-M in controlling membrane egress activity by, perhaps, regulating high oligomers assembly at the plasma membrane.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P124

A RECOMBINANT MEASLES VIRUS BLIND TO SLAM IS A PROMISING CANDIDATE FOR SCIRRHOUS GASTRIC CANCER THERAPY Chieko Kai* 1, Koichiro Shoji1, Hyun-jeong Kwon1, Akihiro Sugai1, Kazuyoshi Yanagihara2, Hideki Yamaguchi3, Tomoko Nakanishi1, Hiroki Sato1, Tomoko Fujiyuki1, Misako Yoneda1 1Laboratory Animal Research Center, The Institute of Medical Science, THE UNIVERSITY OF TOKYO, Tokyo, 2Exploratory Oncology & Clinical Trial Center, National Cancer Center, Chiba, 3 Department of Cancer Cell Research, Sasaki Institute, Tokyo, Japan

Abstract: Virotherapy using oncolytic virus is expected as a novel therapy for cancer. We previously generated a recombinant measles virus (rMV-SLAMblind), which is unable to use SLAM as its principal receptor and lost the MV pathogenicity. We have reported that it infects and kills cancer cells derived from breast, lung, colorectal, and pancreatic cancers dependently on expression of the virus receptor molecule, Nectin-4. (Gene Therapy, 2012; Oncotarget, 2015; Scientific Reports, 2016; Cancer Science, 2016). Gastric cancer is the third most common cause of cancer-related death in the world. Especially, scirrhous gastric cancer (SGC) has a very poor prognosis, and thus novel therapies are desired. In this study, we examined applicability of rMV- SLAMblind for SGC. Nectin-4 expression was observed in approximately 70% of SGC cell lines, and showed severe cytotoxic activity to SGC cell lines highly expressing nectin-4. Intratumoral administration of rMV-SLAMblind to subcutaneous xenograft mouse model with a SGC cell line remarkably suppressed the tumor growth. Furthermore, rMV- SLAMblind intraperitoneally inoculated into peritoneal dissemination xenograft model targeted scattered tumors in the peritoneal cavity, and significantly suppressed the tumor growth and clinical symptoms. These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for treatment of scirrhous gastric cancer, particularly with peritoneal dissemination.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P125

RIFT VALLEY FEVER VIRUS INFECTION OF PREGNANT SPRAGUE-DAWLEY RATS RESULTS IN FETAL INFECTION AND DEMISE Amy Hartman* 1, Cynthia McMillen1 1Center for Vaccine Research, UNIVERSITY OF PITTSBURGH, Pittsburgh, United States

Abstract: Rift Valley fever is an important emerging zoonotic disease in Africa and the Saudi Arabian peninsula. Domesticated livestock are higly susceptible to severe disease when infected with RVFV, and death of fetuses in pregnant animals is extraordinarily high. The effect of RVFV infection on human pregnancy is less clear. Two cases of vertical transmission of RVFV have been described in humans and one cross-sectional study reported a 4-fold increased risk of miscarriage in RVFV+ women. Here, we sought to determine the effect of RVFV in pregnant rats with the goal of developing a reliable rodent model of congenital RVF. Pregnant immunocompetent Sprague-Dawley rats at early (embryonic day 5; ED5) and late (embryonic day 14; ED14) gestational stages were subcutaneously infected with pathogenic RVFV. Pregnant E14 dams with no clinical signs had widespread virus detected in the spleen, liver, lung, brain, uterus, and ovaries at five days post-infection. Some pups were stillborn, and surviving 10-day old pups from these mothers retained 3-log more viral RNA than maternal tissue despite grossly normal physiology of the pups. In infected ED5 rats, virus preferentially homed to the placenta and fetal tissue, in which 1000x more virus was detected than in maternal tissue. Potential readsorption and hemorrhage of the fetuses was evident. This study is the first of its kind to develop a rodent model of congenital RVF and further explore the potential for RVFV to cause congenital disease in humans. This rodent model can serve as a tool to screen antiviral therapies and vaccinations targeting RVFV.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P126

GENOME-WIDE ANALYSES OF FILOVIRUS-HOST CELL PROTEIN-PROTEIN INTERACTIONS Douglas J. LaCount* 1, Veronica J. Heintz1, James P. Connell1, Venkatesh Sivanandam1, Aditi Kesari1, Ling Wang1, Olena Shtanko2, Ann Reyes2, Robert A. Davey2 1Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, 2Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, United States

Abstract: Ebola (EBOV) and Marburgvirus (MARV) cause potentially lethal hemorrhagic fevers in humans characterized by uncontrolled viremia, a systemic pro-inflammatory response, and multi-organ failure. Recent outbreaks highlight the critical need to better understand the interactions of these viruses with host cells. Using a combination of genome-wide yeast two-hybrid (Y2H) screens and siRNA knockdowns, we are identifying critical virus-human protein-protein interactions. We performed 432 Y2H library screens with 190 filovirus bait constructs against three cDNA libraries. We identified 916 unique interactions between filovirus and cellular proteins, of which 365 were reproduced in at least two independent screens. One hundred and two interactions were shared between EBOV and MARV. Enrichment analyses revealed that VP30 targets proteins involved in RNA metabolism, many of which localize to stress granules. We screened 66 cellular proteins that bound to either EBOV or MARV VP30 in siRNA knock down experiments followed by live virus infection. We observed a higher percentage of the cellular proteins identified in the Y2H screens affected virus replication than previously observed in experiments with randomly selected siRNAs. Two distinct phenotypes were observed. Although most siRNAs that affected virus replication caused a decrease, eight sets of siRNAs, including those targeting RBBP6, increased replication. This suggests these cellular factors normally function to inhibit virus replication. Studies to ascertain the mechanism by which virus replication is affected are underway. This study represents the largest effort to date to compare the host factors targeted by EBOV and MARV.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P127

INFLUENZA VIRUSES AND PARAMYXOVIRUSES ENHANCE THE ADHERENCE AND INVASION PROPERTIES OF STREPTOCOCCI BY A SIALIC ACID-DEPENDENT INTERACTION WITH THE CAPSULAR POLYSACCHARIDE Georg Herrler* 1, Fandan Meng1, Jie Tong1, Nai-Huei Wu1, Yuguang Fu1, Nadine Krüger1, Maren Seitz2, Peter Valentin- Weigand2 1Virology, 2Microbiology, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany

Abstract: Viral-bacterial co-infections may result in more severe disease than the respective mono-infections. The underlying mechanisms varies among different pathogens. We report a mechanism involving the interaction of viral hemagglutinins with sialic acids of encapsulated streptococci. The porcine pathogen Streptococcus suis that has a zoonotic potential contains a2,6-linked sialic acid that is recognized by porcine and human influenza viruses. Using precision-cut lung slices (PCLS) as a model for differentiated airway epithelial cells we found that hemagglutinins expressed on the surface of virus-infected cells interact with S. suis resulting in enhanced bacterial adherence. Late in infection, when the airway epithelium is destroyed, the porcine pathogen adhered and invaded subepithelial cells independent of the capsular sialic acid. Group B streptococci (GBS, S. agalactiae), a human pathogen, contains a similar capsular polysaccharide differing only in the linkage type of the terminal sialic acid. GBS contains a2,3-linked sialic acid which is recognized by avian influenza viruses. Adherence to and invasion of airway cells was greatly enhanced when the cells were preinfected by avian influenza viruses. Co-infections by GBS and avian influenza viruses may be rare events. However, for some paramyxoviruses, e.g. human parainfluenzavirus 3 (HPIV3) and mumps virus (MuV) it has been shown that they use a2,3-linked sialic acid as a receptor determinant. We found that adherence to and invasion of airway cells is enhanced when the cells express the HN proteins of either HPIV3 or MuV on the surface. Taken together, bacterial pathogens containing capsular sialic acid residues may use the interaction with virus-infected cells to enhance their adherence and invasion properties which my result in a more severe disease.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P128

SEX-DEPENDENT HOST RESPONSES ASSOCIATED WITH EBOLA VIRUS PATHOGENICITY Atsushi Okumura1, Friederike Feldmann2, Elaine Haddock2, David Threadgill3, Heinz Feldmann2, Angela Rasmussen* 1 1Center for Infection and Immunity, Columbia University, New York, 2Laboratory of Virology, Rocky Mountain Laboratories, NIAID/NIH, Hamilton, 3Veterinary Pathobiology, Texas A&M University, College Station, United States

Abstract: The sexes differ in susceptibility to viral infection and subsequent disease pathogenesis. During the West African Ebola virus disease (EVD) outbreak, female patients had a significantly lower risk of death compared to male patients (p<0.001), despite similar numbers of exposures to sick individuals. Genetic and physiological differences between the sexes may thus dictate host responses to Ebola virus (EBOV) infection that determine disease severity and outcome. We used the Collaborative Cross (CC) mouse model to investigate the relationship between sex and pathogenicity in the context of multiple EVD phenotypes. The CC is a panel of genetically diverse mice that develop background-dependent distinct disease presentations. Both standard CC mice and F1 mice bred from intercrossed CC lines are susceptible to three major outcomes analogous to different presentations of human EVD: survival, lethal EVD, and lethal EVD with hemorrhagic syndrome. Of 19 lines screened for sex specific outcomes, we observed stark differences (in which all the animals of one sex survived while the other sex succumbed to lethal disease) in 2 lines, and measurable sex-specific disease features in 74% of the lines tested, including body weight, viral titers in liver and spleen, kinetics of disease progression, and survival rate. We then used RNAseq to develop sex-specific transcriptomic profiles associated with disease severity and outcome. We identified a network of differentially expressed testosterone-dependent genes related to inflammation, shock, vascular remodeling, and apoptosis that were strongly upregulated in male mice with lethal disease, indicating that distinct sex- specific host responses underlie pathogenesis. We are currently expanding these efforts to identify sex-specific host responses that determine disease severity, as well as performing more detailed studies of sex-dependent mechanisms of pathogenicity.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P130

WHOLE GENOME CRISPR/CAS9-KO SCREEN REVEALS PROTEIN KINASE R (PKR) AS THE KEY PLAYER IN RABIES VIRUS CYTOTOXICITY Alexander Ghanem* 1, Alexandru A. Hennrich1, Maximilian F. Eizinger1, Karl-Klaus Conzelmann1 1MAX V PETTENKOFER-INSTITUTE AND GENE CENTER, LMU MUNICH, Munich, Germany

Abstract: Wildtype neurotropic rabies rhabdovirus (RABV) is typically a non-cytolytic "stealth" virus, while cell culture- adapted attenuated vaccine strains like SAD eventually kill infected cells. Understanding of the differential mechanisms behind RABV cytotoxicity is crucial for the development of vaccines, oncolytic viruses, and rabies vectors for neuronal tracing. To identify cellular factors mediating RABV cytotoxicity, we performed a pooled genetic CRISPR/Cas9 KO-screen. Human HEK293T cells expressing Cas9 were transduced with a genome-wide lentiviral sgRNA library and infected with cytotoxic SAD. Genomic DNA from surviving cells was isolated and analyzed by NGS for enriched sgRNA sequences. Notably, in cells surviving SAD infection, only sgRNAs targeting a single gene (EIF2AK2) encoding protein kinase R (PKR) were highly enriched. Independent knock-out of the EIF2AK2 gene rendered HEK293T and A549 cells resistant to SAD- mediated cell death. Knock-out of the PKR-activating PACT, or of RIG-I and MAVS, which are instrumental in sensing RABV 5’-triphosphate RNAs and inducing interferon, further illustrate that PKR is the sole RNA sensor mediating death in SAD- infected cells. While infection of wt HEK293T cells with SAD and cytotoxic versions of SAD (SAD TR) strongly activated PKR and caused phosphorylation of eIF2α, a biologically selected non-cytotoxic SAD version (SAD Le) did not. Nevertheless, cytotoxic viruses produce more IFN-inducing RNAs than persistent variants. We are currently investigating the contribution of different viral RNA species to the activation of cellular RNA sensors in different cell lines, including stem- cell derived neurons.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P131

FUNCTIONAL ANALYSIS OF A CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS GENOME FROM TICKS IDENTIFIES A GLYCOPROTEIN VARIANT THAT POORLY INFECTS HUMAN CELLS Brian Hua* 1, Florine Scholte1, Valerie Biewener2, Marco Marklewitz2, Christian Drosten2, Stuart Nichol1, Christina Spiropoulou1, Sandra Junglen2, Éric Bergeron1 1Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States, 2Institute of Virology, Charité Universitätsmedizin Berlin, Berlin, Germany

Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread emerging pathogen that can cause severe human disease with case mortality rates up to 30%. CCHFV is a tick-borne virus exhibiting extensive genomic diversity across strains with highly divergent strains known to co-circulate in the same geographical area. It is not fully understood how these differences in genomic sequence regulate virus activity especially in the context of virus transmission to humans. To further investigate CCHFV genomic contribution in the infection of human cells, we obtained the full genomic sequence of a CCHFV strain directly from a tick captured in Malko Tarnovo (MT), Bulgaria. The CCHFV MT strain is closely related to the Greek AP92 strain, which is known to exhibit widespread dissemination but relatively infrequent detection in clinical disease. Using the CCHFV MT genome sequence, we employed reverse-genetics approaches to generate virus-like particles (VLPs) and a plasmid set to rescue recombinant MT CCHFV. Attempts to generate VLPs with the MT glycoprotein precursor (GPC) uniquely resulted in attenuated reporter activity as compared to VLPs generated with the GPCs of 11 strains representing all other CCHFV phylogenetic clades. Consistently, we rescued virus with the MT S and L genomic segments but failed to rescue virus containing the MT M (GPC-encoding) genomic segment. Analysis of chimeric and point mutant GPC constructs revealed that a single amino acid in the MT Gc glycoprotein region, Gly1116, is responsible for poor MT GPC activity in human cells. Our study warrants further functional analyses of CCHFV sequences from tick reservoirs to better understand CCHFV transmission and disease association.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P132

TISSUE TROPISM AND DISSEMINATION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS IN IFNAR KNOCKOUT MICE VISUALIZED BY IN SITU FLUORESCENT IMAGING Stephen R. Welch* 1, Florine E. Scholte1, Jana M. Ritter2, JoAnn D. Coleman-McCray1, Jessica R. Harmon1, Sherif R. Zaki2, Stuart T. Nichol1, Eric Bergeron1, Jessica R. Spengler1, Christina F. Spiropoulou1 1VSPB, 2IDPB, Centers for Disease Control and Prevention, Atlanta, United States

Abstract: Animal models of disease are a vital tool for understanding viral pathogenesis and immunity. The use of reporter viruses in these models allows for direct visualization and identification of infected tissues and cells in the host. Here, we investigated a Crimean-Congo hemorrhagic fever virus (CCHFV, strain IbAr10200) expressing the ZsGreen1 (ZsG) fluorescent protein (10200/ZsG) in the IFNAR knockout mouse model. We also evaluated in vivo a reporter mutant virus in which the non-structural protein NSm ORF was removed from the M segment (10200ΔNSm/ZsG). While functions have been ascribed to the NSm proteins of other species of the Bunyavirales family, nothing is known about the role CCHFV NSm plays during infection. We followed infection by assessing survival, viral loads, and histopathology, and imaged tissues expressing ZsG in situ. We found that disease phenotype and pathology were comparable between the reporter 10200/ZsG virus and wild-type 10200 CCHFV, both showing uniform lethality within 5–6 days. We also found that CCHFV NSm was dispensable both in vitro and in vivo, but the virus lacking the NSm protein exhibited an attenuate phenotype. Disease onset was delayed in mice infected with 10200ΔNSm/ZsG, and 1 of the 5 animals survived infection. In situ visualization confirmed the highly hepatotropic nature of CCHFV, and revealed viral tropism towards previously overlooked tissues and organs. Overall, the lack of attenuation of the 10200/ZsG reporter virus, and the ease of directly visualizing infected tissue and cells, will facilitate further investigations into virus-host dynamics of CCHFV infection in vivo.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P133

HUMAN IMMUNE RESPONSES TO LASSA VIRUS INFECTION IN NIGERIA Lisa Oestereich* 1, 2, Emily Speranza3, 4, David M. Wozniak1, 2, Julia R. Port1, 2, Sabrina Bockholt1, 2, Elisa Pallasch1, 2, Jonas Müller2, Danny Asogun5, Ephraim Ogbaini-Emovon5, Meike Pahlmann1, 2, John H. Connor3, Cesar Muñoz-Fontela1, 2, Stephan Günther1, 2 1Virology, German Center for Infection Research (DZIF), 2Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 3Department of Microbiology, Bioinformatics Program, Boston University School of Medicine, Boston, 4Laboratory of Virology, RML NIH, Hamilton, United States, 5Institute for Lassa Fever Research and Control, Irrua Specialist Teaching Hospital, Irrua, Nigeria

Abstract: Lassa virus (LASV) is a zoonotic pathogen that is endemic in several West African countries. It causes annual outbreaks of Lassa fever (LF) with a case fatality rate of 20-30% in hospitalized patients. LF is on the R&D Blueprint list of the World Health Organization. The pathogenesis of LF is poorly understood, although deregulated host responses presumably play a key role. However a better understanding about the underlaying mechanisms is urgently needed in order to identify currently licensed drugs that might have a beneficial effect on the course of the infection. Together with cooperation partners from the Irrua Specialist Teaching Hospital (ISTH) in Irrua, located in a hyper-endemic LF zone in Nigeria, we investigate the host response in LF. Between 2014 and 2018 more than 1000 samples belonging to more than 300 patients were collected and analyzed. The analyses include serology for viral proteins, virus titer determination, clinical chemistry, hematology, soluble cytokine and chemokine analysis and multiparameter flow cytometry. Here we summarize the data gathered from the immuno phenotyping and the result of a principal component analysis to identify key biomarkers that can predict the outcome of the infection.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P134

IDENTIFICATION OF ESCAPE MUTANTS OF EQUINE INFLUENZA USING POLYCLONAL SERA AND MONOCLONAL ANTIBODIES: A POTENTIAL ANTIGENIC DRIFT WITH AN IMPACT ON VACCINE EFFICACY

Shadia Omar* 1, Neil Bryant1, Debra Elton1 1virology, Animal Health Trust, Newmarket, United Kingdom

Abstract: Equine influenza virus (EIV) is known to antigenic drift similar to other Influenza Viruses which causes vaccine breakout when vaccinated horses are infected with some escape mutants that is significantly distant from the vaccine strains. Antigenic drift occurs by multiple amino acid substitutions in the surface protein haemagglutinin (HA). HA is responsible for initiating entry into upper respiratory tract epithelial cells which involves multiple HAs binding to sialic acids on cells surface, therefore, HA is the main target for neutralizing antibodies and the mutation will enable the escape mutants to evade the antibodies. We attempted to predict future variants of EIV by carrying out serial passage of EIV vaccine recommended strains A/equine/Richmond/1/07, A/equine/South Africa/04/03 in eggs using polyclonal ferret and equine sera. This methodology has been used to map the antigenic sites of human influenza viruses, including H3N2, but has not been applied to influenza viruses from other mammals. We also made a panel of 50 recombinant viruses with additional point mutations in antigenic sites that have occurred naturally in field isolates, using reverse genetics and site-directed mutagenesis, to test the effect of substitutions in the antigenic sites on their own and in combination with additional changes, in order to determine how many changes and of what type are most likely to make an antigenic difference for EIV. The reassortants and recombinant viruses described above were tested by haemagglutination-inhibition assays. We identified single amino acid substitution at residue K156N in antigenic site B and we found that mutations at positions 159, 172 and 189 have altered antigenicity. These methods will improve the current selection process for vaccine strains.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P135

IDENTIFICATION OF AMINO ACIDS THAT ENHANCE VIRUS REPLICATION IN MAMMALIAN HOSTS IN THE PB2 AND PA PROTEINS OF A HIGHLY PATHOGENIC H7N9 INFLUENZA VIRUS ISOLATED FROM A HUMAN Seiya Yamayoshi* 1, Maki Kiso1, Atsuhiro Yasuhara1, Mutsumi Ito1, Yuelong Shu2, Yoshihiro Kawaoka1, 3, 4 1Division of Virology, INSTITUTE OF MEDICAL SCIENCE, UNIVERSITY OF TOKYO, Tokyo, Japan, 2Sun Yat-Sen University, Shenzhen, China, 3University of Wisconsin-Madison, Madison, Wisconsin, United States, 4International Research Center for Infectious Diseases, INSTITUTE OF MEDICAL SCIENCE, UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Highly pathogenic H7N9 viruses emerged in late 2016 and infected humans. The highly pathogenic H7N9 human isolate A/Guangdong/17SF003/2016 (GD/3) possesses HA with human-type-receptor preference and NA with inhibitor resistance, and transmits among ferrets via respiratory droplets. GD/3 also shows high replication capability and high virulence in mice. To understand the role of the viral polymerase complex in the high pathogenicity of GD/3, we investigated its polymerase activity, growth kinetics, and pathogenicity by using a mutagenesis approach. We found that arginine at position 482 and valine at position 588 of PB2 and arginine at position 497 of PA individually enhanced viral polymerase activity in human cells, upregulated virus propagation in human cells, and contributed to efficient replication and high virulence in mice. In combination, these mutations had additive effects. PB2-482R and PB2-588V are located at the ‘cap-binding domain’ and near PB2-627, respectively, whereas PA-482R is not exposed on the protein surface. These findings suggest that PB2-588V is likely involved in ANP32A-dependent high polymerase activity in mammalian hosts (ANP32A is a key host protein for PB2-627K-dependent high polymerase activity). Our data further suggest that PB2-482R and PA-497R contribute to enhanced polymerase activity in other ways. Our findings indicate that the presence of multiple replication-enhancing mutations in a single highly pathogenic H7N9 virus isolate may contribute to its high virulence in mammalian hosts.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P136

COMPARATIVE IN VITRO AND IN VIVO ANALYSIS OF SWINE-ORIGIN H1N1 AND H1N2 INFLUENZA VIRUSES ISOLATED FROM HUMAN CASES BETWEEN 2011 AND 2016. Joanna Pulit-Penaloza* 1, Jessica A. Belser1, Claudia Pappas1, Xiangjie Sun1, Nicole Brock1, Hui Zeng1, Terrence M. Tumpey1, Taronna R. Maines1 1CENTERS FOR DISEASE CONTROL AND PREVENTION, Atlanta, United States

Abstract: Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infection, and are termed variant viruses. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no pre-existing immunity is an ongoing threat to public health. Recently isolated H1 variant viruses were shown to display great genetic diversity and in many instances were antigenically different from human vaccine strains. We utilized in vitro and in vivo approaches to provide extensive characterization of H1N1 and H1N2 variant viruses isolated since the 2009 pandemic. A majority of the tested variant viruses emerged as a result of reassortment between H1N1 2009 pandemic and diverse swine triple reassortant viruses and showed similarities in molecular markers in the HA and polymerase genes with the 2009 pandemic viruses. The H1 variant viruses were well adapted to replicate in a human respiratory cell line and the respiratory tracts of mice and ferrets. However, the variant viruses had HA activation pH thresholds closer to that of most North American swine isolates rather than viruses that had adapted to humans. Consistent with what was previously observed for swine isolates, all of the tested variant influenza viruses were capable of efficient transmission between co-housed ferrets, but the ability to efficiently transmit via respiratory droplets differed between strains. This investigation highlights the need to closely monitor variant viruses for additional molecular changes that could facilitate efficient transmission between humans.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P137

ONCOLYTIC RECOMBINANT MEASLES VIRUS IS A CANDIDATE THERAPEUTIC AGENT FOR REFRACTORY BREAST CANCER Tomoko Fujiyuki* 1, Yosuke Amagai1, Koichiro Shoji1, Akihiro Sugai1, Mutsumi Awano1, Hiroki Sato1, Misako Yoneda1, Chieko Kai1 1Laboratory Animal Research Center, The Institute of Medical Science, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Oncolytic virotherapy is a new approach for cancer treatment. A recombinant measles virus (rMV-SLAMblind) that we have previously generated lost its affinity to a principal receptor of MV, SLAM, and thus lost the MV pathogenicity, whereas it is able to use Nectin-4 on cancer cells. We have previously demonstrated that rMV-SLAMblind infected several breast cancer cell lines using Nectin-4 and showed antitumor activity. Triple-negative breast cancer (TNBC) is known to be aggressive and often relapses with poor prognosis more than other types of breast cancer. Owing to resistance to both hormone and trastuzumab treatments, TNBC often causes recurrence and metastasis. Thus, novel effective therapies for TNBC are needed. In this study, we examined whether rMV-SLAMblind is effective for TNBC. We examined expression level of nectin-4 on the surface of TNBC cell lines with flow cytometry, and found that it was expressed on the surface of 75% of the analyzed cell lines. By inoculating the cells with rMV-SLAMblind, rMV-SLAMblind infected the nectin-4-expressing TNBC cell lines, and showed cytotoxicity in vitro. Furthermore, we examined anti-tumor effect of rMV-SLAMblind in vivo by using mouse xenograft models. Intratumoral administration of the virus suppressed tumor growth. To further assess the effectiveness of rMV-SLAMblind treatment for metastatic cancer, we administered rMV- SLAMblind expressing luciferase via intravenous route. In vivo imaging indicated that the virus replicated selectively in the tumor. In addition, the tumor growth was significantly suppressed. These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for TNBC. We are advancing translational research of rMV-SLAMblind.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P138

SPECIFIC IMMUNOLOGICAL SIGNATURE DEPENDING ON THE DISEASE OUTCOME IN EBOLA VIRUS-INFECTED PATIENTS FROM MACENTA (GUINEA) Stephanie Reynard* 1, 2, Emilie Gloaguen3, Cedric Laouénan3, Jimmy Mullaert3, Natalia Pietrosemoli4, Hugo Varet4, Justine Schaeffer1, 2, Sylvain Baize1, 2 1Unité de Biologie des Infections Virales Emergentes, Institut Pasteur, 2Centre International de Recherche en Infectiologie, Lyon, 3Département d’Epidemiologie, Biostatistiques et Recherche Clinique, Inserm, 4Centre de bioinformatique, biostatistique et biologie intégrative, Institut Pasteur, Paris, France

Abstract: Ebola virus (EBOV), responsible for the West African epidemic in 2014-2015, causes severe hemorrhagic syndrome. The pathogenic events leading to a fatal outcome or to survival remain poorly understood. Samples from 75 EBOV-infected patients and 15 febrile controls admitted in an Ebola Treatment Center (Macenta, Guinea) but who did not receive any specific EBOV therapy, were analyzed to characterize virological and immunological parameters. Transcriptomic analyses were performed on RNA extracted from leucocytes (GeneChip Human ST 2.0, Affymetrix) and 96 analytes were detected in plasma samples using a multiplex assay (Luminex) to investigate the course of the immune response. Viral loads and specific antibody responses were also measured. The transcriptomic analysis revealed that similar immunological pathways were activated for fatal and non-fatal outcomes. However, the protein levels for immunological parameters were consistently different depending on the issue. Survivors presented moderated and controlled cytokine levels associated with an antibody response and a moderated viral load, while fatal outcomes were characterized by an inefficient antibody response and an excessive immunological response unable to control viral dissemination. Moreover, our data revealed a strong correlation between the cytokine profiles from fatal issues and the immunological patterns described in sepsis. In addition, we identified 8 cytokines as robust markers for the disease outcome that could be used for predictive diagnosis and to manage the sepsis-like syndrome.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P139

MEASLES VIRUS V PROTEIN CONTRIBUTES TO THE TRANSPORT OF NUCLEOPROTEIN COMPLEXES AND EFFICIENT VIRAL ASSEMBLY IN NEURONAL CELLS Shotaro Uchida* 1, Hiroki Sato1, Tetsuro Arai1, Fusako Ikeda1, Chieko Kai1, Misako Yoneda1 1Laboratory animal research center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Abstract: Measles virus (MV) is a causative agent of measles, but in rare cases, it also causes fatal neurological disease such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. It has been reported that V protein is critical for its neurovirulence, but its function has not been understood. In this study, we have established a live imaging system for directly observing the transport of nucleoprotein complexes (NCs) of MV, and found that V protein is required for the Rab11-dependent transport of NCs in neuronal cells, which was required for the efficient viral assembly. To understand the molecular mechanism of V protein-dependent transport of NCs, we analyzed host binding proteins, and found that V protein directly binds to the microtubule to intermediate the association of microtubules and NCs. Then, we rescued mutant rMVs whose V proteins lost the affinities to microtubule. The transport of NCs of the mutant viruses were ablated, and they were significantly attenuated in a mouse brain. Our results suggest the novel function of MV V protein which contributes to its neurovirulence.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P140

EVIDENCE OF VIRAL PERSISTENCE IN CYNOMOLGUS MACAQUES THAT SURVIVE PARENTERAL EXPOSURE WITH LASSA FEVER VIRUS Kathleen Cashman* 1, Eric Wilkinson1, Xiankun Zeng2, Jeremy Bearss3, Paul Facemire3, Todd Bell3, Connie Schmaljohn4 1Virology Division, THE GENEVA FOUNDATION / USAMRIID, 2Pathology Division, USAMRIID, 3Pathology Division, 4Office of the Chief Scientists, USAMRIID, Fort Detrick, United States

Abstract: Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a rodent-borne, severe, and often fatal hemorrhagic fever disease endemic to regions of Central and West Africa. Our laboratory is engaged in studies to elucidate the disease progression and pathogenesis of LF in nonhuman primates (NHP), as well as to develop and test pre- and post- exposure medical countermeasures against infection. Typical LF in NHP occurs in two phases: 1) an acute phase, in which NHP develop severe disease signs that sometimes require humane euthanasia approximately 12-18 days post-exposure; and/or 2) a chronic phase in which NHP experience severe disease signs in the expected window, but do not succumb. NHP that experience the chronic disease phase rarely recover. Chronically ill NHP clear LASV from the blood by 21 days post-exposure as measured by plaque assay. Samples of testicular tissue collected from a chronically ill male NHP stained positive for LASV antigen 45 days post-exposure as measured by conventional immunohistochemical (IHC) staining methods, but all other tissues collected from chronically ill NHP were negative for the presence of LASV antigen by study terminus (days 30-45). In addition, two of five chronically ill NHP developed sudden onset sensorineural hearing loss by the end of the studies, a known consequence of LF in approximately 30% of recovering human patients. NHP remained moderately to severely ill, without improvement, until the study endpoints, despite the lack of measurable LASV in serum or tissues. We sought a more sensitive method of identifying LASV in tissues and employed a novel in-situ hybridization method in which LASV-specific nucleic acid probes were used to re-stain tissues in the chronically ill NHP. Using this new method, we were able to identify the presence of LASV antigen in a number of different IHC-negative tissues.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P141

IN SITU DEMONSTRATION OF RIFT VALLEY FEVER DISSEMINATION WITHIN CULEX PIPIENS MOSQUITOES Nicholas Johnson* 1, Sarah Lumley2, Luis Hernandez-Triana1, Dan Horton3, Maria Del Mar Fernandez de Marco1, Laura Hunter2, Kirsty Emory2, Dan Hicks4, Jolyon Medlock5, Roger Hewson2, Anthony Fooks1, Alejandro Nunez4 1Virology, Animal and Plant Health Agency, Addlestone, 2Virology and Pathogenesis Group, Public Health England, Porton Down, 3School of Veterinary Medicine, University of Surrey, Guildford, 4Histology, Animal and Plant Health Agency, Addlestone, 5Entomology, Public Health England, Porton Down, United Kingdom

Abstract: Rift Valley fever phlebovirus (Family Phenuiviridae, Genus Phlebovirus - RVFV) is a zoonotic pathogen of veterinary and medical importance causing a haemorrhagic disease in humans. One of the main vector species is Culex pipiens, an abundant species across Africa and Europe. To investigate the competence of temperate populations of this species we have developed in situ hybridisation probes (RNA Scope) to detect the distribution of RVFV RNA. Culex pipiens mosquitoes (strain Caldbeck) were fed on blood containing RVFV (Strain ZH501, 107 pfu/mL) and maintained at 25oC. An infection rate of 27% (n = 15) was observed at 7 days. In parallel, whole mosquitoes were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained either with a bacterial DapB probe (negative control), mosquito GAPDH probe (positive control) or a RVFV probe directed to the nucleoprotein. Sections were counterstained with haematoxylin. Non-infected mosquitoes showed no staining with the exception of non-specific staining of the corneal lens in all sections. Infected sections showed extensive staining throughout body tissues with particularly strong staining in the basal membrane of the midgut and proventriculus. Extensive dissemination of virus within the head and thorax including tissues such as the ganglion, leg and Johnstone's organ. No staining was observed within the ovarian follicles. This study demonstrates the application of in situ detection of virus RNA to investigate RVFV dissemination within a key vector species. It also suggests that the proventriculus, the region of the oesophagus immediately anterior to the midgut is highly infected and could be a site of virus egress from the mosquito gut. Secondary sites of replication were evident throughout the thorax and likely precede infection of the salivary glands prior to potential transmission.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P142

EUROPEAN SUBGROUP C AVIAN METAPNEUMOVIRUS: PRELIMINARY EXPERIMENTAL INVESTIGATIONS ON THE ROLE OF THE SH PROTEIN IN PATHOGENICITY FOR MUSCOVY DUCKS Nathan Szerman1, Chantal Allée1, Evelyne Lemaitre1, Céline Courtillon1, Michel Amelot2, David Courtois2, Clive Naylor3, Aurélie Leroux4, Pierrick Lucas4, Yannick Blanchard4, Nicolas Eterradossi1, Paul Brown* 1 1VIPAC, 2SELEAC, ANSES, Ploufragan , France, 3Infection Biology, Infection and Global Health, Liverpool, United Kingdom, 4GVB, ANSES, Ploufragan , France

Abstract: Subgroup C Avian metapneumoviruses (AMPV-C) have the broadest avian host range of the four described AMPV subgroups (A, B, C and D) although two distinct lineages have been observed; one in galliformes (viruses isolated from turkeys) and the other in palmipeds (viruses isolated from ducks). AMPV-Cs also have a closer genetic relationship with human Metapneumoviruses. These properties of AMPV-C together with the fact that target species are available as experimental models makes it a key virus in the genus metapneumovirus for studying host tropism and its molecular basis. In the current study the first reverse genetics system for an AMPV C virus from palmiped lineage was developed and used to rescue viruses in which the small hydrophobic (SH) protein or just its ectodomain had been exchanged for those of AMPV-C galliform lineage. NGS analyses revealed that the rescued viruses consisted of a mixed virus stock combining SH modified viruses with correct full length genomes others with one or two nucleotide insertions resulting in a truncated attachment glycoprotein G ORF. Experimental infections of SPF ducks with these SH modified viruses consistently failed to produce clinical signs, as opposed to the unmodified recombinant AMPV-C duck virus, despite similar levels of viral RNA production in the trachea and choanal cleft. These preliminary results demonstrated that the phenotype of a pathogenic AMPV-C duck virus was modified in SPF Muscovy ducks when its full SH ORF or just the ectodomain portion was modified. However it must be considered that SH modified viruses containing G ORF sequence modifications were also present in the rescued virus inoculum. Future and ongoing studies are aimed at assessing G mutations in the pathogenic recombinant AMPV-C copy and at obtaining recombinant SH swap viruses with an intact G ORF.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P143

INFLUENZA A VIRUS INFECTION PROMOTES LOCAL AND SYSTEMIC SUPER-INFECTION BY WEAKENING THE NATURAL SHIELD OF COMMENSAL BACTERIA Soner Yildiz* 1, Beryl Mazel-Sanchez1, Damien Baud1, Patrice Francois1, Mirco Schmolke1 1Department of Microbiology and Molecular Medicine, University of Geneva, CMU, Geneva, Switzerland

