Biological Characterization of Porcine Pegivirus
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University of Veterinary Medicine Hannover Institute of Virology Department of Infectious Diseases Biological characterization of porcine pegivirus THESIS Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD) awarded by the University of Veterinary Medicine Hannover by Johanna Kennedy Koblenz Hannover, Germany 2020 Supervisor Prof. Dr. Paul Becher Supervision Group Prof. Dr. Paul Becher Prof. Dr. Karl-Heinz Waldmann Prof. Dr. Eike Steinmann 1st Evaluation Prof. Dr. Paul Becher Institute of Virology, University of Veterinary Medicine Hannover, Germany Dr. Imke Steffen Institute for Physiological Chemistry, University of Veterinary Medicine Hannover, Germany Prof. Dr. Eike Steinmann Faculty of Medicine, Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany 2nd Evaluation Prof. Benedikt Kaufer, PhD Institute of Virology, Free University of Berlin, Germany Date of final exam 30th March 2020 Once again… for science, obviously. Instant classic. Parts of the thesis have been published previously in: Research article Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M, Baumgärtner W, Becher P, Baechlein C. Genetic variability of porcine pegivirus in pigs from Europe and China and insights into tissue tropism. Sci Rep. 2019 Jun 3;9(1):8174. doi: 10.1038/s41598-019-44642-0 Poster and oral presentations Kennedy J, Baechlein C, Hoeltig D, Becher P. Presence of porcine pegivirus in domestic pigs and