Abstract: Microbiota integrity is essential for a growing number of physiological processes. Importantly, commensal microbes provide a natural shield against invading bacterial pathogens. We recently demonstrated that acute viral infection of the respiratory tract can dramatically alter the composition and abundance of systemic microbiota in a transient fashion. In the respiratory tract we identify a commensal E. coli, increasing in titer specifically in lungs of influenza A virus (IAV) infected animals. Associated with virus induced lung commensal dysbiosis was an increased sensitivity to S. pneumoniae super-infection. Remarkably, exogenously applied E. coli itself was able to promote bacterial super-infection with S. pneumoniae, in absence of IAV infection. In the small intestine, we found transient change in composition of microbiota, as well as quantitative depletion of total bacteria, at both genomic and cellular level after IAV infection. In parallel to depletion of bacterial content, IAV induced disruption of mucus integrity in the small intestine, which in turn increased the risk of pathogen S. typhimurium invasion in a mouse model. Our data suggest that either qualitative or quantitative changes in microbiota as a consequence of an acute IAV infection could increase the risk of bacterial super-infection, by reducing weakening this natural shield of commensals.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P144

AMINO ACID SUBSTITUTIONS IN THE PB2 PROTEIN OF AN H5N1 AVIAN INFLUENZA VIRUS ENHANCE ITS REPLICATION EFFICIENCY AND PATHOGENICITY IN MAMMALIAN HOSTS. Takeaki Imamura* 1, Shinya Yamada1, Kiyoko Iwatsuki-Horimoto1, Reviany V. Nidom2, Setyarina Indrasari2, Kuncoro P. Santoso2, 3, Chairul.A Nidom2, 3, Yoshihiro Kawaoka1, 4 1Division of Virology, The Institute o Medical Science, The University of Tokyo, Tokyo, Japan, 2AIRC Laboratory, Professor Nidom Foundation, 3Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, 4Influenza Research Institute, School of Veterinary Medicine, Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States

Abstract: Highly pathogenic H5N1 avian influenza viruses have been circulating in Indonesia since 2004, and human cases have been reported since 2005. Characterization of highly pathogenic H5N1 avian influenza viruses isolated in Indonesia revealed two phylogenetically related strains belonging to Clade 2.1.3: A/chicken/South Kalimantan/UT521/2010 (UT521) and A/chicken/South Sulawesi/UT541/2010 (UT541). Compared to UT541, UT521 showed higher replication efficiency in cultured human lung epithelial cells (A549 cells) at 33°C and higher pathogenicity in BALB/c mice. Reassortant and mutant viruses of UT521 and UT541 were generated by reverse genetics, and characterization of these viruses revealed that unique amino acids in the polymerase binding protein 2 (PB2) of UT521 conferred its higher replication efficiency and pathogenicity. These findings are beneficial for assessing the risk of avian-to-human transmission of highly pathogenic H5N1 avian influenza field isolates.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P145

LOW-PATHOGENIC AVIAN INFLUENZA VIRUSES HIJACK HOST’S CENTRAL METABOLIC PATHWAYS Léa Meyer* 1, Olivier Leymarie1, Christophe Chevalier1, Evelyne Esnault2, Marco Moroldo1, Bruno Da Costa1, Sonia Georgeault3, Philippe Roingeard3, Bernard Delmas1, Pascale Quéré2, Ronan Le Goffic1 1INRA, Jouy-en-Josas, 2INRA, Nouzilly, 3Université François Rabelais et CHRU de Tours, Tours, France

Abstract: Low-pathogenic influenza viruses are a major cause of infections in poultry farms. Close proximity of the chickens due to holding conditions promotes aerosol transmission of the virus, thus making lung epithelial cells a primary target. We aimed at characterizing the mechanisms ruling low-pathogenic influenza virus infection of a chicken lung epithelial cell line, CLEC213. We used a transcriptomic microarray approach to uncover the cell mechanisms that were affected upon infection. We found that viral infection induced a characteristic signature of immune response, involving pathways such as IL-6, IL-8, and STAT3. Noteworthy we could not detect changes in interferon-stimulated pathways. Unexpectedly we found a strong metabolic signature at the transcriptomic level with the induction of oxidative phosphorylation (OxPhos), a central metabolic pathway. The mitochondrial chromosome codes proteins involved in this pathway, presumably as a means to fine-tune OxPhos activity. Viral infection upregulated every gene of the mitochondrial chromosome. We validated our data by quantitative Polymerase Chain Reaction then quantified OxPhos’s final product, Adenosine Triphosphate (ATP), and observed enhanced production upon viral infection. Viral replication was downregulated in the presence of an OxPhos inhibitor, oligomycin, indicating the virus relies on enhanced ATP production for optimal replication. Viruses have evolved ways to deflect central metabolism to promote their cycle. Influenza infection represents a cost of up to 10% of a cell’s energy pool. Our work thus uncovers a reliance of avian low-pathogenic influenza viruses on OxPhos.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P146

A DONOR-MATCHED HUMANIZED MOUSE MODEL REVEALS A CHIEF ROLE OF THE APC-T CELL INTERFACE IN EBOLA VIRUS DISEASE Monika Rottstegge* 1, 2, Estefania Rodriguez-Burgos3, Paula Ruibal4, Lisa Oestereich2, 5, Elisa Pallasch5, César Muñoz- Fontela1, 2 1Virus Immunology, Bernhard-Nocht-Institute, 2Partner Site Hamburg, German Center for Infection Research, 3Heinrich- Pette-Institute, Hamburg, Germany, 4Leiden University Medical Center, Leiden, Netherlands, 5Bernhard-Nocht-Institute, Hamburg, Germany

Abstract: During the recent Ebola virus disease (EVD) outbreak in West Africa (2013 – 2016), research from our laboratory as well as from other colleagues underscored the role of dendritic cells (DC) and T cells on EVD immunity. These studies showed that proper DC and T-cell-mediated responses were critical for virus clearance and survival. To further explore the relevance of this immune cell interaction, we have developed a humanized mouse model based on transplantation of donor- matched peripheral blood APCs and T cells into HLA-transgenic NSG mice (NSGPBL-Dm).

Here we show that transplantation of donor-matched DCs and T cells in these mice is sufficient to render them highly susceptible to EBOV infection. Mice showed high viremia, weight loss, gastrointestinal bleeding, and hematuria, and therefore reproduced important features of human EVD. Of note, some pathological features including time to death depended on the donor, which suggested that these mice may reproduce to some extent interindividual differences to EBOV infection.

We speculate that this model, which is easy and fast to generate, will facilitate future studies on immunogenetics, antigen presentation and T-cell biology in the context of filovirus and perhaps other human infections.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P147

LYMPHOCYTE HOMING SIGNATURES IN ACUTE LASSA VIRUS INFECTION Julia Port* 1, 2, David Wozniak1, 2, Yemisi Ighodalo3, Jennifer Oyakhilome3, Rita Esumeh3, Jonas Müller2, Stephan Günther1, 2, Lisa Oestereich1, 2, Cesar Muñoz-Fontela1, 2 1German Center for Infection Research (DZIF), 2Bernhard Nocht Institut for Tropical Medicine, Hamburg, Germany, 3Irrua Specialist Teaching Hospital, Irrua, Nigeria

Abstract: Lassa virus (LASV), the etiological agent of Lassa fever, is endemic in several West African countries and causes around 300,000 cases annually with 5,000-6,000 deaths. Transmission occurs seasonally, mostly through direct or indirect contact with the reservoir host species, the multimammate rat Mastomys natalensis. Human to human transmission may also occur, in particular in nosocomial settings. Here we summarize data gathered in hyper-endemic areas in Nigeria from a hospital-based acute Lassa fever patient cohort (January 2017 to March 2018). Patient data on behavior patterns linked to LASV exposure and likely transmission scenarios was collected. We gathered data on cross-sectional and longitudinal T-lymphocyte signatures of tissue homing, which is thought to be associated with the site of antigen encounter. Flow cytometry was used to describe homing signatures by characterization of activated and LASV specific T cells expressing either a “skin” (CLA, CCR4), an “intestinal” (beta 7 integrin, CD49d) or an “inflamed mucosal tissue and respiratory” (CCR3, CD29, CD49a) specific pattern of chemokine receptors and adhesion molecules. Preliminary data indicate distinct homing signatures in activated T cells which are consistent with exposure through respiratory and intestinal mucosae. We speculate that this approach will provide valuable information on T cell homing behavior during acute LASV infection, which can clarify the overall role of T cells in Lassa fever pathogenicity. Placing this data into the context of the epidemiological background could help determine the more prevalent transmission routes, which has implications for public health policies.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P148

MMP9 ASSOCIATES WITH ENDOTHELIAL GLYCOCALYX DEGRADATION DURING HEMORRHAGIC FEVER WITH RENAL SYNDROME Gregory Rankin1, Julia Wigren Byström2, Rasmus Gustafsson3, Magnus Hansson4, Therese Thunberg2, Clas Ahlm2, Anne- Marie Connolly-Andersen* 2 1Department of Public Health and Clinical Medicine, 2Department of Clinical Microbiology, Umeå University, Umeå, 3Dept. of Clinical Neuroscience, 4Dept. of Clinical Chemistry, Karolinska Institutet, Stockholm, Sweden

Abstract: Haemorrhagic fever with renal syndrome (HFRS) is characterized by fever, hypotension, vascular leakage, thrombocytopenia and renal failure. HFRS in Sweden is caused by the Puumala hantavirus. It is spread by viral-infested droppings from bank voles. The health care system has little to offer these patients since there is no antiviral treatment and no vaccine available. We previously showed that a marker of endothelial glycocalyx degradation (syndecan-1) was associated with disease severity and disseminated intravascular coagulation during HFRS. We analysed the levels of other endothelial glycocalyx (GCX) degradation markers (heparan sulfate, soluble thrombomodulin), potential "sheddase": Matrix metalloproteinase 9 (MMP9) and a neutrophil activation/tissue damage marker (neutrophil gelatinase-associated lipocalin, NGAL) in patient plasma collected consecutively following disease onset. We used the generalized estimating equation to analyse the association between endothelial GCX degradation, MMP9, neutrophil activation/tissue damage and HFRS disease outcome (needing oxygen, blood component transfusion, intensive care and renal damage). A total of 44 HFRS patients were included in this study. The levels of MMP9 was significantly associated with endothelial GCX degradation. Neutrophil activation/tissue damage (NGAL) was also significantly associated with MMP9 and endothelial GCX degradation. In addition, endothelial GCX degradation was significantly associated with HFRS disease outcome. Neutrophil activation leading to release of MMP9 could be partly responsible for endothelial GCX degradation during HFRS.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P149

HANTAVIRUS RECEPTORS EXPRESSION AND DISTRIBUTION IN A LETHAL PULMONARY SYNDROME ANIMAL MODEL

Amelina A. Albornoz* 1, Rebecca Brocato2, Carola Otth3, Jay Hooper2, Nicole Tischler1 1Molecular Virology, Fundación Ciencia y Vida, Santiago, Chile, 2Virology, US Army Medical Research Institute of Infectious Diseases, Maryland, United States, 3Virology, Universidad Austral de Chile, Valdivia, Chile

Abstract: Hantaviruses are enveloped viruses which belong to the Hantaviridae family. They are known to cause hemorrhagic fever with renal syndrome (HFRS) and the highly lethal hantavirus pulmonary syndrome (HPS) in humans. Both diseases are related with an increased vascular permeability; yet the mechanisms leading to pathogenesis are not well understood. Among them, the usage of different cell entry factors has been described. Receptors such as the b3 integrin subunit and the receptor for the globular head domain of complement C1q (gC1qR) have been shown to be used by pathogenic hantaviruses in vitro. In this work, we analyze the expression of b3 integrin and gC1qR in an animal model for HPS. For this purpose, we performed qPCR and immunohistochemical studies of lungs and kidneys (as control) at 8 and 12 days post-infection (d.p.i.) with Andes hantavirus. In control and 8 d.p.i animals, both receptors are localized in pulmonary veins while at 12 d.p.i. moribund animals, blood vessel staining of the receptors is weaker. Interestingly, the qPCR results show that the expression of b3 integrin and gC1qR are, in general, reduced in lungs of 8-12 d.p.i. animals, even though the histology of the 8 d.p.i. tissue appears normal with no signs of disease. The decrease of receptors mRNA levels in the lungs at 8 d.p.i. seems to be highly specific since infected kidneys of the same animals show similar receptor mRNA levels compared to controls. Together, these results suggest that the expression of receptors may be an important step involved in vascular permeability and subsequent pathogenesis in the lungs of the HPS model. FUNDING: CONICYT by grants FONDECYT 1181799 and AFB 170004.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P150

BIOMARKER ANALYSIS OF HUMAN EBOLA VIRUS DISEASE UNDERSCORES THE ROLE OF TISSUE INTEGRITY IN SURVIVAL Romy Kerber* 1, 2, Ralf Krumkamp2, 3, Misa Korva4, Toni Rieger1, 2, Stephanie Wurr1, 2, Sophie Duraffour1, 2, Lisa Oestereich1, 2, Martin Gabriel1, 2, Jürgen May2, 3, Tatjana Avšič Županc4, César Muñoz-Fontela2, 5, Stephan Günther1, 2 1Virology, 2German Centre for Infection Research (DZIF), 3Epidemiology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 4Institute of Microbiology & Immunology, University of Ljubljana, Ljubljana, Slovenia, 5Virus Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

Abstract: Background. The largest epidemic of Ebola virus disease (EVD) occurred between 2013-2016 in West Africa. Despite the fact that EVD is a severe viral infection with high case fatality rates (CFR) still little is known about putative immune correlates of outcome. Methods. Cross-sectional as well as longitudinal data on the expression of 54 biomarkers, measured in plasma samples from 180 hospitalized EVD patients, was obtained. Principal component analysis (PCA) was used to summarize the overall expression pattern of biomarkers in immune response. Results. Circulating pro-inflammatory cytokines and chemokines as well as markers of endothelial dysfunction and disseminated intravascular coagulation were highly expressed in EVD patients with fatal outcome. In contrast, biomarkers of gut integrity and T cell response were elevated in survivors. Overall expression profiles revealed no clear differences between fatalities and survivors. Conclusion. Endothelial integrity, gastric tissue protection and T-cell immunity play an important role in EVD survival.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P151

CYTOKINE RESPONSE IN BUNYAVIRUS HEMORRHAGIC FEVERS Misa Korva* 1, Katarina Resman Rus1, Ana Saksida1, Miša Pavletič1, Petra Bogovič2, Xhevat Jakupi3, Isme Humoli4, Jusuf Dedushaj4, Franc Strle2, Tatjana Avšič Županc1 1Virology, University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, 2Department of Infectious Diseases, University Medical Center Ljubljana, Ljubljana, Slovenia, 3Department of Microbiology, 4Department of Epidemiology, National Institute of Public Health of Kosovo, Pristina, -

Abstract: Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers, especially in the Balkans. Infection with Dobrava or Puumala virus and CCHF virus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that host’s immune mechanism is an important factor in optimizing survival rate. Thus, the aim of our study was to investigate cytokine response in patients with VHF to expose possible biomarkers for clinical outcome. For cytokine analysis acute serum samples (first 7 days of illness) were collected from 100 HFRS patients infected with DOBV or PUUV, 70 CCHFV patients and 30 healthy controls. Using Human Cytokine/Chemokine Panel (Milliplex) we have examined 20 cytokines: IL-1α, IL-1b, IL-1RA, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IP-10, IFNα2, IFNγ, GM-CSF, GROα, sCD40L, MCP-1, MCP-3, MIP-1α, MIP-1b and TNFα. In comparison to the control group patient with HFRS or CCHF had significantly increased levels of IL-4, IL-6, IL-10, IL-12p70, IP-10, INFγ, TNFα, GM-CSF, MCP-3, and MIP-1b. Interestingly, HFRS patients had higher concentrations of serum MIP-1α, MIP-1b, which promote activation of macrophages and NK cells. Also, HFRS patients had higher concentrations of IFNγ, where in CCHF patients significantly higher concentrations of IFNα were measured, suggesting activation of different inflammation pathways. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFNα2 and MIP-1α, while GRO, chemokine related to activation of neutrophils and basophils, was downregulated. Our study supports the hypothesis that the major players in the immunopathogenesis of CCHF and HFRS are pro-inflammatory cytokines, which mediate vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P152

MOLECULAR CHARACTERIZATION OF PB1-F2-MEDIATED PATHOLOGY IN INFLUENZA MOUSE MODEL OF INFECTION C Chevalier* 1, F Jamme2, O Leymarie1, B Da Costa1, P Maisonnasse1, M Réfrégiers2, B Delmas1, R Le Goffic1 1VIM, INRA, Jouy-en-Josas, 2DISCO, Synchrotron SOLEIL, St-Aubin, France

Abstract: PB1-F2 is a virulence factor of influenza A virus (IAV) known to increase viral pathogenicity in mammalian host. PB1-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers in IAV-infected cells. Using synchrotron Fourier-transform infrared (FTIR) spectroscopy, we previously evidenced the presence of PB1-F2 fibers in IAV- infected cells and assigned an IR β-aggregated signature at the single-cell level. Using DUV (Deep Ultraviolet) microscopy and taking advantage of the high content of tryptophan residues in the sequence of PB1-F2 (5/90 aa), we showed that the increase of the autofluorescent signal recorded in IAV-infected cells can be correlated with the IR detection of β-aggregates. Here, we used FT-IR and DUV microscopies to prove the presence of PB1-F2 fibers in IAV-infected mice. Mice were infected with a wild-type IAV and its PB1-F2 knockout mutant and monitored at different time post-infection. DUV microscopy was used to map the presence of PB1-F2 β-aggregates within slices of lung tissues of IAV-infected mice. IR spectra were recorded in the regions of interest and subjected to multivariate analysis revealing the presence of β-aggregated structures in mice infected with PB1-F2-expressing IAV. In order to study the correlation between PB1-F2 structure and inflammatory response, NF-KB luciferase transgenic mice were intranasally instilled with monomeric, fibrillated or C- and N-terminal domains of recombinant PB1-F2. Our results clearly show the pro-inflammatory effect of fibrillated PB1-F2 compared to monomeric and non-fibrillated forms. It is noteworthy that only the N-terminal part of PB1-F2, unable to fibrillate, does not provoke any inflammation. Thus, the PB1-F2-induced inflammation is tightly correlated with sequence and oligomerization status of the protein. Graphical Abstract:

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P153

RECOMBINANT GENOTYPE G MUMPS VIRUS EXPRESSING ENHANCED GREEN FLUORESCENT PROTEIN EFFICIENTLY REPLICATES IN PRIMARY HUMAN CELLS AND IS VIRULENT IN COTTON RATS Linda J. Rennick* 1, Sham Nambulli1, Connor G.G. Bamford2, Steven Rubin3, Nicholas A. Crossland4, W. Paul Duprex1 1Department of Microbiology and National Emerging Infectious Diseases Laboratories, Boston University School of Medicine, Boston, United States, 2School of Medicine, Dentistry and Biomedical Sciences, Queen’s University of Belfast, Belfast, United Kingdom, 3Food and Drug Administration, Center for Biologics Evaluation and Research, Silver Spring, 4National Emerging Infectious Diseases Laboratories, Boston University School of Medicine, Boston, United States

Abstract: Mumps, caused by mumps virus (MuV), is a disease characterized by painful inflammatory symptoms, such as parotitis and orchitis. In many cases there is also central nervous system infection leading to meningitis or encephalitis. The mechanism of MuV infection and pathogenesis in humans are not well understood. This information is critical for the design and testing of live, rationally attenuated MuV vaccines, which are necessary due to the global resurgence of mumps (including in highly vaccinated populations) and the discontinued use of some MuV vaccines outside the U.S. due to insufficient attenuation and safety concerns. In this study, a recombinant (r) MuV (rMuVG09) was generated based on a consensus genotype G genomic sequence obtained directly from an unpassaged clinical specimen from a patient infected with MuV during the 2009 outbreak in New York City. An additional transcription unit encoding enhanced green fluorescent protein (EGFP) was introduced between the V/phosphoprotein and matrix genes (position 3) of the genome to generate rMuVG09EGFP(3). Infection of polarised epithelial cell lines and primary differentiated normal human bronchial epithelial cells showed that this virus could infect, and be released, from both ciliated and non-ciliated cells via their apical and basolateral surfaces. Intranasal inoculation of cotton rats with rMuVG09EGFP(3) led to both upper and lower respiratory tract infection with spread in lungs and virus isolation from lung tissue and bronchoalveolar lavage samples. The animals seroconverted by 21 days post-infection. In vivo tracking of infected cells by detection of EGFP fluorescence is an efficient means of identifying primary sites of infection and sites of secondary spread which are critical elements towards gaining an understanding of MuV pathogenesis.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P154

THE HIGHLY PATHOGENIC AVIAN INFLUENZA H5N1 A/CHICKEN/FRANCE/150169A/2015 PRESENTS IN VITRO PROPAGATION CHARACTERISTICS CONSISTENT WITH ITS PREDICTED TROPISM FOR AVIAN SPECIES Pascale Massin* 1, Cécile Guillou-Cloarec1, Claire Martenot1, Eric Niqueux1, Audrey Schmitz1, François-Xavier Briand1, Chantal Allee1, Carole Guillemoto1, Marie-Odile Lebras1, Aurélie Le Prioux1, Katell Ogor1, Nicolas Eterradossi1 1VIPAC, ANSES, Ploufragan, France

Abstract: Avian influenza A viruses (AI) are a major threat to animal and public health. Since 1997, several highly pathogenic (HP) H5N1 avian viruses directly transmitted from poultry to humans caused numerous humans deaths and had a considerable economic impact on poultry market with high mortality rate and massive preventive culling. Surveillance and study of AI H5 viruses are essential to improve knowledge on its persistence, transmission and evolution. During 2015/2016, a HP-AI outbreak occurred in Southwestern France. Different subtypes circulated including a HP-H5N1: A/chicken/France/150169a/2015. Genomic analysis indicated that the virus does not possess the set of determinants known to promote the transmission to humans. However, a number of determinants are present as in other contemporary H5-AI viruses. To assess the absence of zoonotic potential of the HP-H5N1 150169a virus, some host restriction determinants described in the literature have been analyzed: (i) cell receptor-specificity; (ii) temperature sensitivity of viral replication related to primary infection sites: 33°C in human upper respiratory tract and 37°C or 40°C in avian respiratory or digestive tracts, respectively. The receptor binding assay using synthetic sialylglycopolymers showed that the virus preferentially recognizes avian receptor as other avian viruses used as controls. The temperature sensitivity was analyzed through plaque phenotype and multiplication/genomic replication kinetics on mammalian cells (Madin-Darby Canine Kidney cells). A delay in genomic replication and multiplication was observed at 33°C as compared to 37°C. To conclude, our results suggest that HP-H5N1 A/chicken/France/150169a/2015 has an avian phenotype in vitro in accordance with the initial in silico predictions based on genomic markers.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P155

ADAPTIVE MUTATIONS IN INFLUENZA A/CALIFORNIA/07/2009 ENHANCE POLYMERASE ACTIVITY AND INFECTIOUS VIRION PRODUCTION Patrick Slaine* 1, Cara MacRae2, Mariel kleer1, Emily Lamoureux3, Sarah McAlpine4, Michelle Warhuus4, André Comeau3, Craig McCormick1, Todd Hatchette4, Denys Khaperskyy1 1microbiology and immunology, Dalhousie university, halifax, 2The Hospital for Sick Children, University Health Network, Toronto, 3CGEB-Integrated Microbiome Resource (IMR) and Department of Pharmacology, Dalhousie University, 4Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority (NSHA), halifax, Canada

Abstract: Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P156

3D IMAGING OF VIRUS INFECTIONS IN SOLVENT-CLEARED ORGANS Luca Zaeck* 1, Madlin Potratz1, Stefan Finke1 1FRIEDRICH-LOEFFLER-INSTITUT, Greifswald - Insel Riems, Germany

Abstract: The visualization of infection events in tissues and organs using immunolabeling is a key method of modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ samples provides pivotal data on disease development and progression. Until recently, immunolabeling was mostly restricted to thin sections of paraffin-embedded or frozen samples. Because of the limited 2D image plane provided by thin sections, crucial information on the complex structure of respective organs as well as on both the compartment and the surrounding cellular context of the infection environment is lost. Consequently, distinct assertions on topics like epithelial barrier function, infiltration of cells to the site of infection, or directed virus spread in vivo can prove difficult. The introduction of a new solvent-based tissue-clearing technique [1] and its successor uDISCO (ultimate 3D imaging of solvent-cleared organs) [2] as well as the implementation of an applicable immunostaining protocol [3] now provide an efficient tool to study high-volume image stacks of infected organs. Here, we applied uDISCO to both brain and lung tissue samples from animals infected with rabies virus and swine influenza virus, respectively. Confocal laser scanning microscopy paired with custom- built, 3D-printed imaging chambers enabled us to obtain high-resolution image stacks of organ slices as thick as 1 mm in order to gain further insights into the infection environment of respective target tissues.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P157

HUMAN B-CELLS ARE HIGHLY SUSCEPTIBLE TO IN VITRO AND IN VIVO MEASLES VIRUS INFECTION Brigitta M. Laksono* 1, Rory D. de Vries1, Christina Grosserichter-Wagener2, Eline G. Visser3, Pieter L. Fraaij3, Wilhemina L. Ruijs4, Marion P. Koopmans1, Menno C. van Zelm2, Albert D. Osterhaus1, Rik L. de Swart1 1Viroscience, 2Immunology, 3Paediatrics, Erasmus MC, Rotterdam, 4Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands

Abstract: Measles is characterised by transient immune suppression. Studies in non-human primates showed that measles virus (MV) preferentially infects memory T-cells, which express higher level of the cellular receptor CD150 than naive T- cells. Based on these findings, we hypothesised that MV causes ‘immune amnesia’ by infecting and depleting memory lymphocytes. Since limited information was available about CD150 expression and susceptibility of B-cells to MV infection, we investigated the susceptibility of human B-cell subsets to in vitro MV infection and demonstrated that they were more susceptible and permissive to infection than T-cells. To investigate whether infection of B-cells could be observed in naturally infected measles patients, we performed an observational cohort study in unvaccinated children during a measles outbreak in the Dutch Orthodox Protestant community in 2013. We collected single blood samples from acute measles patients or paired blood samples from healthy children before and after measles. Detection of MV-infected cells in peripheral blood mononuclear cells (PBMC) by intracellular staining of the MV nucleoprotein confirmed that MV infected B-cells during prodromal measles. Staining of PBMC isolated from paired blood samples demonstrated a significant reduction in peripheral memory B-cells after measles. Altogether, our data strongly indicate that B-cells are highly susceptible to in vitro and in vivo MV infection and the loss of pre-existing memory lymphocytes contributes to measles-induced immune suppression.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P158

USE OF REVERSE GENETICS TECHNIQUE TO STUDY THE EARLY PATHOGENESIS OF PESTE DES PETITS RUMINANTS VIRUS Muneeswaran Selvaraj* 1, Mana Mahapatra1, Pippa Hawes1, Ricahrd Kock2, Satya Parida1 1Livestock viral disease, The Pirbright Institute, Woking, 2Pathobiology and Population Sciences, Royal Veterinary College, London, United Kingdom

Abstract: Across the developing world, Peste-des-petits ruminants virus (PPRV), places a huge disease burden on agriculture, in particular affecting small ruminant production and in turn increasing poverty in many developing countries. The current understanding of PPRV pathogenesis has been mainly derived from the closely related rinderpest virus (RPV). There are few studies that have focused on the late stages of pathogenesis of PPRV in the field and very little is known about the processes underlying the early stages of pathogenesis. It is believed that PPRV replicates mainly in the epithelial cells of the respiratory and gastro-intestinal tracts before disseminating throughout the host. We hypothesize that PPRV infects immune cells of the respiratory mucosa, but not respiratory epithelial cells and then migrates to the tonsil and local lymphoid organs for primary replication, after which virus enters the general circulation and secondary replication occurs in the epithelium of respiratory and gastro-intestinal tracts. The application of reverse genetics techniques provides a tool to gain a better understanding of the molecular factors underlying virus host range and pathogenesis. Recently we have established reverse genetics system for PPRV and using this we have engineered a GFP tagged PPR virus (rMorrocco 2008 GFP). Further, in the biosafety containment, we have infected targeted animals (goats) with this GFP tagged virus and following this virus in the body of infected goats in 6 hourly interval we could demonstrate that the virus primarily replicates inside the pharyngeal and palatine tonsils and then causes viremia and secondary replication.

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DAMAGING AND SPREADING – pathogenesis Abstract final identifier: P159

ENTRY AND RELEASE OF LASSA VIRUS IN WELL-DIFFERENTIATED PRIMARY BRONCHIAL EPITHELIAL CELLS Helena Müller* 1, Sarah K. Fehling1, Thomas Strecker1 1Institute of Virology, PHILIPPS UNIVERSITY MARBURG, Marburg, Germany

Abstract: Lassa virus (LASV), a member of the family Arenaviridae, is a highly pathogenic hemorrhagic fever virus that can cause severe systemic infections in humans. The primary reservoir is the multimammate rat Mastomys natalensis. Humans are primarily infected through mucosal exposure to virus-containing aerosols of rodent excreta. To advance our knowledge on the molecular mechanisms underlying LASV replication in the respiratory tract, we established well-differentiated primary cultures of human bronchial epithelial cells (HBEpC) grown under air-liquid interface conditions that closely mimic the bronchial epithelium in vivo. Our major findings were: (i) HBEpCs fully supported the entire lifecycle of LASV infection. (ii) LASV can infect polarized bronchial epithelial cells via the apical or basolateral membrane, while progeny virus particles are released predominantly from the apical surface. In vivo, such apical virus shedding from infected bronchial epithelia might support virus transmission via airway secretions. In summary, HBEpC represent a useful cell culture model system for the detailed analysis of LASV-host interactions in the respiratory tract.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P160

THE PAN-EBOLAVIRUS THERAPEUTIC COCKTAIL MBP134 DEMONSTRATES UNIVERSAL PROTECTION FROM PATHOGENIC EBOLAVIRUSES. Zachary Bornholdt* 1, Andrew S. Herbert2, Chad E. Mire3, Shihua He4, Robert W. Cross3, Dafna M. Abelson1, Joan B. Geisbert3, Anna Z. Wec5, 6, Krystal N. Agans3, Bronwyn M. Gunn7, Rebekah M. James2, Marc Antoine de La Vega4, Do Kim1, Eileen Goodwin6, Galit Alter7, Laura M. Walker6, Kartik Chandran5, Larry Zeitlin1, Thomas W. Geisbert3, Xiangguo Qiu4, John M. Dye2 1Mapp Biopharmaceutical, Inc., San Diego, CA, 2USAMRIID, Fort Detrick, MD, 3UTMB, Galveston, TX, United States, 4PHAC, Winnipeg, Manitoba, Canada, 5Albert Einstein College of Medicine, Bronx, NY, 6Adimab, LLC, Lebanon, NH, 7Ragon Institute, Cambridge, MA, United States

Abstract: The 2014-16 Ebola virus (EBOV) outbreak highlighted the absence of available medical countermeasures for filoviruses. Importantly, one vaccine and one therapeutic (ZMapp™) demonstrated clinical efficacy during the outbreak. However, both products are specific for EBOV and have shown no efficacy against Sudan (SUDV) or Bundibugyo (BDBV) ebolavirus which together have caused nine sporadic and unpredictable deadly outbreaks responsible for 645 infections and 289 deaths since 2000. Our approach to address this unmet need was to develop a therapeutic based on fully human monoclonal antibodies derived from a single survivor of the EBOV/Makona outbreak. A thorough down-selection was performed on a 349 mAb panel involving in vitro analyses and in vivo testing across multiple animal models for EBOV, SUDV and BDBV. The down selection resulted in two mAbs being combined into the pan-ebolavirus immunotherapeutic cocktail, MBP134. MBP134 has since proven to be extremely potent against EBOV, SUDV, and BDBV providing protection from EBOV, SUDV and BDBV challenge in non-human primates (NHPs) when administered therapeutically once symptoms were apparent. MBP134 is the first product to demonstrate protective efficacy post-infection with a single dose against every major pathogenic ebolavirus species in NHPs.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P161

ENHANCEMENT OF DNA VACCINES FOR EBOLA VIRUS WITH AN IMPROVED PLASMID DESIGN John J. Suschak1, Charles J. Shoemaker1, Lesley C. Dupuy1, James A. Williams2, Connie Schmaljohn* 1 1USAMRIID, Frederick, MD, 2Nature Technology Corporation, Lincoln, NE, United States

Abstract: We previously demonstrated that a DNA vaccine expressing the codon-optimized Ebola virus (EBOV) glycoprotein (GP) gene provides protective immunity to mice and nonhuman primates. To determine if modifications to the plasmid backbone could improve immune responses to EBOV in mice, we constructed EBOV GP DNA vaccines using minimalized Nanoplasmid™ expression vectors that are smaller than traditional DNA vaccine plasmids, have improved uptake, and persist longer in transfected cells. Mice were vaccinated by intramuscular injection of our standard pWRG7077- based EBOV GP DNA vaccine, an EBOV GP nanoplasmid construct, or EBOV GP nanoplasmids designed to improve innate immune responses by co-expressing a retinoic acid-inducible gene 1 (RIG-I) agonist or the RIG-I agonist and CpG RNA. Additionally, we used the nanoplasmid vectors to launch EBOV GP virus-like particles (VLP) endogenously within the host. We then measured the immunogenicity and protective efficacy of each of these constructs in the vaccinated mice. While the DNA-launched VLPs did not improve immunogenicity, mice vaccinated with the nanoplasmid vaccine developed increased anti-EBOV GP IgG and neutralizing antibody responses as compared to the traditional pWRG7077-based vaccine and had significantly increased populations of EBOV-specific IFN-γ+ and IL-2+ T cells. Humoral and cellular immune responses were further boosted by vaccination with the nanoplasmids co-expressing the innate immune agonist genes. Challenge with mouse-adapted EBOV demonstrated that both the EBOV nanoplasmid and DNA-launched VLP vaccines elicited protective immunity in mice. Our results suggest that the nanoplasmid vectors can improve the immunogenicity of EBOV DNA vaccines.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P162

THE EFFECTS OF OUTSIDE TEMPERATURE IN THE INDUCTION OF ADAPTIVE IMMUNITY TO INFLUENZA VIRUS INFECTION Takeshi Ichinohe* 1, Miyu Moriyama1 1Department of Virology, UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Climate change and global warming increasingly pose a severe problem to public health, as illustrated by the recent outbreaks involving Zika fever, West Nile fever, and malaria. Although climate change may expand the geographical distribution of several vector-borne diseases, the effects of outside temperature in host defense to viral infection in vivo are largely unknown. Here, we demonstrate that warm exposure of mice at 36˚C impaired the virus-specific CD8 T cells and antibody responses against influenza virus infection. In contrast, IgG antibody responses remained intact in warm-exposed mice after intraperitoneal infection with Zika virus or sever fever with thrombocytopenia syndrome (SFTS) virus, or subcutaneous vaccination with inactivated influenza virus. Following influenza virus infection, the warm-exposed mice failed to stimulate inflammasome-dependent cytokine secretion compared with room temperature (RT) or cold-exposed groups. Although the warm-exposed mice did not change commensal microbiota composition, they decreased their food intake and body weight by 10% and increased autophagy in the lung tissue. Induction of autophagy in RT-exposed mice by 24 hours- starvation or rapamycin treatment severely impaired virus-specific CD8 T cells and antibody responses following respiratory influenza virus infection. By using parabiotic mice joining control and starvation mice, we show that signals from ad libitum- fed mice restored the virus-specific CD8 T cell responses in starvation mice. These findings uncover an unexpected mechanism by which outside temperature alters virus-specific adaptive immune responses.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P164

INSIGHTS INTO ANTIGENICITY OF INFLUENZA A(H3N2) VIRUS, 2012-2018 L Gubareva* 1, V Mishin1, P Jorquera1, H Nguyen1, A Chesnokov1, B Mann1, J Barnes1, R Garten1, J Katz1, D Wentworth1 1CDC, Atlanta, United States

Abstract: Influenza A(H3N2) viruses evade immunity by rapidly acquiring changes in the hemagglutinin (HA). Some changes alter HA receptor-binding, precluding the use of HI assay for antigenic analysis of many recent viruses. Hence, next-generation sequencing has become a leading tool for monitoring virus evolution and neutralization assays have supplemented HI antigenic analysis. As existing neutralization assays have limited throughput, alternative methods to ascertain antigenic relatedness between co-circulating viruses and vaccines are desirable. We developed a high content imaging-based neutralization test (HINT) that limits artifacts of antigenic analysis caused by virus culturing. Reference antisera were raised by infecting ferrets with clinical specimens containing viruses representing antigenic groups of recent vaccines. Antigenic relatedness was evaluated using clinical specimens and MDCK-SIAT1 isolates. Antigenic cartography methods were applied. The data showed that gain of a glycan at 158, rather than F159Y, was a major determinant for the antigenic transition from A/TX/50/2012-like to A/HK/4801/2014-like viruses. Glycan loss at 158 can occur upon culturing and it reduces the distance from A/TX/50/2012, while increasing the distance from A/HK/4801/2014. Multiple introductions of F193S (site B) were associated with the increased distance from A/SW/97152963/2013 and A/TX/50/2012. Since the summer of 2017, viruses carrying T135K and/or I192T have been detected in different countries; some viruses showed reduced neutralization (>4-fold) by anti-A/HK/4801/2014-like virus serum, an early sign of antigenic transition. Our data demonstrate that HINT is a valuable new approach for antigenic analysis of A(H3N2) viruses.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P165

IDENTIFICATION OF ESSENTIAL HOST FACTORS FOR EBOLA VIRUS RNA SYNTHESIS USING A GENOME-WIDE SIRNA SCREEN

Scott Martin1, Abhilash Chiramel2, Marie Schmidt3, Yu-Chi Chen1, Nadia Whitt1, Ari Watt2, Eric Dunham2, Kyle Shifflett2, Shelby Traeger2, Anne Leske4, Eugen Buehler1, Cynthia Martellaro2, Janine Brandt3, Lisa Wendt3, Sonja Best2, Jürgen Stech3, Stefan Finke3, Angela Römer-Oberdörfer3, Allison Groseth4, Heinz Feldmann2, Thomas Hoenen* 2, 3 1Division of Preclinical Innovation, NIH, Bethesda MD, 2Laboratory of Virology, NIH, Hamilton MT, United States, 3Institute for Molecular Virology and Cell Biology, 4Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Riems, Germany

Abstract: Ebola virus (EBOV) causes a severe hemorrhagic fever, with no licensed treatment currently available. Antivirals targeting host factors are advantageous since they may target a wider range of viruses, including newly emerging ones, and since development of resistance is unlikely. However, systematic approaches to screen for host factors important for EBOV are hampered by the need to work with this virus at biosafety level 4 (BSL4).

In order to identify such EBOV-interacting host factors, we performed a genome-wide siRNA screen against 21,566 human genes to assess their activity in viral RNA synthesis. As a platform for screening and subsequent hit characterization we used minigenome-based life cycle modelling systems, including transcription-and-replication competent virus-like particle (trVLP) systems. In trVLP systems minigenomes are packaged into VLPs that can infect target cells, but are biologically restricted to cells expressing multiple viral proteins in trans, providing a way to model virtually the entire EBOV life cycle (i.e. particle entry, RNA synthesis, and progeny particle production) over multiple infectious cycles under BSL1 conditions. Identified hits were confirmed using infectious EBOV. Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for RNA synthesis of EBOV. Targeting this pathway with an FDA and EMA-approved small molecule inhibitor showed antiviral activity against EBOV in both life cycle modelling systems and experiments with infectious EBOV, and also showed activity against some other non-segmented negative-sense RNA viruses (NNSVs).

This work demonstrates the power of life cycle modelling systems for conducting large-scale host factor screens for BSL4 viruses, and provides a data set for future analysis examining the role every human gene in EBOV RNA synthesis.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P166

CHARACTERIZATION OF INFLUENZA VIRUS CLINICAL ISOLATES OBTAINED DURING UMIFENOVIR CLINICAL STUDY “ARBITR” Irina Leneva* 1, Irina Falynskova1, Nailya Makhmudova1, Artem Poromov1, Svetlana Yatsyshina2, Victor Maleev2 1I.Mechnikov Research Institute for Vaccines and Sera, 2Central Research Institute for Epidemiology, Moscow, Russian Federation

Abstract: Oral drug umifenovir (Arbidol) is licensed and widely used in Russia for treatment and prophylaxis of influenza A and B infections. We investigated susceptibility of influenza viruses isolated from patients pre- and during administration of umifenovir in phase IV clinical trial ARBITR. In addition, we examined the susceptibility of a panel of reference and NA inhibitor-resistant viruses and their sensitive counterparts to umifenovir. Umifenovir inhibited replication of all reference human influenza A and B viruses that circulated in 2012-2014 seasons. The wild-type influenza viruses and their oseltamivir-resistant mutants were susceptible to umifenovir at similar levels. All 18 isolates obtained before and during therapy with umifenovir in ELISA-cell assay were equally sensitive to umifenovir with IC50 falling in the range of 7.0 to 12,5 ug/ml and similar to IC50 previously observed for laboratory and clinical isolates. Matched isolates of two patients from whom we were able to obtain day 3, 5 and 7 samples were chosen for sequence analysis. No amino acid changes in HA that had previously been identified in vitro as being involved with reduction of susceptibility to umifenovir were observed. None of the viruses isolated before and during therapy with umifenovir displayed reduced susceptibility to NA inhibitors and no mutations that led to an amino acid substitution in the NA were found in studied samples. Umifenovir is effective against influenza viruses of 2012-2014 season and oseltamivir-resistant influenza viruses. No umifenovir-resistance has emerged during therapy of acute influenza infection. Umifenovir administration did not affect the susceptibility of influenza viruses to NA inhibitors.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P167

DEVELOPMENT AND APPLICATION OF ARENAVIRUS LIFECYCLE MODELLING SYSTEMS Anne Leske1, Eric Dunham2, Irke Waßmann1, Kevin Schnepel1, Kyle Schifflett2, Ari Watt2, Heinz Feldmann2, Thomas Hoenen2, 3, Allison Groseth* 1, 2 1Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany, 2Laboratory of Virology, National Institutes of Health, Hamilton, MT, United States, 3Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany

Abstract: Lifecycle modelling systems, which make use of miniature genome analogues (minigenomes) to recapitulate all or part of the viral lifecycle, are valuable tools for studying virus biology. If adequately optimized, they also provide robust systems for screening antiviral compounds and host cell factors regulating virus infection, and for mechanism of action studies. However, despite early successes developing arenavirus minigenomes, little subsequent effort has been focused on refining and optimizing these systems to make them suitable for such applications. To achieve this we developed both fluorescent and luciferase-based Junín and Tacaribe virus minigenomes, which form the basis for corresponding minigenome systems (to model replication and transcription) and transcription and replication-competent virus-like particle (trVLP) systems (which also model budding and virus entry). All these systems can be safely used under BSL1 conditions and thus open up access to technologies and expertise not usually available under BSL4 conditions. In particular, we have applied these tools for drug library screening, where we identified AVN-944 as a novel inhibitor of arenavirus RNA synthesis, as well as for a limited siRNA screen to look for pro- and antiviral factors that are either unique or shared with other hemorrhagic fever viruses. Most recently we have also analysed neutralization of trVLPs containing foreign arenavirus glycoproteins by serum from human Candid#1 vaccinees, the results of which suggest a lack of robust cross-neutralization between even closely related species. Overall our findings highlight the broad utility of arenavirus lifecycle modelling systems for a variety of applications.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P168

VACUOLAR ATPASE INHIBITING NANOPARTICLES EXHIBIT POTENT HOST-TARGETED ANTIVIRAL ACTIVITY AGAINST INFLUENZA VIRUSES You-Ting Chen1, Che-Ming Hu2, Hui-Wen Chen* 1 1Department of Veterinary Medicine, National Taiwan University, 2Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Abstract: Conventional treatments against influenza virus infections are designed to target viral proteins. However, the emergence of new influenza viral strains carrying drug-resistant mutations that can outpace the development of pathogen- targeting antivirals presents a major clinical challenge. Two vacuolar ATPase (v-ATPase) inhibitors, diphyllin, and bafilomycin, were previously identified with a broad-spectrum antiviral activity. However, their poor water solubility and their potential adverse effect may limit its clinical application. The central objective of this project is to generate a novel anti- influenza therapeutic strategy, integrating nanoparticle technology to enhance host-targeting antiviral delivery towards improved drug safety and efficacy. In this study, we have prepared a nanoformulation of diphyllin and bafilomycin, which the size of both nanoparticles were measured 200 nm by the dynamic light scattering and transmission electron microscopy. The drug encapsulation of diphyllin and bafilomycin were analyzed by the high-performance liquid chromatography. The nanoparticles showed to have efficient intracellular delivery in multiple cell lines including Fcwf-4, ARPE-19, and MH-S cells. The nanoformulation of diphyllin also exhibited lower cytotoxicity as compared to the free drugs in MDCK and MH-S cells. Furthermore, the nanoparticles demonstrated prominent anti-influenza activity against H1N1 and H3N2 in vitro. Upon the investigation of in vivo safety study, diphyllin nanoparticles were found well-tolerated in mice, from which the body weight was monitored and blood chemistry parameters were evaluated following intravenous or intranasal administration. Collectively, this work highlights the nanoformulation of v-ATPase blocking compounds as the potential effective host- targeted treatment against influenza.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P169

A VSV-MEASLES CHIMERIC VIRUS CAN TARGET NECTIN-4 POSITIVE BREAST CANCER TUMORS IN AN IMMUNE COMPETENT MOUSE AND IS ENHANCED BY TRANSIENT INHIBITION OF ANTI-VIRAL INNATE IMMUNITY Christopher D. Richardson* 1, Gary Sisson1, Natalie Mishreky1, Angelita Alcos1 1Microbiology & Immunology/Pediatrics, DALHOUSIE UNIVERSITY, Halifax, Canada

Abstract: Our laboratory previously discovered that the tumor marker Nectin 4 (PVRL4) is the epithelial cell receptor for measles virus, and is expressed on many adenocarcinomas. Breast cancer cells expressing Nectin 4 can be infected with measles virus or a vesicular stomatitis virus (VSV) hybrid virus containing the H and F proteins of measles virus. Tumors can be readily infected with measles and VSV-measles viruses that express either green fluorescent protein (eGFP) or firefly luciferase (Luc) reporter genes. Histological analysis of MD468 or patient derived triple negative tumors showed that they expressed eGFP reporter and viral proteins. Although the viruses replicate in the tumors engrafted onto immune deficient mice, the infectious agents were not oncolytic and only slowed tumor progression. To test the contribution of the host immune system, mouse 4T1 and 4T1-Nectin 4 tumors were produced in immune competent BALB/C mice. The highly conserved Nectin-4 protein increased growth and metastasis to produce aggressive 300 cubic mm mouse breast tumors within 6 days. 4T1 and 4T1-Nectin 4 tumors on xenographic (NIH III nude) and syngeneic (BALB/C) mice were injected with measles and VSV-measles viruses and monitored over 24 days. The growth of 4T1-Nectin 4 tumors in the immune competent BALB/C mice was slowed by intra-tumoral injection of measles virus or VSV-measles chimeric virus. Tumor regression with the VSV-measles chimeric virus was further enhanced using either BX795 or vanadate chemical inhibitors, which transiently inhibited anti-viral innate immunity. Infection of tumors was documented using confocal immune microscopy. We concluded that the anti-tumor properties of measles and VSV-chimeric viruses are dependent on both the innate and adaptive host immune systems.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P170

ENHANCING THE IMMUNOGENICITY AND ATTENUATION OF RESPIRATORY SYNCYTIAL VIRUS VACCINE CANDIDATES BY ALTERING NS1 FUNCTION Michael Teng* 1, Kim Tran1, Andrew McGill1, Olivia Harder2, Stefan Niewiesk2 1Internal Medicine, UNIVERSITY OF SOUTH FLORIDA, Tampa, 2Veterinary Biosciences, Ohio State University, Columbus, United States

Abstract: Development of live-attenuated vaccines for respiratory syncytial virus (RSV) has been hampered by identifying candidates that are sufficiently attenuated but retain immunogenicity. One issue is that natural RSV infection itself only induces short-lived immune responses, resulting in recurrent infections. In addition, the likely target population for a live- attenuated vaccine are infants who do not have a completely developed immune system. Therefore, a highly attenuated, but strongly immunogenic, vaccine would be desirable. RSV encodes a small nonstructural protein (NS1) that has multiple functions in the viral life cycle. NS1 inhibits production type I interferon (IFN) responses via inhibition of the RIG-I/MAVS pathway and plays a role in viral RNA synthesis. the IFN antagonism and viral replication functions of NS1 are independent functions of NS1 and could be segregated by mutagenesis. Decreasing the IFN antagonism of NS1 would allow for a live- attenuated virus that has increased immunogenicity due to the immunostimulatory properties of type I IFNs. Thus, we reasoned that the IFN antagonism and viral replication functions of NS1 are independent functions of NS1 and could be segregated by mutagenesis. Through this process, we have identified specific regions and residues in the N- and C-termini of NS1 that are required for each function. Recombinant RSV (rRSV) encoding NS1 point deletion mutations displayed increased IFN production and enhanced replication compared with NS1 deletion rRSV. In addition, these viruses are attenuated and immunogenic in cotton rats. Our studies have allowed us to develop a menu of NS1 mutations that we can use to independently regulate IFN antagonism and viral replication to tune potential vaccine candidates for immunogenicity and attenuation.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P171

FAVIPIRAVIR BUT NOT RIBAVIRIN IS EFFECTIVE AGAINST TWO STRAINS OF CRIMEAN CONGO HEMORRHAGIC FEVER IN MICE David Hawman* 1, Elaine Haddock1, Kimberly Meade-White1, Kyle Rosenke1, Takashi Komeno2, Yousuke Furuta2, Brian Gowen3, Heinz Feldmann1 1NIH/NIAD, Hamilton, United States, 2Research Laboratories, Toyama Chemical, Toyama, Japan, 3Utah State University, Logan, United States

Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a negative sense RNA virus in the Orthonairovirus genus. CCHFV is widely distributed through Africa, Eastern Europe and the Middle East. Infected humans exhibit non-specific symptoms such as fever and myalgia and then progress into the hemorrhagic phase. Case-fatality rates can be as high as 30%. Ribavirin is recommended by the WHO for treatment of CCHFV but its efficacy in humans and animal models is inconclusive. Favipiravir is a promising antiviral for CCHFV and has shown efficacy against CCHFV in a mouse model. To reconcile the inconsistent data on the efficacy of ribavirin and test these antivirals against another isolate of CCHFV, we tested ribavirin and favipiravir against the prototypical CCHFV strain 10200 and the clinical isolate Hoti in our IFNAR-/- mouse model. Against either strain ribavirin treatment was unable to prevent terminal disease in infected mice. In contrast, favipiravir treatment protected 100% of 10200-infected mice even when started as late as 24 hours prior to terminal disease. Against strain Hoti, favipiravir treatment had clinical benefit when started as late as 6 days post-infection, a time when mice are exhibiting severe disease. Interestingly, in two favipiravir-treated mice recrudescence of CCHFV was observed weeks post-infection and several tissues collected from survivors five weeks-post infection showed persistence of CCHFV. Nevertheless, these data suggest that favipiravir is a potent antiviral against CCHFV. We plan to further evaluate favipiravir against CCHFV in our recently developed non-human primate model.

This research was supported by the NIAID Intramural Research Program.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P172

SCALABLE REPLICON-PARTICLE VACCINE PROTECTS AGAINST LETHAL LASSA VIRUS INFECTION IN THE GUINEA PIG MODEL Markus Kainulainen* 1, Jessica Spengler1, Stephen Welch1, JoAnn Coleman-McCray1, Jessica Harmon1, John Klena1, Stuart Nichol1, César Albariño1, Christina Spiropoulou1 1Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, United States

Abstract: Lassa fever is an arenaviral zoonosis that causes a substantial number of human infections in West Africa every year. While Nigeria, Sierra Leone, Guinea and Liberia have long been considered Lassa-endemic countries, evidence for a wider distribution is accumulating. Despite the disease burden, there are so far no approved vaccines to prevent Lassa virus infections. To generate a vaccine candidate with scalability and efficacy benefits typical of live attenuated vaccines, we have used our Lassa reverse genetics system to remove the essential glycoprotein precursor gene from the virus genome, and expressed the glycoproteins in a stable Vero cell line instead. The system produces virus replicon particles (VRPs) that replicate in the first cells encountered, but do not spread due to lack of de novo virus glycoprotein synthesis. We have shown that a single subcutaneous vaccination with the VRPs protects guinea pigs from lethality and clinical signs after challenge with Lassa virus strain Josiah. No residual viral RNA was detected in the vaccinated animals at the end of the experiment. Currently, we are focusing our efforts on further refining and scaling up the VRP production in order to perform future studies in the nonhuman primate Lassa animal model.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P173

REMOVAL OF THE N-GLYCOSYLATION SEQUON AT POSITION N116 LOCATED IN P27 OF THE RESPIRATORY SYNCYTIAL VIRUS FUSION PROTEIN ELICITS ENHANCED ANTIBODY RESPONSES AFTER DNA IMMUNIZATION Annelies Leemans* 1, Marlies Boeren1, Winke Van der Gucht1, Isabel Pintelon2, Kenny Roose3, Bert Schepens3, Xavier Saelens3, Dalan Bailey4, Wim Martinet5, Guy Caljon1, Louis Maes1, Paul Cos1, Peter Delputte1 1Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium, 2Laboratory of Cell Biology and Histology, University of Antwerp, Wilrijk, Belgium, 3Medical Biotechnology Center, VIB, Ghent University, Ghent, Belgium, 4The Pirbright Institute, Surrey, United Kingdom, 5Laboratory of Physiopharmacology, University of Antwerp, Wilrijk, Belgium

Abstract: Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority and development of a prophylactic vaccine is very important in the control of RSV disease burden. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. RSV F is highly conserved among RSV strains and has 5 N-glycosylation sites that are located in the F2 subunit (N27 and N70), the F1 subunit (N500) and the p27 peptide (N116 and N126). To our knowledge, improvements can be made in the understanding of the role of N-glycosylation in the immunogenicity of the RSV F protein. Expression vectors encoding RSV F glycosylation mutants were developed by replacement of the asparagine (N) codon by a glutamine (Q) codon. To study the effect of the loss of one or more N- glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with the different constructs encoding the RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titers were observed following immunization with F N116Q DNA. Upon subsequent RSV challenge, lower viral RNA levels were detected in the F N116Q DNA immunized mice compared with the levels in mice that had been immunized with F WT DNA. Since p27 is assumed to be post-translationally released from the mature RSV F protein after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this N-sequon can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P174

RIBAVIRIN HAS A DEMONSTRABLE EFFECT ON CRIMEAN-CONGO HEMORRHAGIC FEVER VIRAL POPULATIONS AND VIRAL LOAD DURING PATIENT TREATMENT Nicole Espy* 1, Unai Perez-Sautu1, Eva Ramírez de Arellano2, Anabel Negredo2, Michael Wiley1, 3, Sina Bavari1, Marta Diaz Menendez4, Maria Paz Sanchez-Seco2, Gustavo Palacios1 1United States Army Research Institute of Infectious Diseases, Frederick, United States, 2Arbovirus and Imported Viral Diseases Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain, 3College of Public Health, University of Nebraska Medical Center, Omaha, United States, 4High Level Isolation Unit, Hospital Universitario La Paz- Carlos III, Madrid, Spain

Abstract: Next-generation sequencing of viral populations allow researchers to gather evidence of a nucleoside analogue’s mechanism of action in vivo. The use of the guanosine analog ribavirin to treat infections of Crimean-Congo Hemorrhagic Fever virus (CCHFV) has been controversial based on uncertainties on its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case. We collected plasma samples taken from a CCHFV-infected patient before, during, and after a five day regimen of ribavirin, in which CCHF viral load dropped during ribavirin treatment. Viral RNA extracted from these longitudinal samples was deep sequenced to measure subclonal diversity and specific base changes that occurred due to ribavirin treatment. Coverage in each sample trended with viral load, and subclonal diversity increased in each viral segment during treatment. Base changes consisted primarily of T to C, G to A, and C to T transitions and of indels that accumulated preferentially in the CCHFV L segment. These data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo. Although the results are based on a single case, the paucity of other clinical data makes this result relevant for the clinical management of CCHFV-infected patients. Further, this study demonstrates that genomic analysis of viral populations can provide a clinical demonstration of nucleoside analogue activity.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P175

DEVELOPMENT OF BROAD-SPECTRUM PEPTIDOMIMETIC INHIBITORS OF PARAMYXOVIRAL FUSION Victor Outlaw* 1, Ross Cheloha1, Matteo Porotto2, 3, Sam Gellman1, Anne Moscona2, 3, 4, 5 1Department of Chemistry, University of Wisconsin, Madison, 2Department of Pediatrics, 3Center for Host-Pathogen Interaction, 4Department of Physiology & Cellular Biophysics, 5Department of Microbiology & Immunology, Columbia University Medical Center, New York, United States

Abstract: The respiratory pathogens parainfluenza (HPIV3) and respiratory syncytial (RSV) negatively impact global child and elderly health, and effective clinical treatment options are lacking. Viral fusion has emerged as a compelling target for the development of novel antiviral therapeutics. Recently, peptides were reported that inhibit fusion by several related members of the Paramyxoviridae and Pneumoviridae families. While the fusion mechanism is conserved, the individual fusion (F) proteins possess distinct primary sequences; therefore, the ability of a single peptide to disrupt the fusion machinery within multiple viruses is intriguing. Using these inhibitors as a template, we have adopted a three-pronged approach to develop analogs with increased efficacy and enhanced resistance to enzymatic degradation. Protease resistance could improve efficacy by prolonging in vivo half-life; we predicted that beta-amino acid residues would enhance stability. First, to identify the interactions that underlie the broad-spectrum activity of the peptides, we structurally and biophysically characterized inhibitors bound to each distinct viral target. X-ray structures revealed six-helix bundle complexes and exposed key interactions that comprise each unique protein:inhibitor interface. Second, we used structure- guided design principles to incorporate beta residues into the inhibitor sequence to create foldamers that structurally and functionally mimic the diverse secondary structures. These peptide mimics maintain potent activity against viral fusion but exhibit increased proteolytic stability. Finally, we created peptide-lipid conjugates to target the inhibitors to their sites of action at the host membrane and thereby increase potency. These agents combine potent inhibition of viral fusion with proteolytic resistance to lay a foundation for broad-spectrum antiviral therapeutics. Graphical Abstract:

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P176

CORRELATES OF PROTECTION OF A MVA-BASED VACCINE AGAINST EBOLA VIRUS IN A MOUSE MODEL Alexandra Kupke* 1, 2, Asisa Volz3, 4, Erik Dietzel1, 2, Jörg Schmidt1, Hosam Shams-Eldin1, Lucie Sauerhering1, Michelle Gellhorn Serra1, Christiane Herden5, Markus Eickmann1, 2, Stephan Becker1, 2, Gerd Sutter3, 4 1Institute of Virology, Philipps University Marburg, 2German Center for Infection Research, TTU Emerging Infections, Marburg, 3Institute for Infectious Diseases and Zoonoses, Ludwig Maximilians University Munich, 4German Center for Infection Research, TTU Emerging Infections, Munich, 5Institute of Veterinary Pathology, Justus Liebig University Giessen, Giessen, Germany

Abstract: Although several vaccine candidates have been shown to provide protection against Ebola virus (EBOV) infection, none of them has been licensed so far. We have developed two vaccine candidates based on recombinant Modified Vaccinia virus Ankara expressing the EBOV nucleoprotein (MVA NP) or the EBOV glycoprotein (MVA GP). The aim of this study was to determine if virus-neutralizing antibodies and/or CD8+ cytotoxic T cells mediate protection. IFNAR-/- mice were immunized with MVA NP or MVA GP and challenged with EBOV after 65 days. Serum and organ samples were evaluated for viral load by qPCR and organ samples were examined histologically. EBOV-specific antibodies were determined by ELISA and neutralization test. Prior to challenge, CD8+ T cells of MVA NP vaccinated mice were depleted to assess their role for the protective immune response. Mice vaccinated with MVA GP and MVA NP developed high titers of EBOV-specific antibodies. Only antibodies raised in MVA GP vaccinated mice were virus-neutralizing. After challenge with EBOV, the mock-vaccinated mice succumbed to infection, all mice vaccinated with MVA GP or MVA NP survived and showed decreased viral loads in serum and organs as well as reduced organ pathology. Depletion of CD8+ T cells in the MVA NP vaccinated mice resulted in high fatality rates after challenge. This study showed that CD8+ T cells play an important role for the protection of MVA NP vaccinated mice. MVA GP and MVA NP induced a protective immunity and are therefore promising candidates for further development.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P177

ISOLATION OF EGG-ADAPTED INFLUENZA A(H3N2) VIRUS WITHOUT AMINO ACID SUBSTITUTIONS IN THE ANTIGENIC SITES OF ITS HEMAGGLUTININ Tomoko Kuwahara* 1, Emi Takashita1, Seiichiro Fujisaki1, Masayuki Shirakura1, Kazuya Nakamura1, Noriko Kishida1, Hitoshi Takahashi1, Noriko Suzuki2, Yoshihiro Kawaoka3, 4, Shinji Watanabe1, Takato Odagiri1 1Influenza virus research center, National Institute of Infectious Diseases, Tokyo, 2Saitama Institute of Public Health, Saitama, 3Institute of Medical Science, University of Tokyo, Tokyo, Japan, 4University of Wisconsin-Madison, Wisconsin, United States

Abstract: Viruses for seasonal influenza vaccine are generally propagated in embryonated chicken eggs. However, antigenic changes in the hemagglutinin (HA) of recent A(H3N2) viruses are often induced when these viruses adapt to their egg hosts, resulting in antigenic mismatch between circulating and vaccine viruses. This antigenic alteration due to egg- adaptation has been a major concern for A(H3N2) vaccine virus selection and its effectiveness. Recently, we have successfully isolated cell-derived and egg-propagated A/Saitama/103/2014 virus (H3N2) (Saitama) virus, which did not have amino acid (aa) substitutions in HA antigenic sites and retained the antigenicity similar to the original cell propagated virus. In this study, we attempted to characterize the virological features of the Saitama virus. Antigenicity of egg-adapted Saitama virus was similar to most cell-propagated circulating viruses. Genome sequencing revealed that the Saitama virus acquired only one aa substitution in the stem region of HA but multiple substitutions in the upper surface region of neuraminidase (NA). Further investigation revealed that NA of egg-adapted Saitama virus manifested greater avidity to receptors than that of NAs derived from clinical isolate or cell-passaged Saitama viruses. These results suggested that aa substitutions in NA play important roles for Saitama virus to efficiently grow in eggs. Furthermore, the strategy of initial isolation in cells followed by passages in eggs may become a resolution to prevent egg-adapted antigenic change of H3N2 vaccine strains.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P178

BEWARE OF THE STING: THE SUPERPOWER OF MOSQUITO-PRODUCED RIFT VALLEY FEVER VIRUS Felix Kreher* 1, Carole Tamietti2, Dominique Simon3, Marie Flamand2, Xavier Montagutelli3, Alain Kohl1 1CVR, GLASGOW UNIVERSITY, Glasgow, United Kingdom, 2Virology, 3Central animal facility, Pasteur, Paris, France

Abstract: Rift Valley fever (RVF) is an important zoonotic disease in Africa causing a huge economic and public health burden by affecting humans and livestock. Symptoms in humans affected by RVF range from influenza-like to lethal haemorrhagic fever and encephalitis. The causative agent, Rift Valley fever virus (RVFV), is transmitted to its host via the bite of an infected mosquito or via exposure to bodily fluids from infected animals. The host’s adaptive immune system may therefore be confronted with RVFV particles that are either of mosquito or of mammalian origin. Vaccine trials for RVFV usually involve the use of mammalian-produced vaccine candidates and vaccinated animals are challenged with mammalian-produced virus. Levels of neutralising antibodies are often used as a measure of protective adaptive immunity in vaccinated animals since it has been shown that high levels of neutralising antibodies can efficiently protect against viral infections. We found that RVFV particles produced in either mammalian cells or mosquito cells displayed very different sensitivities to neutralising antibodies. It appeared that mosquito-produced virus particles were less sensitive than mammalian-produced virus to neutralisation through both mono and polyclonal antibodies. Antibody-antigen complexes seemed to form much quicker between mammalian produced particles and antibodies than with the mosquito-produced counterpart. The impact of these findings in vivo as well as the contribution that glycosylation of viral particles play, will be discussed. These results are relevant in the light of the development of effective and safe vaccines against RVFV.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P179

A VESICULAR STOMATITIS VIRUS RECOMBINANT EXPRESSING THE OROPOUCHE VIRUS GLYCOPROTEINS ELICITS A NEUTRALIZING ANTIBODY RESPONSE IN MICE. Sarah Hulsey Stubbs* 1, Nischay Mishra2, W. Ian Lipkin2, Sean P. Whelan1 1Microbiology and Immunobiology, Harvard Medical School, Boston, 2Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, United States

Abstract: Oropouche virus (OROV), an emerging orthobunyavirus, found in Central and South America is transmitted to humans from forest animals via insect vectors. Human infection is associated with a debilitating febrile illness – oropouche fever – and more than 30 epidemics affecting >500,000 people have been described since 1960. We report a vesicular stomatitis virus (VSV) recombinant in which the endogenous glycoprotein (G) was replaced with the entire M (medium) segment coding region of oropouche virus, VSV-OROV. Characterization of VSV-OROV, demonstrates that the synthesis of the M polyprotein, including its insertion into the endoplasmic reticulum and proteolytic processing to yield glycoprotein Gn, nonstructural protein NSm, and the fusogen Gc appear normal. VSV-OROV propagates readily in cell culture, incorporates both Gn and Gc into particles and enters cells dependent on their incorporation. Inoculation of mice with purified virus induces an immune response, including eliciting antibodies that specifically neutralize VSV-OROV. Tiled arrays of peptides corresponding to the oropouche structural proteins highlight a significant response to the N-terminal domain of Gc. This region of Gc precedes the domains that are structurally homologous to other class II fusion proteins and is more variable than the region containing the conserved fusion machinery. Work with another orthobunyavirus, bunyamwera replaced the homologous portion of its Gc with the fluorescent protein GFP to produce a replication competent virus in cell culture. Identification of peptides within this apparently dispensable region may expose new immunogenic determinants of the fusogen. To further understand the utility of VSV-OROV as a vaccine candidate, protective efficacy and further comparative serology is needed.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P180

IN VITRO CHARACTERIZATION OF MULTIDRUG-RESISTANT INFLUENZA A(H1N1)PDM09 VIRUSES CARRYING A DUAL AMINO ACID SUBSTITUTION ASSOCIATED WITH REDUCED SUSCEPTIBILITY TO NEURAMINIDASE INHIBITORS Emi Takashita* 1, Seiichiro Fujisaki1, Masaru Yokoyama1, Masayuki Shirakura1, Kazuya Nakamura1, Tomoko Kuwahara1, Noriko Kishida1, Hironori Sato1, Shinji Watanabe1, Takato Odagiri1 1National Institute of Infectious Diseases, Tokyo, Japan

Abstract: In Japan, four neuraminidase (NA) inhibitors––oseltamivir, peramivir, zanamivir, and laninamivir––are approved for the treatment of influenza. Recently favipiravir, a viral RNA-dependent RNA polymerase inhibitor, was approved for influenza pandemic preparedness. During the 2013–2014 and 2015–2016 influenza seasons, we detected three influenza A(H1N1)pdm09 viruses exhibiting enhanced cross-resistance to oseltamivir and peramivir from immunocompromised patients. These viruses possessed dual H275Y/I223R, H275Y/I223K, or H275Y/G147R substitutions in their NA protein, respectively. All three showed reduced susceptibility to zanamivir, and the dual H275Y/I223R and H275Y/I223K mutant viruses showed reduced susceptibility to laninamivir relative to the single H275Y mutant viruses. However, all three dual mutant viruses were susceptible to favipiravir. The NA activity of the dual H275Y/I223R and H275Y/I223K mutant viruses was reduced compared with that of wild-type virus, whereas the dual H275Y/G147R mutant virus showed comparable NA activity to that of wild-type virus. The dual H275Y/G147R mutant virus retained growth capability in vitro comparably to that of wild-type virus, but the dual H275Y/I223R and H275Y/I223K mutant viruses did not. Structural analysis of the NA protein predicted that the I223R and the I223K substitutions destabilized the NA structure, whereas the G147R substitution stabilized it. Thus, the introduction of the G147R substitution into the H275Y mutant NA may compensate for structural disadvantages, resulting in the restoration of viral growth.

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ONE-SHOT IMMUNIZATION USING A MEASLES/LASSA VACCINE FULLY PROTECTS CYNOMOLGUS MONKEYS AGAINST LASSA FEVER Mathieu Mateo* 1, 2, Stéphanie Reynard1, 2, Nicolas Baillet1, 2, Xavier Carnec1, 2, Alexandra Fizet1, 2, Marine Jourdain1, 2, Caroline Picard1, 2, Justine Schaeffer1, 2, Frédéric Tangy3, Sylvain Baize1, 2 1Unité de Biologie des Infections Virales Emergentes, Institut Pasteur, 2Centre International de Recherche en Infectiologie, Lyon, 3Génomique Virale et Vaccination, Institut Pasteur, Paris, France

Abstract: Lassa fever is a major public health issue in Western Africa and there is still no licensed vaccine. Here we have used the Measles virus (MeV) vaccine platform to generate Lassa fever vaccine candidates expressing the Lassa virus (LASV) glycoprotein GPC alone or in combination with the nucleoprotein NP or the matrix protein Z. We demonstrated that NP should be mutated to preserve a strong induction of the type I interferon response and activation of human antigen presenting cells in response to the vector. We ultimately selected two vectors, MeV-NPmut/GPC and MeV-Z/GPC, for further testing in non-human primates. Cynomolgus monkeys were immunized with a single dose of vaccine one month before being challenged with a lethal dose of LASV. The two vectors were safe, did neither replicate in nor shed from vaccinees, and protected all cynomolgus monkeys while controls all died from Lassa fever. Interestingly, MeV-NPmut/GPC conferred almost a sterilizing immunity and animals only experienced a transient elevation in the body temperature but no biological alterations or clinical signs. On the contrary, MeV-Z/GPC -immunized monkeys developed more severe symptoms and a prolonged LASV viremia. Analysis of the immune responses showed that early and robust T cell responses against GPC are critical for enhanced protection after challenge and suggests that T cell immunity against NP may greatly enhance protection. In addition, MeV-based vaccines induce a strong humoral response against MeV and could thus be used as bivalent vaccines to prevent Lassa fever and Measles in endemic areas where both LASV and MeV are circulating.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P182

IDENTIFICATION OF SMALL-MOLECULE VIRAL INHIBITORS TARGETING VARIOUS STAGES OF THE LIFE-CYCLE OF HANTAVIRUSES Sylvia Rothenberger* 1, Giulia Torriani1, Jennifer Mayor1, Gert Zimmer2, Nicole Tischler3, Pierre-Yves Lozach4, Stefan Kunz1, Olivier Engler5 1Institute of Microbiology, CHUV/UNIL, Lausanne, 2IVI, Institute of Virology and Immunology, Mittelhäusern, Switzerland, 3Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile, 4University Hospital Heidelberg, Heidelberg, Germany, 5Spiez Laboratory, Spiez, Switzerland

Abstract: Hantaviruses are rodent-borne viruses associated with severe human diseases. There is currently no licensed vaccine against hantaviruses and therapeutic options are limited to supportive care. Our research projects aim at the development of novel strategies for antiviral therapeutic intervention. In order to target 1) host cell entry, 2) transcription and 3) replication, we have developed cell-based assays suitable for high-throughput screening of small synthetic molecules to identify novel candidate inhibitors. First we have established and validated an efficient production system for VSV pseudotypes bearing the glycoproteins of the prototypic highly pathogenic Old World hantavirus Hantaan and the New World hantavirus Andes. In our screens, we have identified several inhibitory compounds interfering with viral entry that are currently tested against infectious hantaviruses in the high containment laboratories at Spiez Laboratory. Second, to target the viral RNA-dependent RNA polymerase L, we have developed a robust cell-based functional assay of for the HTNV and Puumala virus endonuclease function of L. Finally, as a starting point to develop a minireplicon system for hantaviruses, we established a minireplicon system for the Bunyavirus Uukuniemi that can be implemented for drug screening. Taken together, the inhibitors identified by the cell–based assays may pave avenue for the development of new class of anti- hantavirus drugs for prophylaxis and therapy and are useful “molecular probes” to gain novel insights into the fundamental virology of these important pathogens.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P183

A VACCINE PLATFORM AGAINST ARENAVIRUSES BASED ON A RECOMBINANT HYPER-ATTENUATED MOPEIA VIRUS EXPRESSING HETEROLOGOUS GLYCOPROTEINS Xavier Carnec* 1, Mathieu Matéo1, Audrey Page1, Reynard Stéphanie1, Hortion Jimmy1, Picard Caroline1, Yekwe Elsie2, Ferron François2, Sylvain Baize1 1Institut Pasteur, Lyon, 2AFMB UMR 7257 CNRS, Marseille, France

Abstract: Several Old and New-World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. Protection in non-human primates (NHP) against Lassa fever virus (LASV) was shown possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in the NP of MOPV, we found that this activity was essential for multiplication in antigen presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyper-attenuated strain (MOPVExoN6b) which is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to MOPV and (iii) still promotes a strong type I IFN response. The MOPVExoN6b virus was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV, Lujo, Machupo, Guanarito, Chapare, or Sabia viruses in order to broaden specific antigenicity while preserving the hyper-attenuated characteristics of the parental strain. Our MOPV based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in NHP and fully protected them from a lethal challenge with LASV. Thus, our hyper-attenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P184

MECHANISM OF ANTIVIRAL ACTIVITY OF AN M2 ECTODOMAIN-SPECIFIC MONOCLONAL ANTIBODY AGAINST INFLUENZA A VIRUS Rashid Manzoor* 1, Eguchi Nao1, Yoshida Reiko1, Hiroichi Ozaki2, Hiroko Miyamoto1, Ayato Takada1 1Division of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo, 2Faculty of Agriculture, School of Veterinary Medicine, Tottori University , Tottori, Japan

Abstract: Influenza A virus (IAV) matrix protein 2 (M2) is an effective target for the control of influenza since it is antigenically conserved and expressed abundantly on the infected-cell surfaces. In the present study, we studied inhibitory effects of an M2 ectodomain-specific monoclonal antibody (rM2ss23) using different subtypes of IAVs. rM2ss23 showed broad reactivity to the M2 proteins of tested IAVs. rM2ss23 neither neutralized A/Aichi/2/1968 (H3N2) (Aichi) and A/PR/8/1934 (H1N1) (PR8) viruses nor inhibited the ion channel activity of their M2 proteins; however, it significantly reduced the production of progeny virus particles from Aichi-infected cells. The inhibitory effect was also observed for A/Adachi/2/1957 (H2N2) and A/WSN/1933 (H1N1), but not for PR8 and A/Osaka/2001/2009 (H1N1) IAVs. Using reassortant viruses between Aichi and PR8 strains, we found that the inhibitory effect of rM2ss23 not only required Aichi-M2 protein, but was potentiated in the presence of Aichi HA and M1 proteins. Immunofluorescence (IF) microscopy of infected cells revealed that surface distribution of Aichi and PR8 M2 proteins were similar in the absence of rM2ss23. However, addition of rM2ss23 caused accumulation of Aichi-M2 into large foci on the cell surface but did not affect the cell surface distribution of PR8-M2 protein. It is known that the M2 protein localizes around the IAV budozone and mediates the process of virus budding/release; therefore, IF results suggest that rM2ss23 seems to interrupt the process of budding/release by disturbing the formation of the normal architecture of budozone. In addition, the present study suggests a differential mechanism underlying the virus budding between Aichi and PR8 strains.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P185

OPTIMIZATION OF THE CODON PAIR USAGE OF HUMAN RESPIRATORY SYNCYTIAL VIRUS INCREASES PROTEIN SYNTHESIS BUT PARADOXICALLY DECREASES IMMUNOGENICITY Cyril Le Nouen* 1, Cindy L. Luongo1, Lijuan Yang1, Steffen Mueller2, Eckard Wimmer3, Peter L. Collins1, Ursula J. Buchholz1 1RNA Viruses Section, NIH/NIAID/LID, Bethesda, 2Codagenix, Inc., Farmingdale, 3Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, United States

Abstract: Synonymous codon pairs often are present more or less frequently than statistically expected, which is called codon-pair bias. To explore ways to improve live-attenuated vaccine candidates, we subjected open reading frames (ORFs) of Human respiratory syncytial virus (RSV) to codon-pair optimization (CPO) for eukaryotic (specifically: human) codon pair usage without changing overall codon frequencies or amino acid coding. Four viruses were made, all containing the well- characterized attenuating mutation D1313 for safety: MaxA (CPO NS1, NS2, N, P, M, SH); MaxB (CPO G and F), MaxL (CPO L), and MaxFLC (all ORFs CPO except M2-1 and M2-2). Even the most extensive CPO had no significant effect on multi-cycle replication kinetics in vitro in Vero or in human lung epithelial A549 cells, nor on viral temperature sensitivity or specific infectivity. Three viruses, MaxA, L, and FLC – which in common had CPO of one or more ORFs of proteins of the replication complex – exhibited global increases in viral protein synthesis in single-cycle experiments. In contrast, MaxB, with only the G and F ORFs CPO, exhibited overall decreased viral protein synthesis, and its replication was reduced in single-cycle studies. These effects for MaxB appeared to be due to reduced transcription of the CPO G and F ORFs. Each of the CPO RSVs exhibited marginal reductions in replication in mice and hamsters. Surprisingly, serum RSV-neutralizing antibodies were moderately (MaxA, MaxL) or significantly (MaxFLC) lower compared to the non-CPO control in hamsters. We speculate that the decreased frequency in these viruses of CpG and UpA dinucleotides, agonists of innate immunity, reduces viral immunogenicity. Overall, our study described a previously unknown effect of the codon-pair bias of an RNA virus on the adaptive humoral immune response.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P186

DEFECTIVE INTERFERING VRNA ARE PRESENT IN LIVE ATTENUATED INFLUENZA VACCINE BUT THEY ARE NOT A MAJORITY POPULATION AND DO NOT APPEAR TO DRIVE REDUCED VACCINE EFFECTIVENESS. Sameer Ayaz* 1, David Chapman1, Helen Bright1, Oliver Dibben1 1Flu-MS&T, AstraZeneca, Liverpool, United Kingdom

Abstract: In the 2013/14 and 2015/2016 influenza seasons, the pandemic H1N1 (A/H1N1pdm09) component of the quadrivalent live attenuated influenza vaccine (Q/LAIV) was shown to have reduced clinical vaccine effectiveness (VE). More recently, using RT-PCR techniques, it was published that substantial quantities of defective interfering (DI) vRNA were present in commercial 2014/15 Q/LAIV nasal sprayers. These DI vRNA are truncated genomic RNA segments which are known to play a role in viral replication and innate immunity. It was therefore postulated that the presence of large amounts of DI vRNA in LAIV could impact clinical VE. To address these concerns, we aimed to quantify and structurally describe DI vRNAs in LAIV, followed by evaluating the impact of DI vRNAs on LAIV VE. Initially, we made qualitative assessments of DI vRNA content in formulated Q/LAIV using RT-PCR based methods. This confirmed published observations that DI vRNA do exist in specific Q/LAIV formulations. Then, using nanopore sequencing, we were able to show that the abundance and genetic structure of DI vRNA in RT-PCR products was dependent on LAIV subtype. Subsequently, we developed a novel digital polymerase chain reaction (dPCR) assay to provide absolute quantification of PA DI vRNA relative to its full-length gene segment, in the absence of RT-PCR amplification bias. This demonstrated that, in contradiction to the RT-PCR based observations, DI vRNA did not make up a substantial proportion of the PA gene segment population (5-35%) in any of the LAIV strains tested. Furthermore, our data suggested that there was no correlation between LAIV DI vRNA content and clinical effectiveness. Additional experiments evaluating the precise role of DI vRNA in LAIV replication and innate immunity are ongoing.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P187

HA STABILITY IS NOT THE SOLE DETERMINANT OF LIVE ATTENUATED INFLUENZA VACCINE EFFECTIVENESS Lauren Parker* 1, Lydia Ritter1, Wen Wu2, Oliver Dibben1, Helen Bright1 1Virology, MedImmune/AstraZeneca, Liverpool, United Kingdom, 2Biostatistics, MedImmune/AstraZeneca, Gaithersburg, United States

Abstract: During the 2013-14 influenza season, the quadrivalent live attenuated influenza vaccine (Q/LAIV), was shown to have lower than expected vaccine effectiveness (VE) against circulating A/H1N1pdm09 viruses in the United States. The underlying reason proposed for this was that the A/H1N1pdm09 vaccine strain, A/California/07/2009 (CA09), had a thermally unstable haemagglutinin (HA) protein. Consequently, a new A/H1N1pdm09 candidate strain, A/Bolivia/559/2013 (BOL13), was developed for inclusion in the 2015-16 season Q/LAIV. A key parameter for selection of BOL13 was its more thermostable HA phenotype compared with A/CA09. During the 2015-16 season, Q/LAIV containing BOL13 was found in some studies to have improved but still sub-optimal VE against circulating H1N1pdm09 viruses, and was not recommended for use in the US market in the 2016-17 influenza season. This suggested that improved HA stability had not entirely resolved the reduced VE observed. One hypothesis for this was that, by improving thermal stability, the BOL13 HA protein had been over-stabilised, compromising its activation at the low endosomal pH required for successful viral entry. Here we show that, while the BOL13 HA protein is more stable than that of CA09, thermal and pH stability were comparable to historically efficacious LAIV strains, suggesting that the HA had not been over-stabilised. In addition, studies in which Q/LAIV nasal sprayers were exposed to 33°C for 4 hours, simulating potential heat exposure during distribution, showed that while CA09 viral potency was statistically decreased after 12 weeks at 2-8°C, it remained within product specification. These data suggest that although unfavourable HA protein stability may have contributed to the reduced VE of CA09 in 2013-14, it was unlikely to have affected BOL13 in 2015-16. We conclude that HA stability is not the sole determinant of LAIV effectiveness.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P188

LIVE RECOMBINANT MEASLES-M2 VACCINE CANDIDATES INDUCE BROAD-SPECTRUM PROTECTIVE IMMUNITY AGAINST INFLUENZA VIRUSES Ho H. H. Vo1, Jérémy Brunet1, Claude Ruffié1, Valérie Lorin1, Frédéric Tangy1, Nicolas Escriou* 1 1Génomique Virale et Vaccination, CNRS UMR-3569, INSTITUT PASTEUR, Paris, France

Abstract: With the aim of developing an affordable vaccine against seasonal and emerging / pandemic influenza viruses, we evaluated a strategy based on the expression of influenza M2 ion channel by a vector derived from the live-attenuated Schwarz measles vaccine strain (MV). Indeed, the high conservation of the M2 protein, notably of its 23-amino acid extracellular domain (M2e) among human and animal influenza A subtypes, makes it an attractive candidate for a universal influenza vaccine. As proof of concept, we generated recombinant MVs expressing as additional transcription units either full-length M2 or fusion proteins between the measles virus nucleoprotein and three M2e copies. In mice susceptible to MV, recombinant MVs expressing full-length M2 induced the highest titers of M2e-specific antibodies, which were also shown to recognize the native tetrameric form of M2. Interestingly, the delivery of M2 by the measles vector induced a Th1-biased response and bypassed the genetic restriction of M2-specific responses. Partial protection with reduction in mortality rate and delayed death was obtained when immunized mice were challenged with mouse-adapted influenza A/H3N2 and pandemic A/H1N1v strains. The measles vector was further engineered in order to simultaneously express M2 and either influenza M1 matrix or NP nucleoprotein. These double recombinant viruses induced enhanced cellular responses against conserved influenza antigens in mice, and conferred full protection against challenge. Although further studies are required to document the broadly protective capacity of the M2/M1 and M2/NP combinations, this MV vector-based approach is promising toward developing a recombinant pediatric vaccine with dual potency against measles and seasonal influenza. It could also help achieve wide and efficient vaccine coverage of adults against pandemic influenza viruses.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P189

THE ADENOVIRAL VECTOR AD26 EXPRESSING THE PREFUSION F PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS SHOWS HIGH EFFICACY AND A FAVORABLE SAFETY PROFILE IN PRECLINICAL MODELS AS A PREREQUISITE FOR VACCINE DEVELOPMENT FOR YOUNG INFANTS Leslie Van Der Fits1, Renske Bolder1, Marjolein Heemskerk-van der Meer1, Joke Drijver1, Yolinda van Polanen1, Kevin Yim2, Jorge Blanco2, Hanneke Schuitemaker1, Benoit Callendret1, Eirikur Saeland1, Roland Zahn* 1 1Viral Vaccines, Janssen Vaccines & Prevention, Leiden, Netherlands, 2Sigmovir, Sigmovir Biosystems, Inc, Rockville, United States

Abstract: Respiratory Syncytial Virus (RSV) is a negative strand pneumovirus that causes annual epidemics of respiratory tract infection and is the leading cause of hospitalizations due to respiratory disease in infants. RSV vaccine development for infants is complicated by the immaturity and Th2 bias of the neonatal immune system, and the legacy of vaccine- enhanced respiratory disease (ERD) observed in clinical trials with formalin-inactivated (FI-)RSV in the 1960s. We assayed efficacy and safety of a replication-incompetent Ad26 vector encoding the RSV fusion protein stabilized in prefusion conformation (Ad26.RSV.preF). Immunization of adult and neonatal mice resulted in durable humoral immune responses characterized by high titers of RSV neutralizing antibodies, as well as in durable induction of IFNγ+ CD8+ T cells. Ad26.RSV.preF induced responses were skewed to a favorable Th1 profile, both in adult and neonatal mice. In cotton rats, Ad26.RSV.preF induced high neutralizing antibody titers and protected against RSV challenge. In contrast to FI-RSV that induced severe lung pathology after RSV exposure in mouse and cotton rat models, these features of ERD were not induced after RSV challenge in animals immunized with Ad26.RSV.preF at any of the vaccine doses tested, with a total number of over 300 Ad26.RSV.preF immunized animals tested in mouse and cotton rat RSV models. In summary, Ad26.RSV.preF elicits robust and durable humoral and cellular Th1 skewed responses when given as a single intramuscular immunization. In contrast to FI-RSV, the induced response did not predispose to ERD in two animal models. Hence, this vaccine appears potent and safe in preclinical models, and is currently in clinical development in infants.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P190

HYPOTHIOCYANITE DECREASES INFECTIVITY OF INFLUENZA A AND B VIRUSES INDEPENDENT OF NEURAMINIDASE ACTIVITY SUGGESTING A NOVEL MECHANISM OF VIRAL INACTIVATION Aaron Gingerich* 1, Lauren Widman1, Ralph Tripp1, Balazs Rada1 1Infectious Disease, UNIVERSITY OF GEORGIA, Athens, United States

Abstract: Respiratory viral infections are among the most common illnesses in global human and animal populations. Two of the main culprits are influenza A and B viruses (IV), which cause ongoing epidemics with high morbidity and variable mortality. The ability of IV to rapidly mutate and spread among different avian and mammalian species has allowed the virus to evade current vaccines and antiviral therapies. We have previously described an extracellular oxidative antimicrobial mechanism utilized by the innate immune system to repel IAV. The lactoperoxidase/thiocyanate system produces hypothiocyanite ions that have antiviral properties against IV capable of 4-6 log reductions in viral titers in vitro. Currently, the mechanism of action of this virucidal system is unknown. Our data shows that hypothiocyanite is able to inactivate several IV strains including A and B strains and those resistant to current antiviral therapies including oseltamivir and amantadine. These findings were supported by neuraminidase (NA) activity assays that showed no reduction in viral NA activity when exposed to hypothiocyanite. Additionally, pre/co-incubation of hypothiocyanite prevents infection of A549, MDCK, and primary human tracheobronchial epithelial cells by IV. The ability of influenza to bind to the host cells is also decreased by up to 65% when exposed to hypothiocyanite. These results suggest that hypothiocyanite represents a novel mechanism of IV inactivation utilized by the respiratory innate immune system.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P191

MOLECULAR BASIS OF ANTIBODY-MEDIATED NEUTRALIZATION REVEALS AN IMMUNODOMINANT SITE OF VULNERABILITY ON THE PHLEBOVIRAL SURFACE Elizabeth Allen* 1, Stefanie Krumm2, Steinar Halldorsson1, Angela Elliott3, Victoria Graham4, Karl Harlos1, Ben Brennan3, Richard Elliott3, Dennis Burton5, Juha Huiskonen1, Roger Hewson4, Katie Doores2, Thomas Bowden1 1Division of Structural Biology, University of Oxford, Oxford, 2Department of Infectious Diseases, King's College London, London , 3University of Glasgow Centre for Virus Research, University of Glasgow , Glasgow , 4National Infection Service, Public Health England, Salisbury, 5Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United Kingdom

Abstract: Phleboviruses are emerging pathogens that threaten human and animal health at a wide geographic scale. Targeting of the glycoprotein capsomers displayed on the phlebovirus surface by the antibody-mediated immune response is key for responding to and limiting infection. Here we characterized neutralizing monoclonal antibodies (nAbs) against the Gn glycoprotein displayed on the envelope surface of Rift Valley fever virus (RVFV), a phlebovirus responsible for severe disease upon zoonosis. Structural characterization of the epitopes targeted reveals overlapping binding sites at the head domain of RVFV Gn, and our data indicate that virus neutralization is achieved by preventing the required fusogenic rearrangements of the RVFV capsomer during host cell entry. Comparison of our structures with that of a previously characterized Gn glycoprotein epitope on the related severe fever with thrombocytopenia syndrome virus (SFTSV) reveals the Gn head region as an immunodominant site of vulnerability on the phlebovirus surface.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P192

RESPIRATORY SYNCYTIAL VIRUS (RSV) VACCINES BASED ON PARAINFLUENZA VIRUS 5 (PIV5) AMPLIFYING VIRUS-LIKE PARTICLES PROTECT MICE AGAINST RSV CHALLENGE

Maria C. Huertas-Diaz* 1, Shannon Phan1, Andrew Elson1, Ivette Nuñez1, Huiling Wei1, Kaori Sakamoto2, Biao He3 1Infectious Diseases, 2Pathology, University of Georgia , 3Infectious Diseases, Univerity of Georgia , Athens, United States

Abstract: Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract infections in children under one year of age, yet there are currently no licensed vaccines to prevent the disease. Our lab has developed an amplifying virus-like particle (AVLP) vaccine platform based on parainfluenza virus 5 (PIV5), which allows amplifying of a gene of interest in cells without producing PIV5 progeny. We evaluated the potential of this PIV5-based AVLP system as a potential platform for an RSV vaccine. We incorporated the fusion (F) protein and the transcription factor M2-1 into the PIV5-AVLP backbone (AVLP-F and AVLP-M2-1 respectively) and tested their efficacy in BALB/c mice. Single-dose immunizations with the AVLP-F vaccine candidate induced RSV antigen-specific responses. In addition, both vaccines elicited cell-mediated immune responses after a single dose. Most importantly, each vaccine was able to protect against RSV A2 challenge in the mouse model, with AVLP-F providing sterilizing immunity with a high-dose. Lung histology showed no enhanced pathology in the vaccine groups compared to the control group, indicating that our system did not induce enhanced disease. These results validate the use of the PIV5-based AVLP system as a potential platform for vaccines against RSV infection.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P193

IFN-LAMBDA ACTS AS MUCOSAL ADJUVANT STRONGLY PROMOTING TSLP-MEDIATED IGG1 AND IGA ANTIBODY RESPONSES Liang Ye* 1, Daniel Schnepf1, Jan Becker1, Karolina Ebert2, Yakup Tanriver2, Valentina Bernasconi3, Hans Henrik Gad4, Rune Hartmann4, Nils Lycke3, Peter Staeheli1 1 Institute of Virology, University Medical Center Freiburg, 2Institute of Medical Microbiology, Medical Center University of Freiburg, Freiburg, Germany, 3Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden, 4Department of Microbiology and Genetics, Aarhus University, Aarhus, Denmark

Abstract: Interferon-λ (IFN-λ) is an important component of the innate immune system that primarily acts on epithelial cells and protects against viral infections. Here we demonstrate that IFN-λ can also regulate adaptive immune responses. Mice lacking functional receptors for IFN-λ had only low virus-specific serum IgG1 levels and exhibited impaired follicular helper T cell and germinal center B cell responses after intranasal immunization with a live-attenuated influenza virus vaccine. Bone marrow chimeric mice revealed that IFN-λ enhances antibody production through a mechanism that depends on non- hematopoietic cells. When viral antigen was combined with IFN-λ, immunized mice showed improved antigen-specific IgG1 and IgA responses that resulted in better protection against influenza virus challenge infections. These beneficial effects were only observed after intranasal but not parenteral administration of IFN-λ, suggesting a role for epithelial cells. Thymic stromal lymphopoietin (TSLP), a cytokine known to drive Th2-type immune responses, was essential for the mucosal adjuvant effect of IFN-λ. M cells in the upper airways were the main producers of TSLP in IFN-λ-treated mice that, in turn, boosted antibody production. The TSLP-dependent adjuvant effect of IFN-λ might be employed to improve mucosal vaccine efficacy.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P194

BISPECIFIC FC GAMMA RECEPTOR ENGAGING MOLECULES PROTECT AGAINST INFLUENZA A VIRUS INFECTIONS Dorien De Vlieger* 1, Katja Hoffmann2, Lien Van Hoecke1, Inge Van Molle3, Han Remaut3, Hartmut Hengel2, Bert Schepens1, Xavier Saelens1 1Center for Medical Biotechnology, VIB, Ghent, Belgium, 2University of Freiburg, Institute of Virology, Freiburg, Germany, 3Structural Biology, Vrije Universiteit Brussel, Brussels, Belgium

Abstract: Influenza virus infections cause 3 to 5 million cases of severe illness and 250 000 to 500 000 deaths each year worldwide. The best way to prevent disease is vaccination. However due to the short immune memory and antigenic drift in the viral hemagglutinin and neuraminidase protein, influenza vaccines need to be administered yearly. Therefore, and as a measure against pandemic influenza outbreaks, antivirals are indispensable in the battle against influenza virus infections. Here, we describe the development of a new antiviral strategy based on the use of bispecific single domain antibodies (VHHs) termed BiFEs (Bispecific Fcg Receptor Engaging molecules), that can simultaneously bind an influenza A virus infected cell and an immune effector cell. The BiFEs were constructed by linking a VHH directed against the conserved ectodomain of the influenza M2 protein (M2e) to a second VHH directed against the mouse Fc gamma receptor I (FcgRI), mouse FcgRIV or human FcgRIIIa protein. BiFEs were recombinantly produced in Pichia pastoris. Using a newly developed cell-based activation assay, we demonstrate the specific and highly selective activation of individual FcgRs in the presence of the BiFEs and influenza A virus-infected cells. In addition, the BiFEs promoted phagocytosis of influenza-infected cells by macrophages in a concentration-dependent manner. Importantly, BiFEs directed against M2e and mouse FcgRI or –RIV protected BALB/c mice against challenge with influenza X47 (H3N2) virus. These results, together with the ease of production in yeast and the high stability, demonstrate the potential of the BiFEs as a new antiviral treatment option for influenza virus infections.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P195

A NOVEL METHYLTRANSFERASE-DEFECTIVE RECOMBINANT VESICULAR STOMATITIS VIRUS-BASED ZIKA VIRUS VACCINE EXPRESSING PRE-MEMBRANE, ENVELOPE AND NS1 PROTEIN Anzhong Li* 1, Jingyou Yu1, 2, Mijia Lu1, Yuanmei Ma1, Zayed Attia1, Chao Shan3, Kelsey Craig1, Nirajkumar Makadiya1, Jingyang He1, Xueya Liang1, Miaoge Xue1, Ryan Jennings1, Pei-Yong Shi3, Mark Peeples4, 5, Shan-Lu Liu1, 2, 6, Prosper Boyaka1, Jianrong Li1 1Department of Veterinary Biosciences, 2Center for Retrovirus Research, THE OHIO STATE UNIVERSITY, Columbus, 3Department of Biochemistry &Molecular Biology, Department of Pharmacology &Toxicology, and Sealy Center for Structural Biology &Molecular Biophysics, University of Texas Medical Branch, Galveston, 4Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, 5Department of Pediatrics, College of Medicine, 6Department of Microbial Infection and Immunity, THE OHIO STATE UNIVERSITY, Columbus, United States

Abstract: Zika virus (ZIKV) is a mosquito-borne flavivirus that causes fetal microcephaly, birth defects, Guillain-Barré syndrome, and other severe neurological disorders. Despite major efforts, there is no FDA-approved vaccine for ZIKV. Current efforts to develop ZIKV subunit vaccines have been exclusively focused on pre-membrane (prM) and envelope (E) proteins. However, the role of other viral proteins such as NS1 in the ZIKV-specific immune response has not been explored. Here, we developed a novel methyltransferase (MTase)-defective, attenuated recombinant vesicular stomatitis virus (rVSV)- based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate was completely attenuated in BALB/c and interferon receptor deficient A129 mice, where a single immunization induced ZIKV-specific antibody and T cell immune responses that provided complete protection against ZIKV challenge. Co-expression of prM, E and NS1 induced significantly higher levels of Th2 (CD4+IL-5+ and CD4+IL-10+ cell) and Th17 (CD4+IL-17A+ cell) cytokine responses than prM-E, without NS1. In addition, we found that NS1, alone, is capable of conferring partial protection against ZIKV infection in both BALB/c and A129 mice even though it did not induce neutralizing antibody. These results demonstrate that MTase-defective rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection. In addition, we demonstrate for the first time that NS1 protein plays a regulatory role in ZIKV-specific cellular immune response, and that NS1 alone can confer partial protection from ZIKV infection. In summary, we have developed a safe and highly efficacious MTase-defective VSV-based ZIKV vaccine candidate, which is ready for pre-clinical trials in nonhuman primates on its way to clinical trials.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P196

IDENTIFICATION OF DETERMINANTS THAT CONTROL ANTIVIRAL ACTIVITY OF DEFECTIVE INTERFERING PARTICLES (DIP) AND GENERATION OF TOOLS FOR DIP PRODUCTION Prerna Arora* 1, Najat Bdeir1, Sabine Gärtner1, Martin Schwemmle2, Udo Reichl3, Michael Winkler1, Stefan Pöhlmann1 1Infection Biology Unit, German Primate Center, Goettingen, 2Institute of Virology , Medical Center, University of Freiburg, Freiburg, 3Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany

Abstract: Influenza A virus (IAV) infection constitutes a serious health threat. Errors of the viral polymerase can result in the generation of defective interfering (DI) RNAs. These DI RNAs can be packaged into particles, termed defective interfering particles (DIPs), and can exhibit antiviral activity. A well characterized DIP is DI 244, which contains a large deletion in IAV segment 1. However, it is incompletely understood how DI 244 exerts antiviral activity and systems that allow production of DI 244 in the absence of helper virus have not been reported. The objectives of our study were to identify determinants that control inhibitory activity of DI 244 and to generate cell culture systems for production of DIPs in the absence of helper virus. The analysis of the DI 244 RNA in a mini-replicon system revealed that the truncated PB2 protein encoded by DI 244 was not required for inhibitory activity and addition of extra nucleotides and amino acids to the culture medium failed to rescue segment replication from inhibition by DI 244. In addition, knock-out of RIG-I, MAVS and MDA-5 (cells kindly provided by V. Hornung) did not reduce the inhibitory activity of DI 244. In contrast, the analysis of a set of segment 1 variants with nested deletions revealed that deletion size was inversely correlated with inhibitory activity. Finally we generated 293T and MDCK cell lines stably expressing IAV polymerase proteins or NA and are currently testing whether these cell lines are suitable for DIP production. Our results suggest that the inhibitory activity of segment 1-derived DI RNAs is mainly determined by segment length. Furthermore, we established cell lines that could be suitable for production of DIPs in the absence of helper virus.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P197

RESPIRATORY SYNCYTIAL VIRUS (RSV) ENTRY IS INHIBITED BY SERINE PROTEASE INHIBITOR AEBSF WHEN PRESENT DURING EARLY STAGE INFECTION Winke Van der Gucht1, Annelies Leemans1, Guy Caljon1, Louis Maes1, Paul Cos1, Peter L. Delputte* 1 1Department of Biomedical Sciences, UNIVERSITY OF ANTWERP, Antwerpen, Belgium

Abstract: Host proteases play important roles in many viral activities such as uncoating, viral protein production and disease induction. Therefore, these host proteins are putative targets for the development of antivirals via inhibition of cellular proteases. For viruses such as Ebola, HCV and HIV, host proteases that play essential roles have been described allowing specific protease inhibitors to reduce infection. RSV also utilizes host proteases in its replication cycle but their exact role and potential as antiviral target is unknown. Therefore, we evaluated the effect of AEBSF on RSV infection. AEBSF was tested for its inhibitory effect on viral replication, starting treatment 1h prior to inoculation and was continued for 18h during the assay. AEBSF treatment resulted in a significant decrease of RSV infection in HEp-2, A549 and BEAS- 2B at 0.3mM. An almost complete block in the number of RSV infected cells after 18h of incubation was demonstrated in all cell lines. The inhibitory effect was not only observed using RSV A2 but also with clinical isolates. AEBSF was applied over the different phases of an infection cycle (pre-, peri- and post-inoculation treatment) to determine the phase in which AEBSF blocks infection. AEBSF treatment resulted in a significant decrease in RSV infected cells when present during all phases, during peri- and post-inoculation combined or during only the entry phase of RSV. TEM demonstrated that virus remained attached on the cell after AEBSF treatment, while also fusion (4h) and budding (24h) can be followed in the untreated control. AEBSF is able to inhibit RSV infection and based on our experiments, viral fusion with the host cell is blocked. Ongoing experiments will provide more information on the mechanism of AEBSF inhibition and its potential antiviral activity in in vivo RSV models.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P198

HENIPAVIRUS-LIKE PARTICLES INDUCE BOTH HUMORAL AND CELL-MEDIATED IMMUNE RESPONSE IN PIGS Eileen Stroh1, Lucie Sauerhering2, Andrea Maisner2, Charlotte Schröder1, Marting Groschup1, Ulrike Blohm1, Sandra Diederich* 1 1Friedrich-Loeffler-Institut , Greifswald , 2Philipps University of Marburg, Marburg, Germany

Abstract: Nipah virus (NiV) causes severe respiratory disease in pigs and fatal encephalitis in humans. During the first outbreak, the virus was transmitted from pigs to humans. Seroepidemiological studies indicate an existing potential for such spillover infections. To date, there is no effective vaccine against NiV available. Although most current vaccine studies aim to induce a neutralizing antibody response, it becomes evident that a promising vaccine should target both, the humoral and cell-mediated immune response. Virus-like particles (VLPs) have been shown to activate both arms of the adaptive immune response for many viruses. Aim of this study was to investigate if Henipa VLPs composed of the NiV glycoproteins and Hendra virus (HeV) M protein can induce both humoral and cellular immune response in swine. For this purpose, pigs were immunized 3 times in a 3- week interval with VLPs. Weekly, serum was collected as well as PBMCs isolated and analyzed after antigen-specific restimulation in vitro. Here, Henipa VLPs were able to induce an antigen-specific memory B-cell response in pigs with a derivative humoral response resulting in anti-henipavirus protein-specific antibodies with NiV-neutralizing activity. Further, pigs developed a Henipa VLP-specific cell-mediated immune response with regards to proliferation and cytokine production, mostly driven by γδT cells. To conclude, Henipa VLPs induced both, humoral and cell-mediated immune response in a NiV natural host and thus, are promising vaccine candidates. In the future, the protection of VLPs against NiV challenge in swine has to be studied.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P199

UNIVERSAL INFLUENZA VIRUS VACCINE: TWO DOSES OF ADJUVANTED CHIMERIC HEMAGGLUTININ INDUCE A STALK-SPECIFIC PROTECTIVE ANTIBODY RESPONSE AGAINST INFLUENZA VIRUS IN PRIMED ANIMALS

Badiaa Bouzya1, Florian Krammer2, Corey Mallett3, Raffael Nachbagauer2, Ronan N. Rouxel* 1 1Preclinical, GSK, Rixensart, Belgium, 2Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, 3Preclinical, GSK, Rockville, United States

Abstract: Authors listed in alphabetical order Influenza viruses constantly change their surface glycoproteins, which are the main target of the immune response and this allows the virus to escape pre-existing immunity (antigenic drift). Current hemagglutinin (HA) head-based seasonal influenza virus vaccines have to be re-formulated and re-administered annually to face this challenge. The universal influenza virus vaccine approach based on sequential chimeric HA (cHA) immunization aims at focusing the immune response towards the more conserved and immuno-subdominant HA stalk domain, rather than the immunodominant HA head domain. The cHAs are combinations of 'exotic' head domains, mostly from avian influenza virus subtypes, paired with conserved stalk domains of HAs from seasonal viruses. Due to the conservation of the stalk domain within the same group (e.g., influenza A group 1), these antibodies are able to broadly recognize a wide spectrum of influenza virus strains and subtypes. We have conducted nonclinical immunogenicity studies with monovalent cH8/1N1 and cH5/1N1 inactivated investigational vaccines produced in eggs. Our results showed that sequential immunization of the cHA vaccines formulated with AS03 or AS01 adjuvants induced robust anti-H1 stalk IgG antibody titers in primed BALB/c mice and NZW rabbits. In mice, this vaccination regimen elicited functional anti-H1 stalk IgG antibodies with in vitro ADCC activity in a reporter assay. The induced antibodies cross-reacted with antigenically distant group 1 HA antigens. After passive transfer into naïve mice, sera from mice immunized with adjuvanted cHA vaccines prevented mortality and morbidity upon lethal challenge with a wild type H1N1 virus or a chimeric cH6/1N5 virus. These results support the clinical development of this vaccine approach.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P200

THERAPEUTIC EFFICACY OF A RESPIRATORY SYNCYTIAL VIRUS FUSION INHIBITOR Dirk Roymans* 1, Sarhad S. Alnajjar2, Michael B. Battles3, Panchan Sitthicharoenchai4, Polina Furmanova-Hollenstein5, Peter Rigaux1, Steffen Jaensch6, Johannes P. Langedijk5, Mark R. Ackermann2, Jason S. McLellan7, Sandrine Vendeville1 1Janssen Infectious Diseases, Janssen Pharmaceutica NV, Beerse, Belgium, 2Carlson College of Veterinary Medicine, Oregon State University, Corvallis, 3Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, 4College of Veterinary Medicine, Iowa State University, Ames, United States, 5Viral vaccines, Janssen Vaccines & Prevention, Leiden, Netherlands, 6Computational Biology, Janssen Pharmaceutica NV, Beerse, Belgium, 7Molecular Biosciences, The University of Texas at Austin, Austin, United States

Abstract: Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection (ALRTI) in young children, immunocompromised adults and the elderly. Intervention with small-molecule antivirals specific for RSV presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ- 53718678 bound to RSV fusion (F) protein in its prefusion conformation and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship (SAR) and the pharmaceutical optimization strategy of the series are consistent with the binding mode of JNJ-53718678 and other RSV fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established ALRTI in the animals, even when treatment is delayed until external signs of RSV illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop RSV ALRTI.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P201

INCLUSION OF ANDES VIRUS CAP-SNATCHING ENDONUCLEASE IN THE SEARCH FOR NOVEL ANTIVIRALS Yaiza Fernández García* 1, Carola Bush1, Stephanie Wurr1, Maria Kuzikov2, Philip Gribbon2, Sophia Reindl1, Stephan Günther1 1Bernhard Nocht Institute for Tropical Medicine, 2Fraunhofer-IME SP, Hamburg, Germany

Abstract: Andes virus (ANDV) is one of many potentially fatal segmented single-stranded RNA (ssRNA) viruses that lack effective prophylactic and therapeutic management. It is believed to initiate transcription by using the capped 5´ ends of host cells mRNA as primers. The canonical model for the cap-snatching mechanism, involving a cap-binding and an endonuclease domain, was described for Influenza virus (IFV) in 1981. Since then, both viral protein domains have been targeted for the development of novel antivirals. We have identified the 200 residues at the N-terminus of ANDV L protein as the homologous cap-snatching endonuclease to IFV. Due to difficulties in the production of the soluble wild-type protein, we worked with single amino acid mutants. These variants were assessed for their thermal stability and nuclease activity. The unit exhibited a Mn2+-dependent endoribonuclease with specificity for ssRNA. To evaluate the molecular similarities between the equivalent domains of IFV and ANDV, we solved the structure of mutant K127A. This was the most stable mutant that retained activity. The tridimensional arrangement of atoms revealed an evolutionary conserved active site. Mutant N167A was selected as the best candidate to screen for small molecule drugs. Residue 167 is located away from the catalytic pocket and the putative substrate-binding groove. We observed a dose-dependent reduction of the enzymatic activity by a known inhibitor. Towards the transfer to a high-throughput platform, we adapted a FRET-based nuclease assay already published for IFV. As a result, we can expand the search for compounds that inhibit the transcription machinery not only of IFV but also of a variety of neglected pathogens.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P202

ANTIVIRAL ACTIVITY OF RIBAVIRIN AND FAVIPIRAVIR AGAINST VARIEGATED SQUIRREL BORNAVIRUS 1 Kore Schlottau* 1, Donata Hoffmann1, Martin Beer1 1Friedrich-Loeffler-Institut, Greifswald - Isle of Riems, Germany

Abstract: Borna disease virus (BoDV-1, species Mammalian 1 orthobornavirus), known in Central Europe for centuries, causes a progressive meningoencephalitis, mainly in horses and sheep. The discovery of a novel zoonotic Bornavirus, the variegated squirrel bornavirus 1 (VSBV-1, species Mammalian 2 orthobornavirus), which caused the death of three squirrel breeders and one animal caretaker, clearly shows that agents with anti-bornavirus activity are urgently needed. Ribavirin treatment is temporary striking against BoDV-1, but the effect diminishes quickly after stopping medication. Recently, one report by Tokunaga et al. demonstrated that Favipiravir permanent suppresses BoDV-1 replication and suggested, that Favipiravir may have a strong antiviral activity against a broad range of bornaviruses. Therefore, we wanted to test the effect of Ribavirin and Favipiravir on VSBV-1. We used persistently VSBV-1 and BoDV-1 infected Vero cell cultures and different concentrations of the two nucleoside analogues and monitored the inhibition of bornaviral RNA polymerase by measuring the bornavirus RNA by RT-qPCR at different time-points. It was also tested whether the replication again increases after stopping of the treatment. Consistently with previous reports we detected a concentration- and time-dependent inhibitory effect of ribavirin against BoDV-1 and also against VSBV-1, which was immediately repealed after the treatment was stopped. Favipiravir suppressed the replication of VSBV-1 and BoDV-1 likewise in a concentration- and time-dependent manner but had also a strong influence on cell viability. Therefore, the first trials to use Favipiravir against the new detected VSBV-1 were not successful and we will test for further antiviral agents against bornaviruses in the future.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P203

DEVELOPMENT OF A TEMPERATURE SENSITIVE, INTERFERON-SILENT SENDAI VIRUS FOR DELIVERY OF CRISPR/CAS9 FOR HIGHLY EFFICIENT GENE EDITING IN HEMATOPOIETIC STEM CELLS Ruth Watkinson* 1, Olivier Pernet2, Arnold Park1, Patricia Thibault1, Patrick Hong1, Wannisa Khamaikawin 2, Kory Hamane2, Dong Sung An2, Benhur Lee1 1Microbiology, ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, New York, 2UCLA AIDS Institute, UCLA, Los Angeles, United States

Abstract: To realize the revolutionary therapeutic potential of CRISPR/Cas9-mediated gene editing for hematopoietic stem cell (HSC)-based gene therapy, development of highly efficient, low toxicity delivery systems is critical. Sendai virus is an attractive candidate due to its natural tropism for HSCs, and previous development and safety testing for gene therapy applications. We previously repurposed Sendai virus for highly efficient CRISPR/Cas9-mediated gene editing in human cell lines and primary macrophages (>75% editing without selection). Here we build on these results, demonstrating efficient editing at 2 loci simultaneously by introducing 2 guide sequences to a single virus, expanding the potential applications. We have also introduced mutations that confer temperature sensitivity (ts) into Sendai virus to develop a ‘hit-and-run’ CRISPR/Cas9 strategy to maximize permantent on-target gene editing while minimizing accumulation of off-target indels. ts Sendai retained efficient gene editing at the permissive temperature (34°C), but was cleared from infected cells at physiological (non-permissive) temperature (37°C). Unexpectedly, these mutations also resulted in interferon (IFN)-silent infection even at very high multiplicity of infection - a highly desirable vector phenotype. Using this ts, IFN-silent Sendai, we show efficient (~70%) CCR5 gene editing at the permissive temperature in HSCs. Edited HSCs retain their capacity for colony formation and differentiation and clear Sendai virus infection upon incubation at physiological temperature, indicative of the desired efficient, but transient and low toxicity delivery.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P204

CREATION OF A HIGH THROUGHPUT FUSION ASSAY TO SCREEN RSV INHIBITORS James Kelly* 1, Leanne Logan1, Dalan Bailey1 1Department of Virology, The Pirbright Institute , Woking, United Kingdom

Abstract: Respiratory syncytial virus (RSV) is a major cause of childhood respiratory disease; however, currently there is no licenced vaccine available and the only therapeutic, a monoclonal antibody, is expensive and not widely used. RSV particles enter cells by membrane fusion in a process that was recently shown to involve micropinocytosis. The membrane fusion event is orchestrated by the viral Fusion (F) glycoprotein – a type I integral membrane protein. Separately RSV can spread through induction of direct cell-cell fusion – again driven by F activity. Accordingly, fusion and the activity of the F protein represent ideal targets for the development of novel RSV therapeutics, e.g. small molecule inhibitors or alternative monoclonal antibodies, highlighting the need for robust high-throughput assays to screen fusion and F inhibitors. Using a codon-optimised RSV F and a split-GFP-luciferase reporter construct we have developed and optimised a high- throughput cell-cell fusion assay applicable in a 96 well plate format. The assay has additionally been optimised for endpoint analysis of protein content and cell viability. Combined this allows for high-throughput monitoring of F/fusion antagonists, while also allowing an assessment of the toxicity of any potential inhibitors. The dual reporters permit both GFP-fluorescence and luciferase luminescence detection in live cells, providing a robust and sensitive mechanisms for detecting the kinetics of fusion and F activity. We have optimised cell type, number and substrate time of addition, in order to provide the best analytical tool for future identification of RSV inhibitors.

291

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P205

ELICITATION OF NEUTRALIZING ANTIBODIES AGAINST RVFV USING A RECOMBINANT GN GLYCOPROTEIN.

Stefanie A. Krumm* 1, Elizabeth R. Allen2, Victoria A. Graham3, Dennis R. Burton4, Roger Hewson3, Thomas A. Bowden2, Katie J. Doores1 1Department of Infectious Diseases, King's College London, London, 2Division of Structural Biology, University of Oxford, Oxford, 3National Infection Service, Public Health England, Salisbury, United Kingdom, 4Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States

Abstract: Rift Valley Fever virus (RVFV), a phlebovirus and member of the Phenuiviridae family, is an arthropod-borne emerging zoonotic pathogen causing severe diseases in livestock and humans and is found throughout sub-Saharan Africa. The virus can cause major epidemics and is transmitted via mosquitos or bodily fluids from infected animals to humans causing, in severe cases, haemorrhagic fever or meningoencephalitis with a mortality rate of up to 30%. A humoral response is key for clearing the virus but there is currently no effective vaccine or treatment available for use in humans. The RVFV surface glycoprotein is synthesised as a precursor that is cleaved into two subunits, Gn and Gc, which form an oligomer of dimers on the viral surface. Gn is a major target for neutralising antibodies.

Here we characterise the humoral immune response targeting the RVFV glycoprotein in rabbits immunized with RVFV Gn and describe the elicitation and direct isolation of neutralising antibodies. Presence of neutralising antibodies in rabbit sera was assessed and RVFV Gn binding B cells were isolated using antigen-specific single B cell sorting. Paired antibody heavy and light chain genes were rescued by PCR and cloned into rabbit antibody expression vectors and sequence information of the variable domains obtained. Antibodies were further assessed for Gn binding, neutralisation and epitopes were narrowed down via competition ELISAs. We isolated RVFV Gn neutralising and non-neutralising antibodies highlighting the presence of neutralizing and non-neutralizing epitopes on RVFV Gn.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P206

HDAC6, A NOVEL HOST TARGET FOR BROAD SPECTRUM ANTIVIRAL THERAPY Stéphanie Anchisi* 1, Mirco Schmolke1 1Department of Microbiology and Molecular Medicine, Faculty of medicine, University of Geneva, Geneva, Switzerland

Abstract: Influenza viruses pose a major threat to human and animal health. The WHO estimates up to 5 Mio severe infections, including up to 500’000 fatal infections, occurring annually on a global scale. Influenza viruses have a segmented RNA genome of negative polarity and an error prone RNA dependent RNA polymerase. Therefore, influenza viruses display a high genetic variability, which combined with selective pressure, leads viruses to evolve rapidly to avoid immune recognition. In line with this constant evolution, the production of “universal” vaccine or therapeutics directed against influenza virus antigens is almost impossible.

We are proposing host-directed strategies to counteract virus entry, the first step of viral replication cycle. Due to limited genetic coding capacity, influenza viruses heavily depend on the interaction with host proteins at basically every step of its replication cycle to generate progeny viruses. HDAC6, a histone deacetylase family member, has been previously identified as a proviral host factor supporting influenza virus uncoating and release of genetic material into the host cell cytoplasm (Banerjee et al. 2014). Of note, this study was performed using X31, a lab adapted strain of H3N2 influenza virus.

To better understand the relevance of these findings in clinical settings, we have tested the capacity of different clinical isolates of Influenza viruses to infect human lung epithelial expressing WT or mutant HDAC6. This approach allowed us to identify among clinical isolates a strain-dependent sensitivity to HDAC6. Next, we defined the viral determinants of this sensitivity to HDAC6. Preliminary data led us to focus on the matrix protein M in which we found a mutation that seems to play a role in the dependence of virus on HDAC6 to uncoat.

293

FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P207

ANALYSIS OF STABILITY OF LAIV REASSORTANTS BASED ON A/LENINGRAD/134/17/57 Svetlana Shcherbik* 1, Nicholas Pearce1, 2, Ekaterina Bazhenova3, Natalie Larionova3, Irina Kiseleva3, Larisa Rudenko3, Vivien Dugan1, David Wentworth1, Tatiana Bousse1 1Influenza Division, CDC/NCRID, 2Battelle, Atlanta, United States, 3Institute of Experimental Medicine, St. Petersburg, Russian Federation

Abstract: Live attenuated influenza vaccine (LAIV) reassortants based on cold adapted and attenuated A/Leningrad/134/17/57 (Len/17) virus are used internationally. Herein, we conducted studies to improve the characterization of LAIV vaccines based on Len/17. LAIV reassortants for A/H1N1pdm09 and A/H3N2 subtypes were generated by classical reassortment with Len/17 in embryonated chicken eggs. The temperature and pH stability of these Len/17 based LAIVs were analysed by incubation at various temperatures or pH, and infectivity assays were used to determine impact on viability. H1N1pdm09-Len/17 reassortants that showed preferential binding to α2,3 sialic acid (SA) receptors lost their infectivity at higher pH values compared to related viruses that showed α2,6 SA receptor binding preference, suggesting that sequence changes in H1 hemagglutinin (HA) could affect antigenicity and virus stability. The analysis of stability of Len/17 reassortant recommended for H3N2 vaccine component A/Singapore/INFIMH-16-0019/16 revealed a decreased temperature and pH stability compared to the Len/17-A/Hong Kong/4801/14 reassortant. The effect of specific HA amino acid substitutions on virus stability was tested using reverse genetics generated LAIV reassortants. Overall, the data indicate that substitutions that impact receptor binding also impact virus stability and that the analysis of stability via pH or temperature is useful for further characterizing new LAIV candidates as part of their development.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P208

ESTABLISHMENT OF THE CROSS-CLADE ANTIGEN DETECTION SYSTEM FOR H5 SUBTYPE INFLUENZA VIRUSES USING PEPTIDE MONOCLONAL ANTIBODIES SPECIFIC FOR INFLUENZA VIRUS H5 HEMAGGLUTININ Hitoshi Takahashi* 1, Shiho Nagata1, Takato Odagiri1, Tsutomu Kageyama1 1Influenza Virus Research Center, National Institute of Infectious Diseases, Tokyo, Japan

Abstract: The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Therefore, establishment of a rapid and specific diagnostic test for H5 HPAI virus infection is necessary for infection control in humans. A number of commercially available rapid influenza diagnostic tests have been developed, which are immunochromatography assays for influenza A and B virus infections targeting the viral nucleoprotein. However, these tests cannot distinguish HA subtypes of influenza A virus such as H1, H3, H5, and H7; thus, human seasonal influenza A viruses and avian influenza viruses are indistinguishable. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay (ELISA) using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P209

ANTIVIRAL EFFECTS OF MONOCLONAL ANTIBODIES TARGETING H5N1 INFLUENZA VIRUS HEMAGGLUTININ Reiko Yoshida* 1, Ema Qurnianingsih2, Rashid Manzoor1, Mari Ishijima1, Asako shigeno1, Hiroko Miyamoto1, Manabu Igarashi1, Chairul A. Nidom2, Ayato Takada1 1Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan, 2Avian Influenza-zoonosis Research Center, Airlangga University, Surabaya, Indonesia

Abstract: H5N1 highly pathogenic avian influenza (HPAI) viruses have been circulating in poultry in Asia, the Middle East, and Africa. This virus occasionally infects humans and causes severe pneumonia with high fatality rates. Antibodies against a viral envelope glycoprotein, hemagglutinin (HA), play a critical role in preventing influenza A virus infection. Here, we characterized 21 monoclonal antibodies (MAbs) (14 IgG1, 4 IgG2a, and 3 IgG2b) recognizing H5 HA of a clade 2.3.2 virus, focusing on multiple steps of viral replication and in vivo protective potential. Of these, 6 MAbs had neutralizing activity; 2 (IgG1 and IgG2b) and 4 MAbs (1 IgG1, 1 IgG2a, and 2 IgG2b) recognizing the antigenic sites adjacent to the receptor binding pocket and other sites on the HA1 region, respectively. All of the 4 neutralizing MAbs targeting the antigenic sites near the stalk region showed heterosubtypic reactivity and/or broad cross-reactivity to multiple H5N1 viruses. Passive immunization with these neutralizing MAbs are all protective in a mouse model of H5N1 HPAI virus infection. Interestingly, 2 nonneutralizing MAbs completely protected mice from lethal infection. We found that one of those nonneutralizing MAbs (IgG2a) had antibody-dependent cellular cytotoxicity (ADCC) activity in vitro, and another MAb (IgG1) inhibited budding/release of virus particles from infected cells. Moreover, 2 other nonneutralizing MAbs gave partial protection, but had neither ADCC nor budding/release inhibition activity. These results demonstrate that some nonneutralizing MAbs, as well as neutralizing antibodies, play a pivotal role in protective immunity against H5N1 HPAI virus infection.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P210

STATINS SUPPRESS EBOLA VIRUS INFECTIVITY BY INTERFERING WITH GLYCOPROTEIN PROCESSING Punya Ranjan* 1, Mike Flint1, Éric Bergeron1, Anita K. McElroy2, Payel Chatterjee1, César G. Albariño1, Stuart T. Nichol1, Christina F. Spiropoulou1 1VSPB, CENTRE FOR DISEASE CONTROL AND PREVENTION, Atlanta, 2Division of Pediatric Infectious Disease, University of Pittsburgh School of Medicine, Pittsburgh, United States

Abstract: Ebola virus (EBOV) infection is a major public health concern because of high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013–2016 outbreak in West Africa. Here we report the antiviral effects of statins on EBOV infection in vitro. Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing. Finally, we showed that statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings indicate that statin treatment inhibits EBOV infection and suggest that statins should be explored as part of EVD therapeutics.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P211

IMMUNE INDUCTABILITY OF RECOMBINANT MEASLES VIRUS VACCINES AGAINS NIPAH VIRUS INFECTION.

Che Lin1, Yui Matsunaga1, Shotaro Uchida2, Chieko Kai1, Misako Yoneda* 1 1Institute of medical science, 2Laboratoty animal research center, THE UNIVERSITY OF TOKYO, Tokyo, Japan

Abstract: Nipah virus (NiV) was first discovered in an outbreak in Malaysia 1998 and still continue to re-emerge in India and Bangladesh recently. There is still no cure or vaccine has been approved yet. We have developed a recombinant Measles virus (MV) vaccine expressing NiV G protein which has been proved 100% protection against NiV challenge in hamster model. Since most people have been immunized with measles vaccine in the early childhood, it is necessary to investigate if recombinant measles virus vaccine could elicit immune response in previous immunized people. Aim of the study is to investigate both humoral and cellular immune responses induced by rMV-NiV G in measles virus sero-positive hamsters. Hamsters were first immunized by MV-Ed and then inoculated by rMV-NiV G vaccines. Our results demonstrated that both recombinant measles virus vaccine Edmonston strain and HL strain could elicit anti-NiV G antibody immunity in subjects that have previous immunized with measles vaccine Edmonston strain. For cellular immunity, interferon gamma (IFN- γ) was chosen as an indicator of T cell response. We had successfully produced mouse monoclonal and rabbit polyclonal antibodies against hamster IFN- γ. By using homemade antibodies, hamster IFN- γ ELISpot assays was established.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P212

CRISPR SCREENS TARGETING VIRAL AND HOST GENOMES FOR ANTI-VIRAL COUNTERMEASURE DEVELOPMENT

Edwin Saada1, Jennifer Schwedler2, Yooli Light1, Jessica Techel2, Joe Schoeniger1, Oscar Negrete* 2 1Systems Biology, 2Biotechnology and Bioengineering, SANDIA NATIONAL LABORATORIES, Livermore, United States

Abstract: CRISPR/Cas9 systems have been widely used by researchers for functional genomics screening. In general, these screens harness the ability to specifically mutate DNA sequences, to investigate the role of genes in disease pathways. In collaboration with the Doudna lab, we recently demonstrated that Staphylococcus aureus Cas9 (SaCas9) can recognize and cleave single-stranded RNA by an RNA-guided mechanism that is independent of a PAM sequence in the target RNA. SaCas9 RNA cleavage is also programmable and site-specific, and can be exploited to reduce infection by single-stranded RNA phage. This RNA targeting capability enables RNA-virus directed CRISPR-based screens. Using these rules, we created a tiled guide RNA (gRNA) library, comprised of over 22,000 gRNAs targeting the genome and antigenome RNA strand of Rift Valley Fever virus (RVFV), a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. Utilizing 293T cells stably expressing SaCas9 and anti-RVFV gRNAs, we infected the cells with RVFV strain MP-12 expressing GFP. Cells were sorted for the lack of GFP expression, and subjected to sequencing and statistical analysis that uncovered gRNAs highly enriched in the uninfected population relative to control cells. The results from these validation studies will be presented. In addition to virus-directed screening, we performed host- directed CRISPR screening with the overall goal of targeting both viral sequences and host genes required for infection with the same Cas9 enzyme. Using a genome-wide CRISPR knockout library, we screened 293Ts for mutations that protect from infection by RVFV. This screen identified a suite of enzymes involved in glycosaminoglycan biogenesis. The overall goal of our research is to develop CRISPR-based host and virus-directed countermeasures against emerging RNA viruses to which few treatment options exist.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P213

LABYRINTHOPEPTIN A1 AND A2 EFFICIENTLY INHIBIT CELL ENTRY OF HUMAN RESPIRATORY SYNCYTIAL VIRUS IN VITRO AND IN VIVO Sibylle Haid* 1, Sebastian Blockus1, Svenja M. Wiechert1, Theresa Frenz2, Martin Wetzke3, Hans Prochnow4, Ronald Dijkman5, Bettina Wiegmann6, Marc Stadler7, Marie-Anne Rameix-Welti8, Jean-Francois Eléouet9, W Paul Duprex10, Volker Thiel5, Gesine Hansen3, Mark Brönstrup4, Thomas Pietschmann1 1Experimental Virology, 2Experimental Infection Research, TWINCORE, 3Pediatric Pneumology, Allergy and Neonatology, Hannover Medical School, Hannover, 4Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany, 5Infectious Disease and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland, 6Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, 7Microbial Drugs, Helmholtz Centre for Infection Research, Braunschweig, Germany, 8INSERM U1173 Infection-Inflammation, Université de Versailles Saint- Quentin, Montigny-le-Bretonneux, 9INRA Molecular Virology and Immunology, Université Paris-Saclay, Jouy-en-Josas, France, 10NEIDL, Boston University School of Medicine, Boston MA, United States

Abstract: Human respiratory syncytial virus (hRSV) is the most common cause of lower respiratory tract disease in infants and it is a major risk for severe infections in the elderly. Due to the lack of treatment options there is a need for new antiviral drugs. In our study, we aimed to identify small molecules with antiviral activity against hRSV and to investigate the mode- of-action of selected compounds. Therefore, a cell-based screening system based on firefly-luciferase was used to screen a compound library. Time-of-drug- addition, lentiviral hRSV pseudotype assays, infection competition, virus dilution assays and in vivo experiments were conducted to explore the mode of action of lead compounds. The two lantibiotics Labyrinthopeptin A1 and A2 emerged as potent inhibitors of hRSV infection with IC50 values in the low micromolar range. The antiviral effect could be shown in carcinoma derived lung cells as wells as in physiologically relevant primary human air-liquid interphase cultures (ALI). Application during virus inoculation and therapeutic administration of compounds after hRSV infection reduced virus load in the ALI cultures. Moreover, clinical strains of hRSV subtypes A and B were inhibited. In addition, lentiviral hRSV pseudotypes carrying RSV F proteins with resistance mutations against palivizumab or fusion inhibitors in clinical development were susceptible to Labyrinthopeptin A1 and A2. Time-of-drug- addition as well as the pseudotype assays indicated that both compounds inhibit cell entry of hRSV. In vivo imaging of hRSV-infected mice revealed that intranasal treatment with Labyrinthopeptin A1 or A2 reduces viral load in the upper respiratory tract. In summary, Labyrinthopeptin A1 and A2 efficiently inhibit cell entry of hRSV in vitro. When administered prophylactically they reduce viral load in the upper respiratory tract of mice in vivo.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P214

NEUTRALIZING ANTIBODIES AGAINST CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS DERIVED FROM A HUMAN SURVIVOR J. Maximilian Fels* 1, Noel T. Pauli2, Ana I. Kuehne3, Dafna M. Abelson4, Daniel Maurer2, John M. Dye3, Leslie Lobel5, Zachary A. Bornholdt4, Laura M. Walker2, Kartik Chandran1 1Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, United States, 2Adimab, LLC, Lebanon, United States, 3United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, United States, 4Mapp Biopharmaceutical, San Diego, United States, 5Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Abstract: Crimean-Congo Hemorrhagic Fever Virus (CCHFV), belonging to the Nairoviridae family, is an arbovirus found in parts of Africa, western Asia, and southern Europe. Following zoonotic transmission from ticks of the Hyalomma genus, infection is characterized by fever, fatigue, vomiting, diarrhea and in fatal cases often also hemorrhagic symptoms. There are currently no vaccines or targeted treatments available, leading the WHO to declare CCHFV a Blueprint priority pathogen in 2017. Here, we report on a panel of human monoclonal antibodies (mAbs) derived from a convalescent donor. By engineering a novel sorting antigen based on soluble Gn/Gc we were able to isolate memory B cells specific for CCHFV. Potent neutralizing mAbs, with IC50 in the nanomolar range, were then identified using a virus like particle (VLP) system. The activity of candidate hits was also confirmed using authentic CCHFV. Further, we found that these mAbs possess breadth of neutralization spanning 3 clades of CCHFV strains, highlighting their potential as therapeutics. We are currently probing the mechanism of neutralization of the most promising candidate mAbs, as well as evaluating their in vivo efficacy. [JMF, NTP & AIK contributed equally]

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P215

STRUCTURE OF LASSA VIRUS GLYCAN SHIELD PROVIDES A MODEL FOR IMMUNOLOGICAL RESISTANCE Yasunori Watanabe* 1, 2, 3, Jayna Raghwani4, Joel D. Allen3, Gemma E. Seabright2, Sai Li1, Juha T. Huiskonen1, Thomas Strecker5, Thomas A. Bowden1, Max Crispin3 1Division of Structural Biology, 2Glycobiology Institute, University of Oxford, Oxford, 3Life Sciences, University of Southampton, Southampton, 4Big Data Institute, University of Oxford, Oxford, United Kingdom, 5Institute of Virology, Philipps Unversitat Marburg, Marburg, Germany

Abstract: Lassa virus is an Old World arenavirus endemic to West Africa that causes severe hemorrhagic fever in humans. Currently, there are no efficacious vaccines or treatments available to combat this pathogen. Vaccine development has focused on the envelope glycoprotein complex (GPC) that extends from the virion envelope. The often inadequate antibody immune response elicited by both vaccine and natural infection has been, in part, attributed to the abundance of N-linked glycosylation on the GPC. Here, using a virus-like-particle system that presents Lassa virus GPC in a native-like context, we determine the composite population of each of the N-linked glycosylation sites presented on the trimeric GPC spike. Using a multidisciplinary mass spectrometry and structural biology approach, we reveal the presence of under-processed oligomannose-type glycans, which form punctuated clusters that obscure the proteinous surface of both the GP1 attachment and GP2 fusion glycoprotein subunits of the Lassa virus GPC. These oligomannose clusters are seemingly derived as a result of sterically reduced accessibility to glycan processing enzymes, and limited amino acid diversification around these sites supports their role protecting against the humoral immune response. Combined, our data provide a structure-based blueprint for understanding how glycans render the glycoprotein spikes of Lassa virus and other arenaviruses immunologically resistant targets.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P216

RATIONAL DESIGN OF HUMAN RESPIRATORY SYNCYTIAL VIRUS LIVE ATTENUATED VACCINES BY INHIBITING VIRAL MRNA CAP METHYLTRANSFERASES Miaoge Xue* 1, Rongzhang Wang1, Yu Zhang1, Mijia Lu1, Olivia Harder1, Xueya Liang1, Mark Peeples2, Stefan Niewiesk1, Jianrong Li1 1Department of Veterinary Biosciences, The Ohio State University , 2Center for Vaccines and Immunity, The Research Institute at Nationwide Children's Hospital, Columbus, United States

Abstract: Human respiratory syncytial virus (RSV) is the leading causative agent of pediatric respiratory tract disease worldwide. Currently, there are no vaccines or antiviral drugs to combat it. A live attenuated vaccine is one of the most promising vaccines for RSV. However, it has been a challenge to identify an attenuated RSV strain that has an optimal balance between attenuation and immunogenicity. The 5’ end of the mRNA of pneumoviruses contains a unique cap structure that is typically methylated by guanine N-7 (G-N-7) and ribose 2’-O methyltransferases (MTases). The objective of this study is to rationally design RSV live attenuated vaccines by inhibiting viral mRNA cap MTase. The S- adenosylmethionine binding sites in the MTase region of the large polymerase protein of RSV was mutated to alanine, and recombinant RSV (rRSV) carrying these mutations were recovered from an infectious cDNA clone. All three recombinant viruses (rRSV-G1853A, G1857A, G1853A-G1857A) were defective in mRNA cap methylation and were genetically stable and highly attenuated in cell culture. These recombinant viruses had significant defects in spread and replication in human airway epithelial (HAE) cultures, a ‘near in vivo’ model for RSV infection. Finally, the replication, pathogenesis, immunogenicity, and capacity to induce protection of these rRSVs were examined in cotton rats. The results showed that all three recombinant viruses were highly attenuated in replication in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV infection. In addition, no enhanced pulmonary disease was observed. In summary, these results demonstrate that targeting the viral mRNA cap MTase is a novel, new approach to rationally attenuate RSV for vaccine purposes.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P217

INHIBITION OF INFLUENZA- AND CORONAVIRUSES BY 1,4,4- TRISUBSTITUTED PIPERIDINES

Manon Laporte* 1, Sonia de Castro2, Annelies Stevaert1, Evelien Vanderlinden1, José Cumella2, Federico Gago3, Maria José Camarasa2, Sonsoles Velásquez2, Lieve Naesens1 1Rega Institute for Medical Research, KU Leuven - University of Leuven , Leuven, Belgium, 2Instituto de Química Médica, IQM-CSIC, 3Departamento de Ciencias Biomédicas, Unidad Asociada al IQM-CSIC, Madrid, Spain

Abstract: Circulating influenza- and coronaviruses cause respiratory infections with immense medical and socio-economic burden. Besides, our globalized society is threatened by virulent influenza- and coronaviruses that emerge from zoonotic reservoirs. Examples are influenza H5N1 and H7N9, and SARS and MERS coronaviruses. For the latter, a vaccine or antiviral therapy is lacking. There is a clear need for broader antiviral drugs that cover multiple respiratory viruses. We here report a class of N-1-benzylpiperidine-based dipeptide analogues with dual activity against influenza- and coronaviruses. A list of analogues was synthesized to understand the structure-activity relationship. The lead DICAM180 (a 4-fluorobenzyl analogue) displayed an antiviral EC50 value of 2.3 µM in influenza APR8 (H1N1)-infected MDCK cells with a selectivity index of 21. For human coronavirus 229E, evaluated in human embryonic lung fibroblast cells, DICAM180 had an EC50 value of 7.8 µM and selectivity index of 12. Based on a time-of-addition assay with influenza APR8, the action point of DICAM180 was situated at the endosomal stage within 1 h p.i. Serial virus passaging under DICAM180 yielded resistance mutations in the influenza hemagglutinin, namely substitutions HA1-I324T, HA1-S326V, and HA2-L99F. In a polykaryon assay with H1 HA-transfected Hela cells, DICAM180 inhibited low-pH-induced polykaryon formation. The impact of single DICAM180 associated resistance mutations on membrane fusion and DICAM180 resistance is currently being evaluated. For coronavirus the action point was situated beyond virus entry. Resistance selection and mechanistic studies are ongoing to identify the coronavirus target. The insights gained will enable to rationally optimize the 1,4,4-trisubstituted piperidine scaffold to achieve improved dual inhibitors of influenza- and coronaviruses.

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FIGHTING AND RESPONDING – antivirals and vaccines Abstract final identifier: P218

FLEXIBLE BAT IGG FC QUATERNARY STRUCTURE PRESENTS ORDERED AND UNDER-PROCESSED GLYCANS Victoria Avanzato* 1, Alice Stelfox1, Yasunori Watanabe1, 2, Karl Harlos1, Max Crispin2, Vincent Munster3, Thomas Bowden1 1Division of Structural Biology, 2Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 3Virus Ecology Unit, NIAID, National Institutes of Health, Hamilton, United States

Abstract: Bats serve as the natural host for several zoonotic viruses that cause severe disease in humans, such as Nipah and Hendra viruses, yet demonstrate little to no signs of pathology following infection. To better understand the molecular basis of the native reservoir immune response, we structurally characterized the Fc region of IgG from the black flying fox (Pteropus alecto), a member of the genus known to harbor highly pathogenic henipaviruses, and Seba’s short-tailed bat (Carollia perspicillata). Our structures exhibit the classical homodimeric ‘horseshoe’ organization observed across mammalian species, including humans. Analysis of multiple crystal forms showed varying inter-domain conformations of the Fc, likely due to apparent flexibility between the Cy2 and Cy3 domains of the protein. The Fc proteins were also subjected to small angle X-ray scattering (SAXS) analysis in solution, which supported the substantial inter-domain plasticity observed in the crystal structures. Ordered N-linked glycosylation was observed along the surface of the Cy2 domain in an extensive protein-interacting conformation. Furthermore, compositional analysis revealed that Pteropus alecto IgG Fc presents a higher than expected level of mannose-type glycosylation, suggestive that the surrounding proteineous environment modulates glycan biosynthesis to a greater extent than typically found in other mammalian species. This first structure of a bat IgG Fc provides a platform for evaluating downstream effector functions and will contribute to understanding the unique capacity of bats to serve as a reservoir of these highly pathogenic viruses.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P219

COMPARATIVE LOSS-OF-FUNCTION SCREENS HIGHLIGHT COMMON CELLULAR PATHWAYS REQUIRED BY MUMPS VIRUS FOR REPLICATION IN BATS AND HUMANS Danielle Anderson* 1, Kristmundur Sigmundsson2, So Young Kim3, Xiao Fang Lim1, Brian Ho Wenkae1, Paul Duprex4, Lin- Fa Wang1 1Programme in Emerging Infectious Diseases, 2Programme in Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore, 3Department of Molecular Genetics and Microbiology, Duke University, Durham, 4National Emerging Infectious Diseases Laboratories, Boston University, Boston, United States

Abstract: Bats have been implicated as an important source of emerging paramyxoviruses. The identification of bat-borne paramyxoviruses closely related to known human paramyxoviruses suggests a risk of zoonotic transmission of these viruses. Mumps virus (MuV), a contagious virus of the Rubulavirus genus, was thought to be an exclusive human pathogen with no animal reservoir. Recently, the complete sequence of a mumps-like rubulavirus was obtained from an African bat. To ascertain whether bat cells are capable of supporting the replication of MuV, and to identify cellular proteins involved in the viral life cycle, we performed comparative genome-wide siRNA screens using a human and novel bat siRNA library. Our human MuV siRNA screen was performed in A549 cells, a human lung adenocarcinoma cell line. A custom bat siRNA library was designed to target 18,328 genes of the Pteropus alecto genome. The bat siRNA screen was performed in PaKi cells, a P alecto kidney cell line. The coatomer complex I, a known dependency factor, was identified as required for MuV replication in both human and bat cells. Eukaryotic initiation factor 3 is a multiprotein complex that functions during the initiation phase of eukaryotic translation was also identified as a host factor. SEC61B, an endoplasmic reticulum membrane transporter, was identified as a potential pro-viral protein. KRT 1, a member of the keratin family, was identified as a candidate antiviral protein. This study is the first to use a bat genome-wide siRNA screen and provides a novel overview of cellular proteins and pathways that impact this important pathogen.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P220

MOLECULAR ANALYSIS OF RESPIRATORY SYNCYTIAL VIRUS (RSV) F AND G PROTEINS DURING THE 2015-2017 WINTER SEASONS IN THE US AND VIRAL SUSCEPTIBILITY TO ANTI-RSV MABS Bin Lu1, Hui Liu1, Fiona Fernandes1, Weijia Wang1, Seme Diallo2, David Tabor1, Alexey Ruzin2, Qing Zhu2, Mark Esser2, JoAnn Suzich2, Hong Jin* 1 1MEDIMMUNE, South San Francisco, 2MEDIMMUNE, Gaithersburg, United States

Abstract: RSV is a leading cause of lower respiratory tract infections in infants. Palivizumab is the only approved agent for RSV prophylaxis limited to high-risk infants. MEDI8897, a potent anti-RSV F mAb with extended serum half-life, is under clinical investigation as a candidate for RSV passive vaccination in all infants entering their first RSV season. To monitor current RSV genotypes in circulation and natural polymorphisms in the F protein-binding regions of MEDI8897 and palivizumab, we determined G and F sequences of 1,228 RSV isolates collected in the OUTSMART surveillance program during 2015-16 (n=392) and 2016-17 (n=836) RSV seasons from 25 regional laboratories in the US and Puerto Rico. RSV A (62% in 2015-16 and 45% in 2016-17), RSV B (37% in 2015-16 and 54% in 2016-17), and RSVA/RSVB coinfection (1% in both seasons) were detected. All RSV A and RSV B isolates clustered into ON1 and BA genotypes, respectively. A few distinct substitution polymorphisms in the MEDI8897 binding region (AA62-69 and 196-212) of the F protein were identified in RSV A (residues 65, 68 or 206) and RSV B (residues 66, 209, 210 and double 201/209, 206/209) strains. The frequency of these variants was in the 0.2-2.1% range, except for the 206/209 variant detected at a frequency of ~19% in 2016-17. None of these F polymorphisms had an impact on viral susceptibility to MEDI8897 neutralization. One known palivizumab resistant variant at residue 272 of RSV A was observed in the palivizumab binding region (AA267-275). The ongoing OUTSMART study provides viral susceptibility monitoring for anti-RSV mAbs.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P221

EPIDEMIOLOGICAL INVESTIGATION AND MOLECULAR EVOLUTION OF SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME Xiaoxia Huang1, Lin Liu1, Aqian Li1, Chuan Li1, Shiwen Wang1, Yang Liu1, Wei Wu1, Quanfu Zhang1, Dexin Li1, Mifang Liang* 1 1NATIONAL INSTITUTE FOR VIRAL DISEASES CONTROL AND PREVENTION，CHINA CDC, Beijing, China

Abstract: Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV) in genus Phlebovirus of the family Phenuiviridae, is a newly discovered highly pathogenic infectious disease. From 2010 to 2016, a total of 10917 SFTS cases, including 484 deaths, were reported in Mainland China with the average annual incidence rate of 0.12/lakh and case fatality rate of 4.4%．SFTS cases increased from 107 (2010) to 2949 (2016), but the annual fatality rate decreased from 14% (2010) to 2.5% (2016). The top 7 provinces were Henan, Shandong, Hubei, Anhui, Liaoning, Zhejiang, and Jiangsu, which accounted for more than 99% of the total cases. The period from April to October was the epidemic season. SFTSV samples used in evolutionary analysis distributed in China (443), South Korea (23), and Japan (64). Six genotypes of SFTSV were clearly divided. Genotype F was the dominant epidemic genotype of Japan, South Korea and Zhejiang province of China, genotype A was mainly epidemic in Henan, Hubei, and Anhui, genotype B was main in Shandong and Liaoning, and the genotypes are most abundant in Jiangsu and Anhui provinces. A large number of reassortant and recombinant strains of SFTSV were found. We also found a number of genotype-specific mutation sites, and proposed that two mutations (T501S and P662S) in Glycoprotein of genotype F might be associated with the high pathogenicity of genotype F. Many co-mutation sites and their corresponding branches in the phylogenetic tree were identified as well. It was found that SFTSV originated in the early 18th century, and the most likely origin was from Zhejiang province (about 50% probability). Besides, genotype F is the most primitive genotype.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P222

STRANGE STABLE REPLICATORS GENERATED FROM MUMPS VIRUS CDNA CLONES. Connor Bamford1, Elizabeth Wignall-Fleming1, Richard Randall2, Paul Duprex3, Bertus Rima* 4 1Centre for Virus Research , Glasgow University, Glasgow, 2School of Biology, St Andrews University, St Andrews, United Kingdom, 3Department of Microbiology, Boston University, Boston, United States, 4Centre for Experimental Medicine, Queen's University, Belfast, United Kingdom

Abstract: In rescue experiments based on a clinical isolate, we have isolated (r) recombinant mumps viruses (rMuVs) that carry large numbers of mutations clustered in discrete regions of the genome. In two separate experiments we obtained one rMuV with 19 mutations in the V/P gene and a second, which also contained an extra transcription unit encoding enhanced green fluorescent protein (EGFP) with 32 mutations in the nucleocapsid (N) gene. The vast majority of the mutations (48/51) were synonymous. These specific constellations of mutations are not observed in natural MuV isolates. On passage in Vero cells and EBV transformed human B lymphocytes these mutations were stably maintained even though mutations occurred in other genes during passage. Next generation sequencing (NGS) showed that there was no tendency to reversion to the original consensus sequence. Defective Interfering RNAs accumulated in passage in Vero cells and these were highly susceptible to biased hypermutation. Interestingly, in all samples the level of variation in the EGFP gene was the same as in the viral genes. This is surprising since it is unlikely this gene is under any selective pressure. The stability in repeated high multiplicity passage indicates that these mutation constellations placed the virus on fitness peaks from which it cannot escape. What mechanism(s) gave rise to these mutant viruses and their stability remain open questions of interest to negative strand RNA virologists. Quantum biological effects during T7 transcription of the cDNA may provide an explanation.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P223

EVIDENCE FOR CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS IN MONGOLIA Matthew A. Voorhees1, Susana L. Padilla1, Dulamjav Jamsransuren2, Jeffrey W. Koehler1, Korey Delp1, Dolgorkhand Adiyadorj2, Uyanga Baasandagwa2, Battsetseg Jigjav2, Timothy D. Minogue1, Randal J. Schoepp* 1 1United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, United States, 2National Center for Zoonotic Diseases, Ulaanbaatar, Mongolia

Abstract: To determine the risk of Crimean-Congo hemorrhagic fever virus (CCHFV) for human disease in Mongolia, surveillance for IgG antibody prevalence in humans and virus prevalence in tick vectors in selected regions was undertaken. Testing of 1926 human sera by ELISA demonstrated an overall CCHFV antibody prevalence of 1.4%. A total of 4530 ticks (Hyalomma asiaticum or Dermacentor nuttalli) collected in selected regions of Mongolia were pooled and analyzed by real- time RT-PCR. A single CCHFV positive H. asiaticum tick pool was identified and sequence analysis of a partial S segment sequence clustered with CCHFV isolates from Central Asia while a complete M segment grouped with CCHFV isolates from Africa and Asia/Middle East. Overall, this study provides a better understanding of the risk of CCHFV infection in the human population of Mongolia by confirming virus presence in the tick population and assessing the human CCHFV exposure rate.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P224

TRANSMISSION OF RESCUED BAT HL18NL11 INFLUENZA A-LIKE VIRUS IN EXPERIMENTALLY-INFECTED JAMAICAN FRUIT BATS Tony Schountz* 1, Ashley Malmlov1, Jinhwa Lee2, Jingjiao Ma2, Tawfik Aboellail1, Wenjun Ma2 1Microbiology, Immunoloyg and Pathology, Colorado State University, Fort Collins, 2Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, United States

Abstract: Sequences of two novel influenza A-like viruses, HL17NL10 and HL18NL11, were recently discovered in New World little yellow shouldered fruit bats (Sturnira lilium) and flat-faced fruit bats (Artibeus planirostris), respectively. Field studies indicated these viruses circulate among many species of fruit bats of Central and South America, including Jamaican fruit bats (Artibeus jamaicensis). Infectious viruses have not been isolated from bats, thus HL18NL11 virus was generated by reverse genetics. We intranasally inoculated Jamaican fruit bats with the rescued virus to determine susceptibility and infection dynamics, and to determine if virus could be transmitted to other bats. Moderate levels of virus was detected in rectal swabs of all inoculated bats starting two days post inoculation and lasting for nearly two weeks, but without signs of disease. Virus was detected in the two contact bats, indicating virus transmission had occurred. Notably, these two contact bats exhibited ocular and nasal discharges within 5 days of contact. ELISA and neturalization assay showed moderate to low antibody titers in bats between two to four weeks after inoculation or transmission. This is the first study to demonstrate susceptibility to bat influenza A-like viruses and suggests that viral persistence up to 28 days may occur in some bats, supporting the hypothesis that Jamaican fruit bats may be a natural reservoir host of the HL18NL11 virus. The kinetic profile of infection suggests that bat influenza A-like viruses in bats are similar to avian influenza virus infection of waterfowl. Currently, additional infection studies to further characterize the virology and immunology of Jamaican fruit bats against HL18NL11 are under way.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P225

PATHOGENICITY OF NIPAH VIRUS BANGLADESH IN EXPERIMENTALLY INFECTED PIGS Samantha Kasloff* 1, Anders Leung2, Brad Pickering1, Cory Nakamura3, Greg Smith1, Brad Collignon3, Carissa Embury- Hyatt3, Darwyn Kobasa2, Hana Weingartl1 1Special Pathogens Unit, National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, 2Special Pathogens Program, Public Health Agency of Canada, 3National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, Canada

Abstract: Nipah henipavirus is a Risk Group 4 pathogen responsible for hundreds of human deaths in Southeast Asia since its discovery in 1998. While pigs played a central role in virus spillover to farm workers in the initial Malaysia epidemic, subsequent outbreaks with a novel variant in Bangladesh resulted from direct transmission from bats to people, with no confirmed involvement of spillover hosts. Despite suggested epidemiological and serological evidence, the role of pigs in transmission of NiV Bangladesh remains unknown. Landrace piglets were experimentally infected with NiV Bangladesh to study acute and long-term phases of disease. As early as 2DPI, qRT-PCR confirmed shedding in oral, nasal, and rectal secretions. Post-mortem analysis at pre-determined experimental endpoints revealed similar patterns of viral replication to previous findings with NiV Malaysia, with early dissemination from the upper respiratory tract to the brain, and subsequent spread through the respiratory tract, associated lymphatic tissues, and additional visceral organs. Interestingly, experimentally infected animals had no clinical signs, viremia was undetectable throughout the study and only low level neutralizing antibody titers were measured by 28 days post-infection. We provide the first report on susceptibility of pigs to Nipah Bangladesh, demonstrating productive replication, dissemination, and shedding following oronasal infection. Results obtained highlight the potential role of pigs in future Nipah virus transmission events and the need for continued and enhanced surveillance in pigs in Bangladesh and other endemic or at-risk regions.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P226

GENETIC COMPATIBILITY AND VIRULENCE OF REASSORTANTS DERIVED FROM H9N2 AND H5N1 AVIAN INFLUENZA VIRUSES CO-CIRCULATING IN EGYPT Yasuha Arai* 1, Madiha S. Ibrahim2, Takaaki Nakaya1, Yohei Watanabe1 1Department of Infectious Diseases, Kyoto Prefectural School of Medicine, Kyoto, Japan, 2Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt

Abstract: Avian influenza (AI) virus subtype H9N2 was first identified in Egypt in 2011. Circulation of the H9N2 viruses in Egyptian poultry in the presence of AI virus subtype H5N1 poses a more significant threat to the poultry industry and human public health in this area. Previous surveillance study reported numerous co-infection cases of the H5N1 and H9N2 viruses in Egyptian poultry. Egypt is now regarded as a hot spot of AI virus evolution. Therefore, one concern is a possible emergence of novel H5N1-H9N2 reassortants with a high infectivity to humans after their reassortment event, as observed in the emergence cases of H7N9, H10N8 and H5N6 viruses in China. To evaluate the potential public risk of the reassortants derived from these viruses, we used reverse genetics to generate possible 63 H5N1 reassorants derived from Egyptian H5N1 and H9N2 viruses, containing the H5N1 surface protein genes, and evaluate their compatibility, replication ability in vitro and mouse virulence in vivo. These hybrid viruses showed an extremely high genetic compatibility. In human airway cells, two-thirds of the hybrid viruses replicated more efficiently than did H5N1 parental virus. Notably, several hybrid viruses replicated to the levels that was markedly higher than that of both parental viruses. These hybrid viruses also showed a more virulent phenotype in mice than did H5N1 parental viruses, with several logs higher titer in lungs. Our results demonstrate that reassortment between Egyptian H5N1 and H9N2 could generate distinct hybrid viruses with a higher infectivity and virulence to humans. Our findings highlight the importance of active surveillance programs to monitor the emergence of H5N1 reassortant viruses containing H9N2 internal genes in Egypt.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P227

MULTIPLEX PCR-BASED NEXT-GENERATION SEQUENCING OF HANTAVIRUSES IN HUMANS AND RODENTS Won-Keun Kim* 1, Jin Sun No1, Jeong-Ah Kim1, Seung-Ho Lee1, Dong Hyun Song2, Daesang Lee2, Se Hun Gu2, Sunhye Park2, Seong Tae Jeong2, Heung-Chul Kim3, Terry A. Klein3, Michael R. Wiley4, Patrick S. Chain5, Gustavo Palacios4, Jin-Won Song1 1Korea University, Seoul, 2Agency For Defense Development, Daejeon, 365th Medical Brigade, Seoul, Korea, Republic Of, 4U.S. Army Medical Research Institute of Infectious Disease , Maryland, 5Los Alamos National Laboratory, New Mexico, United States

Abstract: Hantaviruses (Genus Orthohantavirus, Family Hantaviridae, and Order Bunyavirales) cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome in humans. Endemic infections of hantaviruses are responsible for annual 150,000 clinical cases with fatality rates from 1 to 35% around the world. The rodent-borne hantaviruses, Hantaan (HTN) and Seoul (SEO) viruses, induce mild to severe HFRS with significant morbidity and mortality in the absence of good medical care. Next-generation sequencing (NGS) is a robust approach to delineate genomic sequences and characteristics of the virus in endemic outbreaks. However, acquisition of viral genome sequences from clinical samples is a challenge due to low copies of viral RNA. Here, multiplex PCR-based NGS was developed and applied to whole-genome sequencing of hantaviruses from HFRS patient and rodent specimens. Multiplex PCR was performed by using the primer set designed to amplify 150 bp-length reads. Nearly whole-genome sequences of hantaviruses were completed by using Illumina MiSeq. In combination with whole-genome sequences of HTNV from HFRS patients and rodents, phylogeographic analysis described the spatial and epidemiological links between clinical cases and their sources, showing geographic clustering of the strains with the HTNV circulating in rodents on each of their exercising location. These results may indicate the most likely site where the patient was infected with HTNV. The phylogeny of SEOV revealed well- defined genotypes from East Asia, Southeast Asia, Western Europe, and North America. Overall, multiplex PCR-based NGS was a potent method to obtain nearly whole-genome sequences of low copy hantavirus RNA from clinical specimens and rodents. This study provides significant insights for the whole-genome sequencing, genomic-based surveillance, and genetic diversity of hantaviruses.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P228

PHENOTYPIC EVOLUTION OF NEURAMINIDASE OF INFLUENZA A(H1N1)PDM09 VIRUS Carles Martínez-Romero* 1, 2, Wenjuan Du3, Raquel Muñoz-Moreno1, 2, Meiling Dai3, Gaalia Strupinsky1, Erik de Vries3, Adolfo García-Sastre1, 2, 4, C. A. M. de Haan3 1Microbiology, 2Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, United States, 3Infectious Diseases and Immunology, Virology Division, Utrecht University, Utrecht, Netherlands, 4Medicine, Division of Infection Diseases, Icahn School of Medicine at Mount Sinai, New York, United States

Abstract: The emergence of the new pandemic influenza virus in 2009 (A(H1N1)pdm09) and the abundance of sequences available allows us to analyze in detail the genetic and phenotypic drift of such a pandemic virus. In the present study we focused on the neuraminidase (NA) protein, which is essential for virion release from cells and decoy receptors and is an important target for antiviral drugs. Phylogenetic analysis of the NA protein sequence revealed a cumulative number of fixed amino acid substitutions over the years. These mutations led to changes in protein expression levels, antigenicity, and specific sialidase activity in vitro. Mutations in NA were also analyzed in the context of virus particles. We generated a set of recombinant influenza viruses with selected amino acid substitutions in the NA protein to mimic the adaptations in four different isolates using the A/California/04/2009 strain as backbone. Mutations affected incorporation of NA in virus particles and replication in different mammalian cell models of influenza infection, including differentiated normal human bronchial epithelial (NHBE) cells. Our results indicate that enzymatic activity of NA may be affected via several different mechanisms, but that it is maintained within a certain bandwidth rather than to evolve to maximal sialidase activity. These findings provide novel insights in the evolution of the enzymatic activity of NA after the introduction of a pandemic virus in the human population and might contribute in the prediction of future adaptations in these viruses.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P229

IDENTIFICATION OF MUTATIONS IN GENES CODING FOR SURFACE F AND G PROTEINS OF HUMAN RESPIRATORY SYNCYTIAL VIRUS ISOLATED FROM CHILDREN TREATED WITH PALIVIZUMAB Julia Dina* 1, Astrid Vabret1 1Virology, University Hospital of Caen, Caen, France

Abstract: Palivizumab is a respiratory syncitial virus (RSV)-neutralizing monoclonal antibody clinically used for the prevention of severe RSV infections in high-risk infants. Palivizumab acts by blocking the fusion step of virus replication. Mutants resistant to palivizumab were isolated in vitro and also in children with RSV infection while receiving palivizumab. The aim of this study was to analyze the complete F and G genes sequences coding the surface glycoproteins of RSV isolates collected from patients receiving palivizumab.RSV isolates were obtained from nasopharyngeal swabs of high-risk infants treated with palivizumab at the University Hospital of Caen between october 2011 and april 2016 and having presented a RSV-breakthrough during the treatment or in the six months after. Among the 273 infants treated with palivizumab during the period of the study, 15 (8,4%) have presented a RSV infection during their treatment or in the six months after. Seven RSV/A and 8 RSV/B were identified by real-time PCR.For the RSV/A analysis, phylogenetic trees were constructed using 6 RSV/A detected, one control RSV/A and 42 reference sequences. The hRSV/A isolated in 2014 or after were identified in the ON1 cluster. When they were detected in 2011 they clustered with the GA2 genotype. None RSV/A was detected between 2011 and 2014. The analysis of complete F genes alignements of hRSV/A shows several mutations out of the liaison site of palivizumab. We found one mutation in the liaison site, the N276S mutation. This was previously described as a mutation conferring partial resistance to palivizumab in vitro and in vivo. This mutation was also identified in the viruses collected from the control population. This study allowed us to characterize mutations of RSV in case of palivizumab failure.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P231

ANALYSIS OF THE ZOONOTIC POTENTIAL OF INFLUENZA D VIRUS FOR HUMANS Melle Holwerda* 1, 2, Jasmine Portmann1, 2, Hanspeter Stalder1, 2, Gert Zimmer1, 2, Ronald Dijkman1, 2 1Institute for Virology & Immunology, Bern and Mittelhäusern, 2Department of Infectious diseases and Pathobiology, Bern, Switzerland

Abstract: Influenza D virus (IDV) is a new genus in the Orthomyxoviridae family and was first detected in pigs with influenza- like symptoms in 2011. IDV-directed antibodies were detected in a broad range of livestock like camelids, cattle and small ruminants, suggesting that the virus has a wide host tropism. Interestingly, IDV-directed antibodies were also found in humans, albeit only in those with occupationally exposure to livestock. To elucidate the zoonotic potential of IDV for humans, we infected well-differentiated human airway epithelial cells (hAECs) from several donors with IDV and analyzed the viral replication kinetics and cell tropism at 33°C as well as 37°C. In addition, we assessed the induction of the innate immune response during the course of infection by qRT-PCR. Upon infection, apical washes were collected every 24 hours to monitor the production and secretion of viral progeny using qRT-PCR and virus titration. It turned out that IDV is able to efficiently replicate in hAECs at both 33°C and 37°C. Moreover, immunofluorescence analysis showed that IDV has a preferential cell tropism for ciliated cells, which is similar to what we have observed for the related Influenza C virus. Finally, analysis of the innate immune response revealed a pronounced upregulation of traditional interferon-stimulated genes such as IFIT1, MxA and OAS1, as well as interferon lambda (IFN-λ) 1, 2 and 3 at 48 and 72 hours post infection. These results demonstrate that IDV replicates efficiently on hAECs with a preference for ciliated cells and induces an innate immune response during the late stage of infection. These findings might explain why humans with occupationally exposure to livestock have developed antibodies directed to IDV.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P232

BAYESIAN EVOLUTIONARY ANALYSIS BY SAMPLING TREES APPLIED TO EXTENDED SEQUENCING OF MEASLES VIRUS FOR MOLECULAR EPIDEMIOLOGY OF OUTBREAKS. Helene Schulz* 1, 2, Joanne Hiebert3, Xenia R. M. R. Lemos4, Paola C. Resende Silva4, Marilda M. Siqueira4, Alberto Severini1, 3 1Medical Microbiology and Infectious Diseases, Universiy of Manitoba, 2Viral Zoonosis, 3Viral Exanthemata and Sexually Transmitted Diseases, PUBLIC HEALTH AGENCY OF CANADA, Winnipeg, Canada, 4Respiratory and Measles Laboratory, Oswaldo Cruz Foundation/FIOCRUZ, Rio de Janeiro, Brazil

Abstract: Background: Endemic circulation of measles virus in the Americas has been interrupted since 2002 and all cases result from importation from endemic areas. Ongoing elimination efforts have contributed to the decreasing diversity of measles virus genome, making it impossible to distinguish independent importations from local transmissions when relying on traditional WHO- recommended genotyping targets. Therefore a higher-resolution genotyping or whole genome sequencing approach is needed. Objectives: 1) develop an algorithm to determine the relatedness of cases in outbreaks based on the established molecular clock rate 2) analyze the sequences for hypervariable regions for development of high-resolution genotyping test. Methods: Whole genome sequencing by Sanger and next-generation sequencing of a large Canadian and a large Brazilian outbreak followed by Bayesian Evolutionary Analysis by Sampling Trees (BEAST). Results: Based on whole genome sequencing, the 1000nt M/F non-coding region (MF-NCR) accounts for most of the variability seen in outbreak-associated viruses. BEAST revealed a clock rate of 3x10-3 and 7x10-4 changes/nucleotides/year for the MF-NCR and whole genome, respectively. Based on the node ages, the main outbreak is distinguished from other unrelated importations. Conclusion: BEAST analysis of whole genome sequencing and extended genotyping of measles virus will empower epidemiological investigation as global measles elimination is approached and the diversity of the measles genome decreases.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P233

SEROLOGICAL INVESTIGATIONS OF LABORATORY-CONFIRMED AND SUSPECTED EBOLA PATIENTS DURING THE LATE PHASE OF THE EBOLA OUTBREAK IN SIERRA LEONE Yang Liu1, Wei Wu1, Aqian Li1, Dexin Li1, Jun Liu1, Mifang Liang* 1 1NATIONAL INSTITUTE FOR VIRAL DISEASES CONTROL AND PREVENTION，CHINA CDC, Beijing, China

Abstract: Sierra Leone in West Africa was one of the three hyperendemic countries during 2014-2015 Ebola epidemic. The data on serological investigation of Ebola virus (EBOV) infection in the late phase of the outbreak were still lacking. 877 blood specimens from 694 Ebola virus disease (EVD) suspected cases were delivered to the Sierra Leone-China Friendship Laboratory from March to December 2015. Specimens were tested by real-time PCR for viral RNA detection, ELISA and Luminex multiple analysis system (Luminex) were performed to evaluate Ebola-specific antibody levels subsequently. Both ELISA and Luminex methods showed consistent and comparable results in this study (p>0.05). Time distribution analysis of serological features in all EVD suspected patients from the late phase showed a decline trend in overall for positive detection of viral RNA or IgM/IgG antibodies, viral RNA positive rate dropped to zero at earlier time, while the disappearance of IgM and IgG antibodies showed a hysteretic situation and contributed 3.8% and 17.8% positive rates for IgM and IgG detection respectively among 663 viral RNA negative patients, this indicated asymptomatic or previous Ebola infection and certain population immunity to the virus infection. 18 of 31 confirmed EVD patients (58.0%) raised IgM antibody responses and nearly all (30/31,97%) developed IgG antibodies. The dynamic analysis of viremia and antibody responses in eight longitudinal EVD patients showed the typical reversed trends of the declining viral load and rising IgM and/or IgG responses in 2 weeks. The study prompts the understanding of serological features in both confirmed and suspected patients in the late phase of outbreak.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P234

DETECTION OF HANTAVIRUS RNA IN HUMAN SAMPLES IN GEORGIA Gvantsa Chanturia* 1, Roena Sukhiashvili1, Nato Kotaria1, Nazibrola Chitadze1, Giorgi Chakhunashvili1, Paata Imnadze1 1National Center for Disease Control and Public Health (NCDC), Tbilisi, Georgia

Abstract: Hantavirus infection is commonly detected in Georgia - the country located in south Caucasus region. Due to complexity of the geography, ecology varies in different regions of the country. Hantavirus cases are registered at the western, more humid part of Georgia, close to Black Sea. The Georgian National Center for Disease Control and Public Health (NCDC) is responsible for conducting surveillance of this pathogen using serological and molecular techniques. The Lugar Center for Public Health Research as a part of NCDC conducts laboratory investigation to facilitate surveillance in the country using routine serology and more advanced molecular approaches including next generation sequencing on Illumina’s MiSeq platform. In addition DTRA funded project for investigation of hantavirus was recently launched at NCDC. According to the last five years of statistical data the annual number of cases varied from two in 2013 to eight in 2014. Seven sporadic cases (with one lethal) were detected and confirmed using western blot technique at 2017. In addition, recently established real-time PCR was applied on eight ELISA IgM positive human sera samples from seven patients and four of them (three patients) were RNA positive on Hantavirus Dobrava. For further characterisation, RNA positive clinical samples were directly sequenced using Illumina’s MiSeq platform. The partial S, M and L segments were elucidated. The data analysis and phylogenetic is in the progress. The work enables molecular epidemiological monitoring of hantavirus and will be useful to understand transmission chains leading to better disease control in the future.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P235

GENOMIC CHARACTERIZATION OF VIRUSES IN DROMEDARY CAMELS FROM UNITED ARAB EMIRATES Suxiang Tong* 1, Yan Li1, Salama Al Muhairi2 1CDC, Atlanta, United States, 2ADFCA , Abu Dhabi, United Arab Emirates

Abstract: Camels can potentially serve as a source for zoonotic transmission of viruses to humans due to the role by camels in the emergence and transmission of MERS-CoV. It is important to understand the viruses that camels harbor and their zoonotic potential. The metagenomic study of NP swabs from camels in Abu Dhabi identified sequence reads of potentially novel virus species or strains. In this study we performed full genome sequencing to further characterize these viruses. To fill the gaps in the sequences generated by NGS for generating their full genome sequences, PCR primers were designed based on the sequences obtained from NGS. The amplicons obtained from RT-PCR or PCR were sequenced by Sanger sequencing. Genome annotation and phylogenetic analysis were done to further characterize the virus genomes. We obtained close-to-full genome sequences of 3 recently discovered camel viruses: camel Crimean-Congo hemorrhagic fever virus (CCHFV), camel parainfluenza virus 3 (PIV3), and camel parainfluenza virus 4 (PIV4). The nucleotide identities of camel CCHFV, camel PIV3 and camel PIV4 to the closest relatives are about 87% to CCHFV strain SPU103/87, 85% to bovine PIV3 isolate TVMDL20, and 80% to HPIV4b strain 04-13, respectively. Annotation and characterization of the genomes predict similar ORFs and genomic features shared by the other viruses in the same taxonomical group. These genomic sequences provide data for a more accurate taxonomical classification of the novel camel viruses. We propose that the camel CCHFV, camel PIV3 and camel PIV4 viruses as novel strains. Because we observe viruses in camels which are related to those that are known to cause disease in humans, we expect that camels may pose a risk as an intermediate species for transmitting viruses to humans.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P236

MOLECULAR DETECTION OF PUUMALA ORTHOHANTAVIRUS: STRUGGLING WITH HIGH NUCLEOTIDE SEQUENCE VARIABILITY Stephan Drewes1, Susanne Röhrs2, 3, Elfi Schlohsarczyk4, Stefan Fischer1, Wolfgang Fritzsche5, Florian Binder1, Gerald Heckel6, 7, Rainer G. Ulrich* 1 1Institute of Novel and Emerging Infectious Diseases, 2Institute for Diagnostic Virology, FRIEDRICH-LOEFFLER-INSTITUT, Greifswald-Insel Riems, 3IDT Biologika GmbH, Greifswald, 4Institute of Veterinary Pathology, Justus-Liebig-University Giessen, Giessen, 5Leibniz Institute of Photonic Technology, Jena, Germany, 6Institute of Ecology and Evolution, University of Bern, Bern, 7Swiss Institute of Bioinformatics, Genopode, Lausanne, Switzerland

Abstract: Puumala orthohantavirus (PUUV), member of the family Hantaviridae, is the most common causative agent of hemorrhagic fever with renal syndrome (HFRS) in Europe. PUUV is a negative-stranded RNA virus with a trisegmented genome. The S-segment encodes two open reading frames (ORF) for the nucleocapsid (N) protein and a non-structural (NSs) protein. To date, eight genetic lineages of PUUV have been described, all associated with the bank vole (Myodes glareolus) as reservoir. Our investigations in Germany showed that PUUV of the Central European lineage is associated with the presence of the Western bank vole phylogroup. Human PUUV infections occur only in western and southern areas of Germany. In contrast, PUUV-infected bank voles in Poland belong to the Carpathian and Eastern evolutionary lineages. Bank voles collected in Germany during 2010-2013 revealed the occurrence of PUUV sequence types of the N- and NSs-ORF with temporal and/or spatial variation. The nucleotide sequence divergence within the S segment of PUUV strains reached 20% causing problems to select primer and probe sequences for PUUV real-time RT-PCR. Currently, we are evaluating several real-time RT-PCR assays for detection of PUUV strains of different origin. Perspectively, we intend to design a diagnostic platform allowing comprehensive and timesaving molecular bed-side and pen-side diagnostics of PUUV and pathogens causing similar symptoms.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P237

SYSTEMATIC INVESTIGATION OF NOVEL LINEAGES OF ENDOGENOUS BORNAVIRUS-LIKE ELEMENTS IN VERTEBRATE GENOMES Masayuki Horie* 1, 2, Shohei Kojima2, 3, Bea C. B. Garcia2, 3, Dong-Yun Kim2, 3, Yahiro Mukai2, 3, Nicholas F. Parrish4, Keizo Tomonaga2, 3 1Hakubi Center for Advanced Research, 2Institute for Frontier Life and Medical Sciences, 3Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 4Section of Surgical Sciences, Vanderbilt University Medical Center, Nashville , United States

Abstract: Endogenous bornavirus-like elements (EBLs) are sequences derived from bornaviruses in eukaryotic genomes. These EBLs serve as precious informational sources to understand ancient bornaviruses as well as the co-evolution between bornaviruses and their hosts. Recently, two novel lineages of exogenous bornaviruses were discovered from carpet pythons and sharpbelly fish. Because these viruses are distantly related to the previously known bornaviruses (genus Orthobornavirus), these viral sequences could enable discovery of novel EBLs. By a tBLASTn screening using those viral sequences as queries, we found many novel EBLs (e.g. 10 novel EBLs in human) in eukaryotic genomes, including EBLs derived from the P gene, from which no EBLs have previously been reported. Phylogenetic analyses revealed that bornaviruses and EBLs are divided into 3 lineages regardless of their host species. We also estimated the insertion dates of EBLs in primates, suggesting that the endogenizations occurred at several points during primate evolution. Next, we performed in silico analyses for the newly identified EBLs in human. We found that at least two of the newly identified human EBLs are expressed as RNA: one is expressed as ncRNA, while the other is, interestingly, located in the 3’ UTR of SLC4A8 mRNA. This type of EBL has not been reported yet, and thus this finding raises a novel hypothesis that EBLs might be involved in control of gene expression as 3’ UTR of mRNA encoding other genes. Taken together, this study provides novel insights into the biological significance of EBLs as well as the virology of bornaviruses.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P238

FUNCTIONAL KUMASI VIRUS SURFACE GLYCOPROTEIN G IS EXPRESSED IN HIGH-ORDER OLIGOMERS Laura Behner* 1, Louisa Zimmermann1, Marc Ringel1, Andrea Maisner1 1PHILIPPS UNIVERSITY MARBURG, INSTITUTE OF VIROLOGY, Marburg, Germany

Abstract: Hendra virus (HeV), Cedar virus (CedV) and Nipah virus (NiV) represent henipaviruses originating from fruit bats in Australia and Asia. In recent years, also African bat henipaviruses were identified at the nucleic acid level. As live viruses could not be isolated so far, the two surface glycoproteins G and F were functionally characterized to investigate their potential to support virus entry and spread by cell-cell fusion in non-bat cell types. Compared to other henipavirus G proteins, surface expression and fusion-helper function of the receptor-binding G protein of Kumasi virus (KV), the prototypic Ghanaian bat henipavirus, was found to be clearly reduced due to a delayed KV G export from the ER. Since defects in the oligomerization of receptor-binding glycoproteins can be responsible for a limited surface transport, we studied the oligomerization pattern of KV G. In contrast to HeV and NiV G proteins which are known to be expressed at a balanced dimer-tetramer ratio, KV G almost exclusively formed stable tetramers or high-order oligomers. KV G protein also showed less stringent requirements for defined stalk cysteines to form dimers and higher oligomers compared to NiV G protein. Interestingly, any changes in the oligomeric pattern negatively affected the fusion-helper function of KV G protein although neither surface expression nor receptor binding was affected. This indicates that the formation of mostly higher oligomeric KV G forms is not a deficiency responsible for ER retention, but is rather a basic structural feature which is essential for the bioactivity of this African bat henipavirus glycoprotein.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P239

SEROLOGICAL EVIDENCE FOR THE CIRCULATION OF EBOLAVIRUSES IN SWINE IN WEST AFRICA Kerstin Fischer* 1, Alimou Camara2, Juliet Jabaty3, Cécile Troupin2, Roland Suluku4, Thomas Hoenen1, Noel Tordo2, Martin Groschup1, Sandra Diederich1 1Friedrich-Loeffler-Institut, Greifswald, Germany, 2Institut Pasteur, Conakry, Guinea, 3Sierra Leone Agricultural Research Institute, Makeni, 4Njala University, Njala, Sierra Leone

Abstract: Infections with ebolaviruses have resulted in numerous outbreaks of severe hemorrhagic fever in humans and other primates. There is molecular and serological evidence for a zoonotic origin of ebolaviruses in wildlife. Concerning livestock, pigs were identified as a susceptible host for Reston virus (RESTV; species Reston ebolavirus), with reported transmission to humans in the Philippines. Under experimental conditions, pigs were also shown to be susceptible to Ebola virus (EBOV) infection. However, there is no evidence of natural EBOV infection in pigs to date. To investigate a potential role of pigs in the EBOV biology, porcine sera were collected from Guinea and Sierra Leone from 2016 to 2017. Areas that were heavily affected by the recent EBOV epidemic were especially targeted, as well as communities with known close contact between livestock and the surrounding wildlife population. Porcine sera were screened for the presence of EBOV- specific antibodies in an ELISA based on the EBOV nucleoprotein (NP). Reactive sera were further tested for (cross-) reactivity in immunoblot analysis against EBOV, Sudan virus (SUDV), and RESTV NP. To determine neutralizing activity of the sera, we established a neutralization test based on transcription and replication-competent virus-like particles which allows modeling of EBOV infection under biosafety level 1 conditions. So far, we found at least three sera to be positive in the EBOV NP ELISA as well as in immunoblot. Interestingly, two of the three sera showed cross-reactivity with RESTV NP and/or SUDV NP while none of them had neutralizing activity. Our results suggest the circulation of ebolaviruses in swine in West Africa that are antigenically related but not identical to EBOV, and could represent undiscovered ebolaviruses with unknown pathogenic and/or zoonotic potential.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P240

SEROLOGICAL AND MOLECULAR EVIDENCE FOR CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS CIRCULATION IN TICKS, CATTLE, AND HUMANS IN ZAMBIA Masahiro Kajihara* 1, Martin Simuunza2, Akina Mori-Kajihara1, Ngonda Saasa2, Yongjin Qiu1, Yoshiki Eto1, Ryo Nakao3, George Dautu4, Bernard M. Hang’ombe2, Yasuko Orba1, Hirofumi Sawa1, Shuetsu Fukushi5, Shigeru Morikawa5, Masayuki Saijo5, Jiro Arikawa6, Aaron Mweene2, Mwaka Monze7, Victor Mukonka8, Ayato Takada1, Kumiko Yoshimatsu6 1Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan, 2School of Veterinary Medicine, University of Zambia, Lusaka, Zambia, 3Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 4Central Veterinary Research Institute, Lusaka, Zambia, 5National Institute of Infectious Diseases, Tokyo, 6Graduate School of Medicine, Hokkaido University, Sapporo, Japan, 7University Teaching Hospital, 8Zambia National Public Health Institute, Lusaka, Zambia

Abstract: Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease with a high mortality rate in humans. While the disease is widely found in Africa, Europe, and Asia, distribution patterns and genetic diversity of CCHF virus (CCHFV) are not fully understood especially in African countries where no CCHF cases were reported until now. To assess potential risks of CCHFV infection in non-endemic areas, cross-sectional epidemiological studies in humans, cattle, and ticks were conducted in Zambia that has never experienced CCHF outbreaks. Through serological screening of 747 and 1,047 human and cattle serum samples, respectively, by an indirect immunofluorescent assay, CCHFV nucleoprotein- specific IgG antibodies were detected in 12 (1.6%) and 88 (8.4%) samples of local people and cattle, respectively. Out of 290 Hyalomma ticks, the principal vector of CCHFV, collected from 7 districts, CCHFV genomes were detected in 11 samples from 5 districts. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between CCHFVs of African and Asian origins. The present study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts including humans.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P241

REVERSE GENETICS PLATFORM FOR IMPROVED CLONING AND EFFICIENT RESCUE OF MEASLES VIRUS FROM CDNA Soroth Chey1, Juliane Stosch1, Mario Hönemann1, U.G. Liebert* 1 1Institute of Virology, Leipzig University, Leipzig, Germany

Abstract: Reverse Genetics is a technology that makes it possible to produce RNA virus from its complementary DNA (cDNA). Standard protocols are laborious and inefficient for negatice-strand RNA viruses. In order to increase the recovery efficiency of measles virus (MV), a plasmid platform with novel features compared to standard rescue systems was established. Key components of the platform include (1) placement of CMV-derived RNA polymerase II promoter and bacteriophage T7 promoter in unidirection on the same plasmid; (2) addition of three G nucleotides immediately after T7 promoter; (3) use of two ribozymes, namely hairpin hammerhead ribozyme and hepatitis delta virus ribozyme. Full-length antigenomic cDNA of various MV strains was inserted into the novel plasmid backbone. Both, wild-type and vaccine-derived MV was easily rescued from their respective cloned cDNA with high efficiency. It is assumed that the platform is useful for the rescue of other mononegavirales including Ebola and Borna virus.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P242

THE CHARACTERISTICS OF RECENT CLINICAL ISOLATES OF HUMAN METAPNEUMOVIRUS IN JAPAN Naganori Nao* 1, Ko Sato2, Fumio Seki1, Junya Yamagishi3, Nishimura Hidekazu2, Makoto Takeda1 1Virology III, National Institute of Infectious Diseases, Musashimurayama, 2Virus Research Center, Clinical Research Division, Sendai Medical Center, Sendai, 3Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan

Abstract: [Introduction] Human metapneumovirus (HMPV) was first isolated in 2001. HMPV has been a major etiological agent of acute respiratory infections in humans. The viral genome is a non-segmented negative-sense RNA, which encodes nine proteins, including three surface glycoproteins: F, SH, and G proteins. Recently, HMPV strains with 180- or 111-nucleotide duplication in the G gene (180nt-dup and 111nt-dup, respectively) were detected in Japan and Spain. [Materials and Methods] Clinical samples collected in Japan in recent years were tested for HMPV by RT-PCR. Nucleotide sequences of F, SH, M2, and G genes of 43 HMPV strains were sequenced by MiSeq (Illumina). Infectious HMPVs were isolated from HMPV positive samples. Full genome sequences of 7 HMPV strains were sequenced by PacBio RS II (Pacific Biosciences). The infectivity of selected clinical isolated strains was analyzed in various types of cell lines and compared with that of GFP-expressing 4 recombinant HMPV strains, which have been commonly used for HMPV studies. [Results and conclusions] The direct PCR detection of clinical samples showed that among 43 HMPV strains, 20 strains had 180nt-dup in the G gene. The 180nt-dup was retained in the genome of isolated viruses. No nucleotide duplication was detected in other genes. All analyzed clinical isolated strains showed a similar infectivity phenotype. The phenotype was most similar to that of GFP- expressing JPS02-76 strain (Shirogane et al. 2008). They showed the highest infectivity to Vero E6 cells, moderate infectivity to BEAS-2B and A549 cells, and low infectivity to HeLa and MNT-1 cells. Further studies are being conducted in our laboratory to reveal the functional advantages of 180- or 111-nt dup in the G gene.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P243

IDENTIFICATION OF NEW ENDEMIC REGION FOR CCHFV IN GEORGIA Roena Sukhiashvili* 1, Ketevan Sidamonidze1, Gvantsa Chanturia1, Giorgi Chakhunashvili1, Randal Schoepp2, Connie Schmaljohn2, Paata Imnadze1 1National Center for Disease Control and Public Health, Tbilisi, Georgia, 2U.S. Army Medical Research Institute of Infectious Diseases, Frederick, United States

Abstract: Crimean-Congo hemorrhagic fever (CCHF) is a widespread disease that causes severe viral hemorrhagic fever outbreaks, with a case fatality rate of 10–40%. In Georgia, the first case was detected in 2009. Since then, new cases have been detected and were confirmed with the highest number (30) registered in 2014. National Center for Disease Control and Public Health (NCDC) of Georgia is responsible for conducting surveillance of CCHF with routine testing of patient samples using serological and molecular methods as well as monitoring and the field surveillance of vectors. For the study, field samples from previous DTRA funded project were used. A total of 386 pooled tick samples collected on animals and/or tick drags from three different regions of east Georgia were assessed by qPCR for the presence of CCHFV RNA. Of the 386 samples 25 were identified as positives using US Army Medical Research Institute of Infectious Diseases (USAMRIID) protocol. All positive samples were from two regions: Samtskhe-Javakheti and Kvemo Kartli. Of these Kvemo Kartli is the newly identified hotspot for the CCHFV. Positive samples were retested with different protocol and different set of primers used for routine testing at NCDC and only three were detected as positive from both region. Testing revealed that USAMRIID protocol provided for the nucleic acid testing of CCHFV was more sensitive. Next generation sequencing is ongoing for further characterization of positive samples. With this work, a new endemic region for CCHF was identified in Georgia. This information is extremely valuable to health care providers, residents and travelers to the newly identified endemic region to prevent or reduce the risk of disease.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P244

CHARACTERIZATION OF H5N1 AVIAN INFLUENZA VIRUS QUASISPECIES WITH ADAPTIVE HEMAGGLUTININ MUTATIONS FROM SINGLE-VIRUS INFECTIONS OF HUMAN AIRWAY CELLS Yohei Watanabe* 1, 2, Yasuha Arai1, 2, Madiha S. Ibrahim3, Tatsuo Shioda1, Yasuo Suzuki4, Takaaki Nakaya2 1Department of Viral Infection, Research Institute for Microbial Diseases, Osaka University, Osaka, 2Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt, 4College of Life and Health Sciences, Chubu University, Kasugai, Japan

Abstract: Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we comprehensively analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in alpha2,6-linked sialic acid binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational changes for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged, but could not outcompete variants with less HA thermostability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results have implications for understanding the mechanism(s) of AI virus adaptation to infect humans and for providing a clearer perspective both on AI virus adaptation to infect humans and on pandemic control strategies.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P245

DEVELOPMENT AND QUALIFICATION OF REAL-TIME RT-PCR ASSAYS FOR THE DETECTION AND DISCRIMINATION OF INFLUENZA B VICTORIA LINEAGE K162N163 DELETION VIRUSES Bo Shu* 1, Janna Murray1, Kai-Hui Wu1, Ji Liu1, Christine Warnes1, LaShondra Berman1, Shannon Emery1, Rebecca Garten1, David Wentworth 1, Stephen Lindstrom1 1Influenza Division,, Centers for Disease Control and Prevention, Atlanta, United States

Abstract: Influenza B viruses are divided into two lineages, B/Victoria/2/87-like (VIC) and B/Yamagata/16/88-like (YAM) that co-circulate annually. In 2016, VIC viruses that were antigenically distinct from the WHO recommended vaccine virus, B/Brisbane/60/2008, were detected in the United States. Genetic analysis of these viruses confirmed a deletion of 6 nucleotides in the hemagglutinin (HA) gene resulting in 2 amino acid (AA) deletions at positions 162 and 163 (K162N163). As of February 2018, viruses with these 2 AA deletions were detected in the Americas and Europe. Also, VIC viruses with a 3 AA deletion (K162N163D164) were detected in China and Laos in 2017. Here, real-time RT-PCR (rRT-PCR) assays targeting the deletion region of the HA gene were designed to detect and differentiate VIC K162N163 deletion viruses (VIC_KN_Del) and other VIC viruses without the deletion (VIC_No_Del), respectively. Analytical performance studies demonstrated the sensitivity of VIC_KN_Del and VIC_No_Del assays to be comparable to that of the influenza B typing (InfB) and genotyping (VIC) assays of the CDC Influenza Virus Real-Time RT-PCR Panel- Influenza B Lineage Genotyping Panel. Cross reactivity was not observed with the VIC_KN_Del assay against VIC viruses without the deletion mutation, nor with the VIC_No_Del assay against VIC K162N163 deletion viruses. Also, both assays did not react with VIC K162N163D164 deletion and YAM viruses. The CDC VIC_KN_Del and VIC_No_Del rRT-PCR assays described here are intended to be used in conjunction with CDC Influenza B Lineage Genotyping Panel to serve as a method for identification of VIC K162N163 deletion viruses from other currently circulating influenza B viruses. The CDC Influenza B/Victoria HA Gene Deletion Panel is available for WHO National Influenza Centers from the CDC International Reagent Resource.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P246

PERFORMANCE EVALUATION OF THE UPDATED CDC REAL-TIME RT-PCR INFLUENZA B LINEAGE GENOTYPING PANEL Kai-Hui Wu* 1, Ji Liu1, Janna Murray1, Bo Shu1, Christine Warnes1, David Wentworth1, Stephen Lindstrom1 1INFLUENZA DIVISION, CENTERS FOR DISEASE CONTROL AND PREVENTION, Atlanta, United States

Abstract: The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel-Influenza B Lineage Genotyping Kit is a real- time RT-PCR(rRT-PCR)assay designed for discrimination of influenza B/Victoria(VIC)and B/Yamagata(YAM)lineage viruses. Recently circulating influenza B viruses have acquired mutations in the oligonucleotide primer and probe regions of the hamagglutinin gene that require modification of the VIC and YAM assays to ensure comprehensive detecion. Analytical sensntivity was eavluated by demonstrating the limit of detection(LOD)of the B genotyping assays against benchmark strains, B/Nevada/03/2011(VIC)and B/Texas/06/2011(YAM), as well as recent strains, B/Maryland/15/2016(VIC)and B/Texas/81/2016(YAM). Viral RNAs were 5-fold serially diluted and each dilution tested in triplicate. Twenty influenza B viruses representing different geographic locations were tested at concentration near the established LOD to demonstrate inclusivity of the assays. Assay specificity was demonstrated by testing influenza B viruses of different genotypes, as well as human and animal influenza A viruses and other common non-influenza human respiratory viruses. The LOD for the updated assays were calculated to be the lowest concentration of influenza virus(EID50/mL)at 1.0 1.7 1.5 which al replicates tested positive. The LOD for the VIC assay was 10 -10 EID50/mL, while that for YAM assay was 10 - 3.5 10 EID50/mL. The VIC and YAM assays correctly detected twenty viruses near their established LOD and demonstrated no cross reactivity with influenza B viruses of the other genotype at higher titer. Exclusivity testing further showed that the updated VIC and YAM assays did not react with sixteen non-influenza human respiratory viruses. Analytical evaluation of the updated CDC Influenza B Lineage Genotyping Kit demonstrated improved ability to detect recent circulating influenza B viruses.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P247

DIFFERENT EFFECTS OF TWO MUTATIONS ON THE INFECTIVITY OF EBOLA VIRUS GLYCOPROTEIN IN NINE MAMMALIAN SPECIES Yohei Kurosaki* 1, Mahoko Takahashi Ueda2, Yusuke Nakano3, Jiro Yasuda1, Yoehio Koyanagi4, Kei Sato3, 4, So Nakagawa2, 5 1Institute of Tropical Medicine, Nagasaki University, Nagasaki, 2Micro/Nano Technology Center, Tokai University, Hiratsuka, 3Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 4Department of Infectious Disease Control, Institute of Medical Science, The University of Tokyo, Tokyo, 5Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan

Abstract: Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, whereas Reston virus (RESTV) causes lethal disease only in non-human primates. The envelope glycoprotein (GP) is responsible for virus entry to host cells. We previously identified two codon sites under positive selection in EBOV GP, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014–2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, the A82V mutation in Makona GP caused a small increase in viral infectivity compared with the T544I mutation in human cell lines. These findings suggest that mutation fixation in EBOV GP may be associated with their increased infectivity levels. However, it remains unclear how these two amino acid residues affect the infectivity of Ebolavirus species in various hosts. We examined infectivity of EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species using pseudotyped virus. I544/545 increases viral infectivity in all host species, whereas V82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann–Pick C1. Our findings indicate that the effect of the V82/83 residue on viral infectivity differs among both Ebolavirus and host species, whereas the residue I544/545 of EBOV and RESTV GPs increases viral infectivity in various host species.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P248

STRUCTURAL BASIS FOR DIVERGENT RECEPTOR TROPISM OF EMERGING UNCLASSIFIED PARAMYXOVIRUSES Alice Stelfox* 1, Ilona Rissanen2, Thomas Bowden1 1Division of Structural Biology, UNIVERSITY OF OXFORD, Oxford, United Kingdom, 2Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

Abstract: Paramyxoviruses that do not classify into any of the established paramyxovirus genera continue to emerge worldwide. The initial stage of viral entry into a host cell is facilitated by the ‘G’ glycoprotein displayed on the surface of these so called ‘orphan paramyxoviruses’. The molecular specificity underlying this recognition event is a key determinant of cell and species tropism, however, the high level of sequence diversity amongst paramyxoviral attachment proteins impedes predictions of receptor usage from amino acid sequence alone. We have previously demonstrated structure-based analyses as a powerful tool to probe paramyxoviral glycoprotein functionality, and sought to utilize X-ray crystallography to structurally classify orphan paramyxoviral glycoproteins. Here, we report crystal structures of the attachment glycoprotein structures from Mossman, Nariva, beilong and J paramyxoviruses (MosPV, NarPV, BeiPV and JPV, respectively), and reveal that these glycoproteins structurally categorise independently from established paramyxoviral hemagglutinin-neuraminidase (HN), hemagglutinin (H), and G glycoprotein architectures. Molecular rationale for independent host receptor usage is provided through comparative analysis with the receptor binding sites of well-characterised paramyxoviruses (e.g. Nipah virus, measles virus, mumps virus). Our progress investigating how these observations impact the unique receptor-mediated entry pathways is also presented. These data expand our appreciation of the diversity of paramyxoviral receptor-binding architectures and indicate new paradigms for paramyxovirus host-cell recognition and entry.

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EMERGING AND EVOLVING – viral discovery, emergence and evolution Abstract final identifier: P249

SOMATIC REPROGRAMMING IN PTEROPUS BAT CELLS TO DEVELOP NEW MODEL TO STUDY HIGHLY PATHOGENIC VIRUSES Noémie Aurine* 1, Julien Fouret2, Clémence Kress3, Christian Jean3, Catherine Legras-lachuer2, Branka Horvat1, Bertrand Pain3 1CIRI, INSERM, Lyon, 2ViroScan3D, Trévoux, 3SBRI, INRA, Bron, France

Abstract: Bats are natural reservoirs for a large range of emerging viruses that cause lethal diseases in human and domestic animals, but remain asymptomatic in bats. Among bats, Pteropus are reservoir of Nipah virus (NiV), a highly pathogenic zoonotic virus. NiV causes regular outbreaks in human causing acute encephalitis and pneumonia with high mortality. Understanding the host reservoir-pathogen interactions relies on the availability of relevant models such as susceptible cell types, likely to be infected in natural context. However, up to date, most of the studies use immortalized primary cells that are not natural target of the virus. In order to obtain more relevant cell phenotypes, the somatic reprogramming approach was applied to various Pteropus primary cells as initial substrates. The reprogrammed cells, first developed in murine and human models, have a capacity for self-renewal and differentiation in different cell lineages. Using a novel combination of 3 reprograming factors, we have generated new lines of reprogrammed cells exhibiting stem cells features. We characterized these cells at transcriptomic level and demonstrated their very high susceptibility to NiV (up to 90%) compared with the low level of infection of the initial primary bat cells. The development of this original model opens new perspectives to study virus-host interactions by identifying factors controlling either susceptibility or restriction to infection by NiV and other highly pathogenic viruses hosted by bats.

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Abstract Authors Index

Arai Yasuha P226,P244 Abdullah Nurshariza P003 Arikawa Jiro P240 Abelson Dafna P160,P214 Arnold Catherine P070 Aboellail Tawfik P224 Arora Prerna P196 Abou Hamdan Abbas P013 Aslam Sadaf 30 Ackermann Mark P200 Asogun Danny P133 Adams Robert 83 Attia Zayed P195 Adiyadorj Dolgorkhand P223 Aurine Noémie 88,P249 Agans Krystal P160 Avanzato Victoria P218 Aguilar Hector 8 Avšič Županc Tatjana P150,P151 Ahlm Clas 41,P148 Awano Mutsumi P137 Albariño César P172,P210 Ayaz Sameer P186 Albe Joseph 64 Ayllón Juan 30 Albertini Aurélie 4,P013 Azarm Kristopher P009,P021 Albornoz Amelina P149 Baasandagwa Uyanga P223 Alcos Angelita P169 Babusis Darius 70 Alfonso Jose 65 Bach S. 18 Alfson Kendra 59 Badham Matthew P116 Alkutkar Tanwee 5 Bailey Dalan P003,P173,P204 Allee Chantal P154 Baillet Nicolas P181 Allée Chantal P142 Baillie J. Kenneth P076 Allen Elizabeth P191,P205 Baillon Laury 28 Allen Joel P215 Baize Sylvain P138,P181,P183 Alnajjar Sarhad P200 Bakkers Mark P016 Altamura Louis P070 Bakkum-Gamez Jamie P031 Alter Galit P160 Balasubramaniam Vinod 30 Altmeyer Ralf P039 Ballif Bryan 48,P112 Amagai Yosuke P137 Bamford Connor G.G. P153,P222 Amarasinghe Gaya 50,P030 Bankwitz Dortothea P020 Amelot Michel P142 Bannister Roy 70 An Dong Sung P203 Barclay Wendy 20,71,P091 Anantpadma Manu 17 Barker Sarah 6 Anchisi Stéphanie P206 Barnes J P164 Andersen Kristian P088 Barr John P022 Anderson Danielle 69,P219 Barrett Chelsea 7 Andrade Jorge P078 Basler Christopher 17,P073 Andrejeva Jelena P072 Batra Jyoti 17 Andrews S 76 Battles Michael P200 Anhlan Darisuren P111 Baud Damien P143 Aquino Dolly 61 Bauer David 66,P056,P066,P Aragão David P073 115 Arai Tetsuro P139 Baumeister Stefan P036 Bavari Sina 70,P174

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Bavro Vassiliy P003 Blohm Ulrike P198 Bazhenova Ekaterina P207 Blondel Danielle 46 Bdeir Najat P196 Blouquit-Laye Sabine P039 Bearss Jeremy P140 Bloyet Louis-Marie 24,88,P038,P058 Becker Jan P193 Boal Carvalho Ines 36 Becker S. 18 Bockholt Sabrina P133 Becker Stephan 31,P036,P045,P Boczkowska Beata 59 047,P065,P113, Boeren Marlies P173 P176 Bogdanow Boris P059 Beer Martin 1,90,P202 Bogovič Petra P151 Behner Laura P065,P110,P238 Bolder Renske P189 Bell Todd P140 Bonaventure Boris 37 Belot Laura 4,P013 Bondugula Pavani 80 Belser Jessica P136 Boon Adrianus 19,50 Benitez Asiel Arturo 30,P078 Börgeling Yvonne P111 Benner Chris P068 Bornholdt Zachary P160,P214 Bennett Richard P012 Botten Jason 47,48,P112 Benton Donald 3 Bouillier Camille P039 Bergeron Éric 40,P131,P132,P Bourhis Jean-Marie P026 210 Berghöfer Susanne P113 Bousse Tatiana P207 Berman LaShondra P245 Bouzya Badiaa P199 Bernabé Alejandro 65 Bowden Thomas 10,74,P009,P018 ,P019,P021,P20 Bernasconi Valentina P193 5,P191,P215,P2 Bernbaum John P102 18,P248 Bertin Nicolas P076 Boyaka Prosper P195 Bertran Andre P046 Bragagnolo Gabriel 26 Best Sonja P165 Brakenhoff Just 75 Bharrhan Sushma 6 Brandt Janine P165 Bhatia Sangeeta P102 Brechbühl Daniel P005 Bhella David 43 Brémont Michel 28 Biacchesi Stéphane 28 Brennan Ben P191 Biedenkopf Nadine 18,P036,P047 Brennan Benjamin P042 Biewener Valerie P131 Briand François- P154 Bignon Eduardo 9 Xavier Binder Florian P236 Bright Helen 81,P186,P187 Binder Marco 35 Brinkmann Constantin P065,P093 Birch Jamie P003 Brocato Rebecca P149 Björkström Niklas 41 Brock Nicole P136 Blackledge Martin 44 Brock Nicole 80 Blanchard Yannick P142 Brodsky Leonid P067 Blanco Jorge P189 Brönstrup Mark P213 Blanco-Melo Daniel 30 Brooke Chris 26 Blest Henry P022 Brown Michael 91 Blockus Sebastian P020,P213 Brown Paul P142

337

Bruce Emily 47,48,P112 Chatterjee Payel P210 Bruchez Anna P071 Cheloha Ross P175 Brummelkamp Thijn 6 Chen Cindy P078 Brunet Jérémy P188 Chen Hui-Wen P168 Brunotte Linda 35 Chen Li P071 Brunton Bethany P031 Chen Wei P059 Bryant Neil P134 Chen You-Ting P168 Buchholz Ursula Chen Yu-Chi P165 Buchholz Ursula 79,91,P185 Chesnokov A P164 Buehler Eugen P165 Chevalier C P152 Bukreyev Alexander 32 Chevalier Christophe P145 Burt Dave P117 Chevret Didier 28 Burton Dennis P191,P205 Chey Soroth P241 Bush Carola P201 Chiodelli Paola P014 Butler Noah P079 Chiramel Abhilash P165 Byrd-Leotis Lauren P004 Chitadze Nazibrola P234 Caballero Ignacio P102 Christen Michael 49 Cabot M P001 Chung Liliane P084 Cadagan Richard P108 Cihlar Tomas 70 Calder Lesley 3 Ciminski Kevin 1,90 Calderon Brenda 89 Clohisey Sara P076 Caljon Guy P173,P197 Coleman-McCray JoAnn P132,P172 Calle Daniel 65 Collignon Brad P225 Callendret Benoit P189 Collins Peter 79,91,P185 Callery Jessica P163 Comeau André P155 Camara Alimou P239 Conceicao Carina P117 Camarasa Maria José P217 Connell James P126 Cambier Seppe P014 Connolly- Anne-Marie P148 Cantoni Diego P081 Andersen Cao Dongdong P029 Connor John P133 Cao Jing Jing P039 Conzelmann Karl-Klaus 27,P130 Cardone Christophe P027,P055 Cooper Shaun 81 Carnec Xavier P181,P183 Cop Christian 65 Carninci Piero P076 Corti Davide 3 Caroline Picard P183 Cos Paul P173,P197 Carrion Ricardo P163 Courtillon Céline P142 Cashman Kathleen P140 Courtois David P142 Cattaneo Roberto 2,25,P067 Craig Kelsey P195 Chaimayo Chutikarn P063 Crepin Thibaut P026 Chain Patrick P227 Crépin Thibaut P040 Chakhunashvili Giorgi P234,P243 Cressey Tessa 22 Chanda Sumit P068,P069 Crispin Max P215,P218 Chandran Kartik 5,6,P006,P017,P Cross Robert P160 160,P214 Crossland Nicholas P153 Chang Max P068 Crowe Jonathan 81 Chanturia Gvantsa P234,P243 Cumella José P217 Chapman David P186 Cummings Richard P004

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NSV 2018, Verona – Abstract Book

Cusack Stephen P033 Digard Paul P076,P084,P117 Cusi Maria P092 Dijkman Ronald 36,P061,P213,P Da Costa Bruno P040,P145,P152 231 Dadonaite Bernadeta P115 Dina Julia P229 Dai Meiling P228 Diskin Ron 82 Danet Nicolas P097 Dolnik Olga P113 Dang Loan P112 Donchet Amélie P026 Danzy Shamika 89 Donohue Ryan 2,25 Dautu George P240 Donohue Ryan P067 Davey Robert 17,P126 Doores Katie 74,P191,P205 Davis C. Todd 80 Doyle Tomas 37 Davison Andrew 23 Drewes Stephan P236 de Castro Sonia P217 Drijver Joke P189 de Haan C. A. M. P228 Drosten Christian P131 De Jesus Paul P068 Du Wenjuan P228 de La Vega Marc Antoine P160 Ducatez Mariette P026 de Swart Rik 55,60,P157 Dugan Vivien 80,P207 De Vlieger Dorien P194 Dumont Claire 88 de Vries Erik P228 Dunagan Megan P063 de Vries Rory 55,60,P157 Dunham Eric P165,P167 de Wit Emmie 66 Duprex W. Paul 55,P049,P153,P 213,P219,P222 Debeljak Franka 39 Dedushaj Jusuf P151 Dupuy Lesley P161 Del Mar Maria P141 Duraffour Sophie P150 Fernandez de Dutch Rebecca 7 Marco Dutia Bernadette P084 Delgado Jennifer 59 Dutta Mukta 32 Delmas Bernard P026,P040,P145 Dye John 6,P160,P214 ,P152 Ebert Karolina P193 Delp Korey P223 Edwards Megan P073 Delputte Peter P173,P197 Ehlert Birthe P118 Deng Tao P107 Eickmann Markus P176 Desco Manuel 65 Eisenhauer Philip 47,48 Desfosses Ambroises 44 Eizinger Maximilian 27,P130 Deval Jerome 22 El Omari Kamel P018 Devignot Stéphanie P089 Eléouët Jean- P027,P028,P039 Di Han 80 Francois ,P051,P055,P21 Diallo Seme P220 3 Diamond Michael 19 Eletto Davide 1 Diaz Menendez Marta P174 Elliott Angela P191 Diaz-Colunga Juan 33 Elliott Richard P191 Dibben Oliver 81,P186,P187 Elsie Yekwe P183 Diederich Sandra P198,P239 Elson Andrew P192 Diehl WE P001 Elton Debra P134 Dietzel Erik P176 Embury-Hyatt Carissa P225

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Emery Shannon P245 Forwood Jade P073 Emory Kirsty P141 Fouchier Ron P041 Enchery François 88 Fouret Julien P249 Engler Olivier P007,P008,P182 Fraaij Pieter 60,P157 Escriou Nicolas P188 Francois Patrice P143 Escudero Pérez Beatriz P129 François Ferron P183 Esnault Evelyne P145 Frenz Theresa P213 Esneau Camille P027 Fritzsche Wolfgang P236 Espy Nicole P174 Fu Yuguang P127 Esser Mark P220 Fujikura Daisuke P010 Esumeh Rita P147 Fujisaki Seiichiro P177,P180 Eterradossi Nicolas P142,P154 Fujiyuki Tomoko P124,P137 Eto Yoshiki P240 Fukushi Shuetsu P240 Facemire Paul P140 Fukuyama Satoshi 68 Falcon Ana 33,65 Fuller Jack P022 Falynskova Irina P166 Furmanova- Polina 75,P200 Fan Haitian 14,P056 Hollenstein Fan Rebecca 66 Furuta Yousuke P171 Fay Elizabeth 42 Furuyama Wakako P010 Fearns Rachel 22 Gabriel Martin P150 Fedeli Chiara 11 Gad Hans Henrik 56,P193 Fehling Sarah P118,P159 Gaglia Marta 34 Feldmann Friederike P128 Gago Federico P217 Feldmann Heinz P010,P128,P165 Gaillard Vanessa P028 ,P167,P171 Galao Rui 39,P075 Fels J. Maximilian 6,P214 Galinski Mary P079 Feneant L P001 Galloux Marie P027,P028,P039 Fernandes Fiona P220 ,P055 Fernández García Yaiza P201 Gamblin Steven 3 Ferren Marion 61 Gandolfo Claudia P092 Fiege Jessica 42,58 Ganser-Pornillos B P001 Filgueiras-Rama David 65 Ganti Ketaki 89 Finke Stefan 49,P121,P156,P Gao Chao P004 165 Gao Yunrong P029 Fischer Kerstin P239 Garcia Bea P098,P237 Fischer Stefan P236 García-Sastre Adolfo 1,19,30,P068,P0 Fitzpatrick Elizabeth P085 69,P108,P228 Fix Jenna P055 Garcin Dominique 56 Fizet Alexandra P181 Garten Rebecca P164,P245 Flamand Marie P178 Gärtner Sabine P196 Fletcher Robert 71 Gast Matthieu P122,P123 Flint Mike P210 Gaucherand Lea 34 Fodor Ervin 14,66,P023,P034 Gaudin Yves 4,46,P013 ,P056,P066,P11 Geffers Robert 62 5 Geisbert Joan P160 Fontana Juan P022 Geisbert Thomas P160 Fooks Anthony P141 Gellhorn Serra Michelle P176 Forst Christian 30

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Gellman Sam P175 Grosserichter- Christina P157 Generous Alex 2 Wagener Georgeault Sonia P145 Grothe Stephanie P111 Gerhardt Dawn P012 Grünweller A. 18 Gerlier Denis 88,P038,P058 Gu Quan P032 Gerlt Vanessa 35 Gu Se Hun P227 Gerold Gisa 11 Guajardo- Gabriel P108 Ghanem Alexander P130 Contreras Ghedin Elodie 54 Guardado-Calvo Pablo 9 Giavedoni Luis P163 Gubareva L P164 Gilbert Robert 10 Guenther Stephan P033 Gillich Nadine P105 Guillemoto Carole P154 Gilman morgan 75 Guillou-Cloarec Cécile P154 Gilmore Jamie P114 Guito Jonathan P070 Gingerich Aaron P190 Gumpper Ryan P024 Gloaguen Emilie P138 Gunn Bronwyn P160 Gogrefe Nadja P033 Günther Maria P121 Goldfarb Daniel 43 Günther Sira 1 Goldhill Daniel 71 Günther Stephan P133,P147,P150 ,P201 Gomba Laura 70 Gunzer Matthias 68 Gómez Medina Sergio P129 Gupta Shawon 41 Gonçalves- Daniel P003 Gusovsky Fabian 32 Carneiro González- Mariana P065 Gustafsson Rasmus P148 Hernández Gutiérrez Celia 65 Goodbourn Steve 23,P072 Gutsche Irina 44 Goodwin Eileen P160 Habjan Matthias P083,P103 Gori Savellini Gianni P092 Haddock Elaine P128,P171 Gorka Marco 90 Haid Sibylle 62,P020,P213 Goujon Caroline 37,P091 Hale Benjamin 1 Gould Joseph P057 Halldorsson Steinar P191 Gowen Brian P171 Halwe Sandro P036 Graham Stephen P003 Hamane Kory P203 Graham Victoria P191,P205 Han Julianna P078 Green Todd P057 Hang’ombe Bernard P240 Greninger Alexander 61 Hansen Gesine 62,P213 Greune Lilo P111 Hansson Magnus P148 Gribbon Philip P201 Hara Koyu P052 Griffin Diane 83 Harder Olivia P170,P216 Griffiths Anthony 59,P054,P163 Harlos Karl 10,P191,P218 Grimes Jonathan 14,P034,P048 Harmon Jessica P132,P172 Groschup Martin P239 Harrison Oliver 2 Groschup Marting P198 Harrison Stephen P050 Groseth Allison P165,P167 Hartman Amy 64,P125 Hartmann Kristin P129

341

Hartmann R. 18 Hoffmann Markus P015,P065,P093 Hartmann Rune 56,P193 Hogg Alison 70 Hassan Ebrahim 90 Hologne Maggy P058 Hatchette Todd P155 Holwerda Melle P061,P231 Hawes Pippa P158 Honce Rebekah 54 Hawksworth Amy 81 Honda Tomoyuki P098 Hawman David P171 Hönemann Mario P241 Hayashizaki Yoshihide P076 Hong Patrick P203 He Biao P043,P192 Honig Barry 2 He Chuan 16 Hooper Jay P149 He Jingyang P195 Horie Masayuki P044,P105,P237 He Shihua P160 Horie Ryo P090 Heaton Michael P003 Horton Dan P141 Heckel Gerald P236 Horvat Branka 88,P249 Heemskerk-van Marjolein P189 Horwitz Joshua P050 der Meer Hoshi Miho P096 Heikel Gregory P084 Hosie Margaret P003 Heimburg- Jamie P004 Hossain Jaber 80 Molinaro Hou Jingyi P059 Heiner Anja P110 Hover Samantha P022 Heins Gudrun P059 Hu Che-Ming P168 Heintz Veronica P126 Hua Brian 40,P131 Hengel Hartmut P194 Huang Xiaoxia P221 Hengrung Narin 14,P034 Huertas-Diaz Maria P192 Hennrich Alexandru P130 Hughes David P072 Hensley Lisa P012 Huiskonen Juha 10,P019,P191,P Hepojoki Jussi 10 215 Herbert Andrew 6,P160 Hulsey Stubbs Sarah P016,P179 Herden Christiane P176 Hultquist Judd 17,P068 Hernandez-Triana Luis P141 Hume David P076 Herold Susanne 67 Humoli Isme P151 Herren Michael P011 Hunter Laura P141 Herrler Georg P015,P064,P127 Huong Tra P106 Hertz Emil P047 Husic Immanuel P059 Hewson Roger P022,P141,P191 Hutchinson Edward 43,P023,P032 ,P205 Hüttl Sarah P015 Hicks Dan P141 Hwang Soon-Bong P087,P099 Hidekazu Nishimura P242 Iampietro Mathieu 32 Hiebert Joanne P232 Ibrahim Madiha P226,P244 Hill Laura 81 Ichinohe Takeshi P080,P086,P162 Hirai Yuya 15 Igarashi Manabu P010,P209 Ho Wenkae Brian P219 Ighodalo Yemisi P147 Hodara Vida P163 Ikeda Fusako P096,P139 Hodges Erin 80 Iketani Sho 61 Hoenen Thomas P165,P167,P239 Imamura Takeaki P144 Hoffmann Donata 90,P202 Imnadze Paata P234,P243 Hoffmann Katja P194 Indra Indrajyoti 2

342

NSV 2018, Verona – Abstract Book

Indrasari Setyarina P144 Jourdain Marine P181 Iqbal Munir P117 Journo Chloe 88 Irie Takashi P120 Jovan Nikolic 4 Ishijima Mari P209 Joyner Chester P079 Ito Morihiro P002 Junglen Sandra P131 Ito Mutsumi P135 Kaderali Lars 62 Iwatsuki- Kiyoko P144 Kadzioch Nicole P122,P123 Horimoto Kageyama Tsutomu P208 Jabaty Juliet P239 Kai Chieko P053,P090,P096 Jacobs Nathan 21 ,P124,P137,P13 Jae Lucas 6 9,P211 Jaensch Steffen P200 Kainulainen Markus P172 Jahrling Peter P012 Kajihara Masahiro P240 Jakupi Xhevat P151 Kandasamy Matheswaran P078 Jalife Jose 65 Kant Jet 57,73 James Rebekah P160 Kapczynski Darrell P117 Jamme F P152 Karakus Umut 1 Jamsransuren Dulamjav P223 Karlsson Erik 54 Jang Gwendolyn 17 Karron Ruth 79 Jang Yunho 80 Kashiwagi Takahito P052 Jangra Rohit 5,6,P006,P017 Kasloff Samantha P225 Jasim Seema P117 Katsura Hiroaki 68 Jean Christian P249 Katz Aliza 82 Jee Youngmee P087,P099 Katz J P164 Jegaskanda S 76 Katze Michael 32 Jennings Ryan P195 Kawabata Ryoko P120 Jensen Malene 44 Kawaguchi Atsushi P060,P101 Jeong Seong Tae P227 Kawaoka Yoshihiro 45,68,77,P035,P Jia Nan P004 135,P144,P177 Jigjav Battsetseg P223 Keizo Tomonaga 87 Jimenez-Morales David P068 Kelly James P204 Jimmy Hortion P183 Kelly Jamie P003 Jin Hong P220 Ken Maeda 87 Johnson Adam 80 Keogh Elissa 75 Johnson Joshua P071 Keown Jeremy 14 Johnson Nicholas P141 Kepler Tom 63 Johnson Rebecca 86 Kerber Romy P150 Johnson Reed P102 Kesari Aditi P126 Jones Harrison 75 Khamaikawin Wannisa P203 Jones Joyce 80 Khaperskyy Denys 34,P155 Jonsdottir Hulda P061 Killip Marian 66 Jonsson Colleen P085 Kim Heung-Chul P227 Jordan Paul 22 Kim Do P160 Jordan Robert 70 Kim Dong-Yun P237 Jorquera P P164 Kim Jeong-Ah P227

343

Kim Preston 50 Krumkamp Ralf P150 Kim So Young P219 Krumm Stefanie 74,P191,P205 Kim Won-Keun P227 Kruse Thomas P047 King Benjamin 47,48 Kuehne Ana 6,P214 Kiseleva Irina P207 Kuhn Jens 84 Kishida Noriko P177,P180 Kujawa Michael 64 Kiso Maki 77,P135 Kunz Stefan 11,85,P007,P008 Klaus Joseph 48 ,P182 kleer Mariel P155 Kupke Alexandra P176 Klein Terry P227 Kurosaki Yohei P247 Kleinfelter Lara 6,P017 Kutluay Sebla 50 Klena John P172 Kuwahara Tomoko P177,P180 Klingstrom Jonas 41 Kuzikov Maria P201 Klingström Jonas P037 Kuznetsova Irina 67 Klinkhammer Jonas 56 Kwon Hyun-jeong P124 Kobasa Darwyn P225 Lackenby Angie 71 Koboyashi Yuki 87 LaCount Douglas P126 Kochektova Irina 2 Lacy Martha P031 Kock Ricahrd P158 Lacy-Hulbert Adam P071 Koehler Alexander P045 Laederach Alain P115 Koehler Jeffrey P223 Lagaudrière- Cécile 46 Gesbert Kohl Alain P042,P178 Kojima Shohei P044,P105,P237 Lakdawala Seema 52 Kolesnikova Larissa P036,P045,P110 Laksono Brigitta 60,P157 Komatsu Yumiko P044 Lamoureux Emily P155 Komeno Takashi P171 Lanckacker Ellen 75 Kondoh Tatsunari P010 Langat Pinky 71 Koopmans Marion 60,P157 Langedijk Johannes 75,P200 Kormelink Richard P046 Langer Klaus P111 Kortekaas Jeroen 57,73 Langlois Ryan 42,58 Korva Misa P150,P151 Laouénan Cedric P138 Kotaria Nato P234 Laporte Manon P014,P217 Koup Richard 32 Larionova Natalie P207 Koyanagi Yoehio P247 Lassoued Safa P055 Kozuka-Hata Hiroko P035 Lau Simone P103 Krähling Verena 31,P036,P065 Lauber Chris 62 Krämer-Kühl Annika P093 Lazert Carine P038,P058 Krammer Florian 30,P199 Le Bars Romain 46 Krasemann Susanne P129 Le Goffic Ronan P028,P040,P145 ,P152 Kreher Felix P178 Le Nouen Cyril 91,P185 Krementsov Dimitry 48 Le Prioux Aurélie P154 Krempl Christine P020 Lebras Marie-Odile P154 Kress Clémence P249 Ledwith Mitch 19 Krey Thomas P020 Lee Benhur P009,P021,P203 Krischuns Tim 35 Lee Daesang P227 Krogan Nevan 17,P068 Lee Ilseob 30 Krüger Nadine P015,P065,P127

344

NSV 2018, Verona – Abstract Book

Lee Jinhwa 90,P224 Liu Guanqun P062 Lee Joo-Yeon P087,P099 Liu Hui P220 Lee SangJoon P060,P101 Liu Ji P245,P246 Lee Seung-Ho P227 Liu Jun P233 Lee Yoontae P078 Liu Lin P221 Leemans Annelies P173,P197 Liu Qiang P062 Legrand Pierre 4 Liu Shan-Lu P195 Legras-lachuer Catherine P249 Liu Xiaoshu P098 Lejal Nathalie P040 Liu Yang P221,P233 Lelek Mickael P039 Livingston Brandi 54 Lemaitre Evelyne P142 Ljunggren Hans-Gustaf 41 Lemos Xenia P232 Lobel Leslie P214 Leneva Irina P166 Locher Samira P005 León Daniel 65 Lockhart Robert 81 Leroux Aurélie P142 Logan Leanne P204 Leske Anne P165,P167 Logan Nicola P003 Lessoued Sana 26 Londrigan Sarah 38 Letko Michael P010 Long Jason 20 Leung Anders P225 Long Joshua 66 Leung Daisy 29 Longhi Sonia P038,P058 Leymarie Olivier P145,P152 Lopes Tiago 68 Leyrat Cédric P048 Lopez Lastra Marcelo P100 Li Anzhong 16,P195 Lorin Valérie P188 Li Aqian P221,P233 Lovett Sean P070 Li Chuan P221 Lowen Anice 21,89 Li Dexin P221,P233 Lozach Pierre-Yves P182 Li Jianrong 16,P195,P216 Lu Bin P220 Li Rong 6 Lu Mijia 16,P195,P216 Li Sai 10,P019,P215 Lu Yao P062 Li Weike P024 Lu Zhike 16 Li Yan P078,P235 Lubaki Ndongala 32 Liang Bo P029 Luban Jeremy P001 Liang Mifang P221,P233 Lübke Anastasia P093 Liang Xueya 16,P195,P216 Lucas Pierrick P142 Liebert U.G. P241 Ludwig Alexander P106 Liekens Sandra P014 Ludwig Stephan 35,P111 Light Yooli P212 Lumley Sarah P141 Lim Xiao Fang P219 Luo Ming P024,P043 Lin Che P211 Luongo Cindy 79,91,P185 Lin Wen-Hsuan 83 Lycett Samantha P084,P117 Lin Xudong 80 Lycke Nils P193 Linder Aline 88 Lythgoe Katrina P003 Lindstrom Stephen P245,P246 Ma Jingjiao P224 Lipkin W. Ian P179 Ma Wenjun 90,P224

345

Ma Yuanmei 16,P195 Mathieu Cyrille 61,88 Macchietto Marissa 42 Matsumoto Yusuke P094,P095 Mackman Richard 70 Matsunaga Yui P211 MacRae Cara P155 Maurer Daniel P214 Maes Louis P173,P197 Maury Wendy P079 Mahapatra Mana P158 Mawhorter Michael 22 Mahlakõiv Tanel 56 May Jürgen P150 Maines Taronna 80,P136 Mayor Jennifer P007,P008,P182 Maisner Andrea P065,P110,P198 Mazel-Sanchez Béryl 36,P143 ,P238 Mazur Magdalena P046 Maisonnasse P P152 Mazur Steve P102 Makadiya Nirajkumar P195 McAlpine Sarah P155 Makhmudova Nailya P166 McCarty Thomas 91 Makhsous Negar 61 McCormick Craig 34,P155 Makino Akiko 15,P105 McDermott A 76 Maleev Victor P166 McElroy Anita P210 Maleki Kimia 41 McGill Andrew P170 Malim Michael 37 McKellar Joe P091 Mallett Corey P199 McLellan Jason 75,P200 Mallory Raburn 81 McMillen Cynthia P125 Malmlov Ashley P224 McMullan Laura 40 Mancio Silva Liliana P102 Meade-White Kimberly P171 Manhart Whitney P102 Mecate-Zambrano Angeles P111 Manicassamy Balaji P078 Medina Rafael P108 Mankertz Annette 69 Medlock Jolyon P141 Mankouri Jamel P022 Mehle Andrew 19 Mann B P164 Meliopoulos Victoria 54 Manzoor Rashid P010,P184,P209 Mena Ignacio 30 Marcos-Villar Laura 33 Meng Fandan P127 Marklewitz Marco P131 Mérour Emilie 28 Markman Matthew 42 Meyer Annika P118 Marsh Glenn 86 Meyer Léa P145 Martellaro Cynthia P165 Michlewski Gracjan P084 Martenot Claire P154 Milles Sigrid 44 Martin Scott P165 Minogue Timothy P223 Martinet Wim P173 Mirazimi Ali P089 Martínez-Alonso Mónica P066 Mire Chad P160 Martínez-Romero Carles 30,P228 Mishin V P164 Martin-Sancho Laura P068 Mishra Anamika P117 Maruyama Junki P010 Mishra Nischay P179 Marzi Andrea P010 Mishreky Natalie P169 Masayuki Horie 87 Mistry Bhakti 20 Massin Pascale P154 Mitchell Tim P003 Mate Maria 26 Mittler Eva 5,6 Mateo Mathieu 2,P097,P181 Miyamoto Hiroko P010,P184,P209 Matéo Mathieu P183 Miyamoto Sho P114 Mateu-Petit Guaniri 80 Moffat Jason 6

346

NSV 2018, Verona – Abstract Book

Moldenhauer Anna-Sophie P065 Nagata Shiho P208 Momose Fumitaka P119 Nagendra Kartikeya 22 Moncorgé Olivier 37,P091 Nagle Elyse P070 Monreal I Abrrey 8 Nair Naina 43 Montagutelli Xavier P178 Nakagawa So P247 Monze Mwaka P240 Nakamura Cory P225 Moreno Hector 11 Nakamura Kazuya P177,P180 Moreno Borrego Hector 85 Nakanishi Tomoko P124 Mori-Kajihara Akina P240 Nakano Masahiro 45,P114 Morikawa Shigeru P240 Nakano Yusuke P247 Morikawa Yuko P119 Nakao Ryo P240 Moriyama Miyu P080,P086,P162 Nakatsu Yuichiro 92 Moroldo Marco P145 Nakaya Takaaki P226,P244 Moscona Anne 61,P175 Nambulli Sham 55,P049,P153 Mostoslavsky Gustavo P102 Nans Andrea 3 Mottram Timothy P042 Nao Eguchi P184 Moulton Hong P068 Nao Naganori P242 Mu D P001 Navaratnarajah Chanakha 2 Mueller Florian 48 Naylor Clive P142 Mueller Steffen P185 Negredo Anabel P174 Muena Nicolás 6 Negrete Oscar P212 Muhairi Salama Al P235 Nehlmeier Inga P065,P093 Mühlberger Elke P102 Nehls Julia P065 Mukai Yahiro P237 Neil Stuart 39,P075 Mukonka Victor P240 Nekhai Sergei P027 Mullaert Jimmy P138 Nelson Emily P129 Muller Claude 78 Nemirov Kirill P097 Müller Helena P159 Nemitz Sabine 49 Müller Jonas P133,P147 Nersisyan Stepan P067 Müller-Guhl Jürgen P129 Netter Hans 86 Muñoz Alía Miguel Ángel 78 Neumann Gabriele 68 Muñoz-Fontela César P129,P133,P146 Ng Melinda 6 ,P147,P150 Ng Weng P018 Muñoz-Moreno Raquel 30,P228 Nguyen H P164 Munster Vincent P010,P218 Nichol Stuart 40,P049,P131,P Muramoto Yukiko 45,P114 132,P172,P210 Murphy Lita P117 Nidom Reviany V. P144 Murphy Nicole P012 Nidom Chairul P209 Murphy Shona P066 Nidom Chairul.A P144 Murray Janna P245,P246 Nieto Amelia 33,65 Mweene Aaron P240 Niewiesk Stefan P170,P216 Nachbagauer Raffael 30,P199 Nikolic Jovan 46 Naesens Lieve P014,P217 Nilsson Jakob P047 Nagata Kyosuke P060,P101 Niqueux Eric P154

347

Nishida Andrew 32 Pallasch Elisa P133,P146 Nishio Machiko P002,P094,P095 Pan James 6 No Jin Sun P227 Panis Maryline 30 Noda Takeshi 45,P114 Pappas Claudia 80,P136 Nojima Takayuki P066 Parida Satya P158 Nolden Tobias 49 Park Arnold P203 Noppen Sam P014 Park Eun Mee P087 Nordhoff Carolin 35 Park Eunmee P099 Noton Sarah 22 Park Sun Whan P099 Nouchi Takashi P095 Park Sunhye P227 Noujaim Sami 65 Park Sun-Whan P087 Nunez Alejandro P141 Parker Lauren P187 Nuñez Ivette P192 Parodi Laura P163 Nyanguile Origène P028 Parrish Nicholas P098,P237 Nyborg Andrew 81 Pascual Gabriel 75 Odagiri Takato P177,P180,P208 Patterson Jean P012,P163 Odongo L P001 Pauli Noel P214 Oestereich Lisa P133,P146,P147 Pavletič Miša P151 ,P150 Pavlovich Stephanie 63 Ogbaini-Emovon Ephraim P133 Paz Sanchez- Maria P174 Ogor Katell P154 Seco Ohta Keisuke P094,P095 Pearce Nicholas P207 Oishi Kohei P035 Peeples Mark 16,P195,P216 Okumura Atsushi P128 Pegan Scott 40 Oldstone Michael P088 Peng Kah-Whye P031 Olinger Gene P071 Pereira Nelson P055 Oliva-Martin Maria Jose 66 Perez Jasmine P078 Omar Shadia P134 Perez-Sautu Unai P174 Orba Yasuko P240 Pernet Olivier P203 Oreshkova Nadia 73 Peteranderl Christin 67 Ortega Victoria 8 Petit Chad P057 Örvell Claes P015 Pfaffinger Verena 27 Osterhaus Albert 60,P157 Pfaller Christian 2,25,P067 Otth Carola P149 Pfeffermann Kristin 69 Outlaw Victor P175 Pfeiffer Sebastian P045 Oyakhilome Jennifer P147 Phan Shannon P192 Oyama Masaaki P035 Phipps Kara 21 Oymans Judith 57,73 Picard Caroline P181 Ozaki Hiroichi P184 Pichlmair Andreas P083,P103 Packiriswamy Nanda P031 Pickering Brad P225 Padilla Susana P223 Pietrosemoli Natalia P138 Page Audrey P097,P183 Pietschmann Thomas 62,P020,P213 Pahlmann Meike P133 Pino Karla P100 Pain Bertrand P249 Pinschewer Daniel 74 Paki Katharina P059 Pintelon Isabel P173 Palacios Gustavo 63,P070,P174,P Pinto Rute P084 227 Plattet Philippe P011,P122,P123

348

NSV 2018, Verona – Abstract Book

Plemper Richard 69 Reading Patrick 38 Pleschka Stephan 67 Réfrégiers M P152 Pohlmann Anne 90 Reguera Juan 12,P033 Pöhlmann Stefan P065,P093,P196 Reichl Udo P196 Pollpeter Darja 37 Reid St Patrick P097 Poon Leo 66 Reiko Yoshida P184 Poromov Artem P166 Reimann Keith P163 Porotto Matteo 61,P175 Reindl Sophia P033,P201 Port Julia P133,P147 Remaut Han P194 Porter Brittany 34 Renner Max P034,P048 Porter Danielle 70 Rennick Linda 55,P049,P153 Portmann Jasmine P231 Resende Silva Paola P232 Potratz Madlin P156 Resman Rus Katarina P151 Prathyumnan Shibily P092 Rey Félix 9 Prevelige Peter P057 Reyes Ann P126 Prochnow Hans P213 Reynard Stephanie P138,P181 Proudfoot Nick P066 Richard Charles- P027,P039,P051 Pryce Rhys P009,P018,P021 Adrien ,P055 Pulit-Penaloza Joanna P136 Richardson Christopher P169 Punch Emma P022 Rieger Toni P150 Pybus Oliver 10 Rigaux Peter P200 Qiu Shihong P057 Rima Bert 61 Qiu Xiangguo P160 Rima Bertus P222 Qiu Yongjin P240 Rincheval Vincent P027,P039 Quéré Pascale P145 Ringel Marc P110,P238 Quigg-Nicol Marlynne P084 Risalvato Jacquline P043 Qurnianingsih Ema P209 Rissanen Ilona 10,P019,P248 Rada Balazs P190 Ritschel Tina 75 Raghwani Jayna 10,P215 Ritter Lydia 81,P132,P187 Ramanathan Palaniappan 32 Rodriguez Gloria 59 Rambo Robert 14,P009 Rodriguez Estefania P129 Burgos Rameix-Welti Marie-Anne P027,P039,P213 Rodriguez- Estefania P146 Ramírez de Eva P174 Burgos Arellano Rodriguez- Ariel P068 Ran Wei 1,90 Frandsen Randall Elizabeth 23 Rogers Kai P079 Randall Richard 23,P072,P222 Rohde Cornelius 31,P047 Randall Rick 66 Röhrs Susanne P236 Ranjan Punya P210 Roingeard Philippe P145 Rankin Gregory P148 Römer- Angela P165 Rasmussen Angela P128 Oberdörfer Rastrojo Lastras Alberto 85 Ronca Roberto P014 Rauschenberger Bianka P033 Roose Kenny P173 Raut Ashwin P117 Rosenke Kyle P171 Raux Hélène 4

349

Rosenthal Maria P033 Sato Ko P242 Rosenthal Peter 3 Sato Moritoshi 92 Rossman Jeremy P081,P116 Sato Ryo P044 Rothenberger Sylvia P007,P008,P182 Sauerhering Lucie P110,P176,P198 Rottstegge Monika P129,P146 Sawa Hirofumi P240 Rouxel Ronan P199 Sayler Katherine 85 Roymans Dirk 75,P200 Schaeffer Justine P138,P181 Rubin Steven P153 Schepens Bert P173,P194 Rudenko Larisa P207 Schewe Kasia 81 Ruffié Claude P188 Schierhorn Kristina 39,P075 Ruggli Nicolas P005 Schifflett Kyle P167 Ruibal Paula P129,P146 Schindler Michael P065 Ruigrok Rob 44,P026,P040 Schloer Sebastian 35 Ruijs Wilhemina 60,P157 Schlohsarczyk Elfi P236 Ruiz-Cabello Jesús 65 Schlottau Kore P202 Russel Altagracia P041 Schmaljohn Connie P140,P161,P243 Russell Charles 53 Schmidt Alexander P111 Russell Stephen 78,P031 Schmidt Jörg P176 Russier Marion 53 Schmidt Marie P165 Ruzin Alexey P220 Schmitz Audrey P154 Ryou Jungsang P087,P099 Schmolke Mirco 36,P143,P206 Saada Edwin P212 Schnepel Kevin P167 Saasa Ngonda P240 Schnepf Daniel 56,P193 Sachs David 30 Schoehn Guy 44 Sadewasser Anne P059 Schoeniger Joe P212 Saeland Eirikur P189 Schoepp Randal P223,P243 Saelens Xavier P104,P173,P194 Scholte Florine 40,P131,P132 Sahin Mehmet 74 Schountz Tony 90,P224 Saijo Masayuki P240 Schramm Antoine P038,P058 Sakabe Saori P088 Schreiber André P111 Sakaguchi Takemasa P120 Schreyer Jocelyn 20 Sakamoto Kaori P192 Schröder Charlotte P198 Saksida Ana P151 Schuitemaker Hanneke P189 Saliba Emmanuel 62 Schultz-Cherry Stacey 54 Samacoits Aubin 48 Schulz Helene P232 Sanchez-Aparicio Maite P068 Schulz Thomas 62 Sanchez-Lockhart Mariano 63,P070 Schulze Jessica 67 sandin sara P106 Schwaderlapp Marilena 56 Santibanez Sabine 69 Schwedler Jennifer P212 Santos Rodrigo 32 Schwemmle Martin 1,19,90,P196 Santoso Kuncoro P144 Scrima Nathalie 46 Satkamp Laura 17 Seabright Gemma P215 Sato Hiroki P053,P090,P096 Sedano Laura P040 ,P124,P137,P13 Seitz Maren P127 9 Seki Fumio P242 Sato Hironori P180 Selbach Matthias P059 Sato Kei P247 Sellers Scott 70

350

NSV 2018, Verona – Abstract Book

Selvaraj Muneeswaran P158 Sizun Christina P027,P055 Sergeeva Alina 2 Sjaastad Louisa 42 Serna Martin Itziar 14,P034 Skehel John 3 Severini Alberto P232 Slaine Patrick P155 Sha Ky P071 Sloan Elizabeth 43,P032 Shams-Eldin Hosam P176 Slough Megan 6,P006 Shan Chao P195 Smith Greg P225 Shang Qiao P057 Smith Kate P073 Shapiro Lawrence 2 Smith Lisa P163 Shareef Afzaal 22 Smith Nikki P117 Sharps Jane 66,P034 Smith Terry 43 Shaw Megan P068 Soetens Eline P104 Shcherbik Svetlana P207 Solà Riera Carles 41 Shean Ryan 61 Solis Loretto P100 Shen Steven 42 Soloveva Veronica 70 Shi Pei-Yong P195 Song Dong Hyun P227 Shi Tianlai P111 Song Jin-Won P227 Shifflett Kyle P165 Spence Jennifer P017 Shigeno Asako P010,P209 Spengler Jessica P132,P172 Shindo Keiko 45,P114 Speranza Emily P133 Shioda Tatsuo P244 Spiropoulou Christina 40,P049,P131,P Shirakura Masayuki P177,P180 132,P172,P210 Shirley David 48 Spiropoulou Christina Shoemaker Charles P161 Spitaels Jan P104 Shohei Kojima 87 Spronken Monique P041 Shoji Koichiro P124,P137 Stadler Marc P213 Short Kirsty P041 Staeheli Peter P060,P193 Shrestha Neeta P011 Stäheli Peter 56 Shtanko Olena 17,P126 Stalder Hanspeter P231 Shu Bo P245,P246 Staller Ecco 20 Shu Yuelong P135 Stass Robert 10,P019 Sidamonidze Ketevan P243 Stech Jürgen P165 Sidhu Sachdev 6 Steel John 21,89 Siegers Jurre 66 Stein David P068 Sigmundsson Kristmundur P219 Steinhauer David P004 Silva Filo 36 Steinmann Eike 62 Silvia Ashley P054 Stelfox Alice P218,P248 Simon Dominique P178 Stéphanie Reynard P183 Simuunza Martin P240 Stertz Silke 1 Siqueira Marilda P232 Stevaert Annelies P014,P217 Sisson Gary P169 Stevens James 80 Sitterlin Delphine P039 Stone Ian 42 Sitthicharoenchai Panchan P200 Stosch Juliane P241 Sivanandam Venkatesh P126 Strecker Thomas P118,P159,P215

351

Strle Franc P151 Tellier Michael P066 Stroeh Luisa P020 Teng Michael P170 Stroh Eileen P198 Tenoever Benjamin 30,93,P078 Strupinsky Gaalia P228 Thamamongood Thiprampai 1,19 Stuart Lynda P071 Thibault Patricia P203 Subbarao K 76 Thiel Volker P061,P213 Sugai Akihiro P053,P124,P137 Thompson Robin P003 Sugita Yukihiko 45 Thor Sharmi 80 Sugrue Richard P106 Thornburg Theresa 2 Sukhiashvili Roena P234,P243 Threadgill David P128 Sullivan Brian P088 Thunberg Therese P148 Suluku Roland P239 Tilston-Lunel Natasha 55,P049 Sun Xiangjie 80,P136 Tischler Nicole 6,9,P007,P008,P Superti-Furga Giulio P083,P103 149,P182 Suschak John P161 Todt Daniel 62 Sutter Gerd P176 Tomiou Andru P076 Suzich JoAnn P220 Tomonaga Keizo 15,P044,P098,P Suzuki Noriko P177 105,P237 Suzuki Yasuo P244 Tong Jie P127 Swanson Chad 39,P075 Tong Suxiang P235 Szemiel Agnieszka P074 Tordo Noel P239 Szerman Nathan P142 Torriani Giulia 11,P007,P008,P Szymanska K P001 182 Jonathan 63,P070 Tabor David P220 Towner Dana 50 Tachedjian Mary 86 Townsend Shelby P165 Tahara Maino 92 Traeger Kim P170 Takada Ayato P010,P184,P209 Tran ,P240 Tran Vy 19 Takadate Yoshihiro P010 Trantcheva Iva 70 Takahashi Hitoshi P177,P208 Tripathi Shashank P068,P069 Takahashi Ueda Mahoko P247 Tripp Ralph P190 Takamatsu Yuki P036 Troupin Cécile P239 Takashita Emi P177,P180 Troyanovsky Regina 2 Takeda Makoto 92,P242 Troyanovsky Sergey 2 Takimoto Toru P063 Tsimbalyuk Sofiya P073 Takizawa Naoki 51 Tsurudome Masato P002,P094 Tamhankar Manasi Ajit P012 Tumpey Terrence 80,P136 Tamietti Carole P178 Tynell Janne P037 Tan boon huan P106 Uchida Shotaro P090,P139,P211 Tangy Frédéric P181,P188 Ueki Hiroshi 68 Tani Kenzaburo 92 Ulrich Rainer P236 Tanriver Yakup P193 Urata Shuzo 72,P118 Tauziet Marine 37 Vabret Astrid P229 Taylor Matthew 2 Vafadarnejad Ehsan 62 te Kamp Verena 49 Valentina Rezelj Veronica P032 te Velthuis Aartjan 66,71 Valentin-Weigand Peter P064,P127 Techel Jessica P212 van de Water Sandra 73

352

NSV 2018, Verona – Abstract Book

Van Der Fits Leslie P189 Wang Rongzhang P216 Van der Gucht Winke P173,P197 Wang Shiwen P221 Van Hoecke Lien P194 Wang Weijia P220 van Keulen Lucien 57,73 Wang Xi P059 Van Knippenberg Ingeborg P032 Wang Zhongde 6 Van Molle Inge P194 Warhuus Michelle P155 van Polanen Yolinda P189 Warnes Christine P245,P246 Van Riel Debby 66 Warren Travis 70 van 't Wout Angelique 75 Waßmann Irke P167 van Zelm Menno 60,P157 Watanabe Hiroshi P052 Vanderlinden Evelien P014,P217 Watanabe Shinji P177,P180 Vanstreels Els P014 Watanabe Yasunori P215,P218 Varet Hugo P138 Watanabe Yohei P226,P244 Varjak Margus P042 Watkinson Ruth P203 Vasilijevic Jasmina 65 Watt Ari P165,P167 Velásquez Sonsoles P217 Weary Taylor 59 Vendeville Sandrine P200 Webb Stacy 7 Vera Otarola Jorge P100 Weber Friedemann P083,P089,P103 Vervelde Lonneke P117 Wec Anna 6,P160 Vetter Barbara P059 Wei Huiling P192 Vijay Rahul P079 Weidner Jessica 70 Vijayakrishnan Swetha 43 Weingartl Hana P225 Villalon-Letelier Fernando 38 Weir Marion 48 Visser Eline P157 Welch Stephen 40,P049,P132,P Vo Ho P188 172 Vogel Dominik P033 Wendt Lisa P165 Voigt Kathleen P015 Wentworth David 80,P164,P207,P 245,P246 Volchkov Viktor P097 Volchkova Valentina P097 Wetzke Martin 62,P213 Volz Asisa P176 Wheatley A 76 von Messling Veronika 69 Whelan Sean 24,P016,P050,P 179 VonDollen John 17 White J P001 Voorhees Matthew P223 Whitt Nadia P165 Vrijens Pieter P014 Wichgers Schreur Paul 57,73 Wachowius Marco 27 Widman Lauren P190 Wadia Jay 75 Wiebusch Lüder P059 Wagner Armin P018 Wiechert Svenja P020,P213 Walker Alex P056 Wiegmann Bettina 62,P213 Walker Laura P160,P214 Wignall-Fleming Elizabeth 23,P222 Walker Olivier P058 Wigren Byström Julia P148 Wang I-Hsuan 68 Wiley Michael P174,P227 Wang Li 80 Wilkinson Eric P140 Wang Lin-Fa P219 Willemsen Joschka 35 Wang Ling P126 Willett Brian P003,P074

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Williams Evan P085 Yim Kevin P189 Williams Graham 50 Yokoyama Masaru P180 Williams James P161 Yoneda Misako P053,P090,P096 Williamson Anthony 75 ,P124,P137,P13 Wilson Harry 39,P075 9,P211 Yoshida Asuka P090,P120 Wimmer Eckard P185 Yoshida Reiko P010,P209 Winkler Michael P093,P196 Yoshikawa Rokusuke P077 Wirchnianski Ariel 6 Yoshimatsu Kumiko P240 Wise Helen P076,P084,P117 Younan Patrick 32 Wittwer Kevin 69 Young Dan 23 Wixler Ludmilla P111 Yu Jingyou P195 Wolf Matthias 45 Yumine Natsuko P094 Wolff Thorsten 67,P059 Zaeck Luca 49,P156 Wong Pamela 70 Zahn Roland P189 Wong Terianne 80 Zaki Sherif P132 Wozniak David P133,P147 Zamarreño Noelia 33,65 Wu Kai-Hui P245,P246 Zamora J. Lizbeth 8 Wu Nai-Huei P064,P127 Zeitlin Larry P160 Wu Wei P221,P233 Zeltina Antra 10,74,P018 Wu Wen P187 Zeng Hui P136 Wuerth Jennifer P083 Zeng Xiankun P140 Wurr Stephanie P150,P201 Zengel James P043 Wylie Kristine 50 Zenklusen Daniel 47,48 Wyss Marianne P011 Zhang Quanfu P221 Xiao Yue P107 Zhang Scott 16 Xue Miaoge 16,P195,P216 Zhang Yu P216 Yahiro Mukai 87 Zhao Boxuan 16 Yaita Kenichiro P052 Zhou Bin 80 Yamada Shinya 77,P144 Zhou Yan P062 Yamagishi Junya P242 Zhu Qing P220 Yamaguchi Hideki P124 Ziegler Christopher 47,48,P112 Yamamoto Yutaro 15 Zimmer Christophe 48,P039 Yamayoshi Seiya 77,P035,P135 Zimmer Gert 11,P005,P007,P Yanagihara Kazuyoshi P124 008,P182,P231 Yanai Mako P044,P105 Zimmermann Louisa P238 Yang Genyan 80 Zurbriggen Andreas P011 Yang Guohua 53

Yang Lijuan 91,P185 Yang Wei P064 Yasuda Jiro 72,P077,P118,P 247 Yasuhara Atsuhiro 77,P135 Yatsyshina Svetlana P166 Ye Liang 56,P193 Yen Hui-Ling 66 Yewdell J 76 Yildiz Soner P143

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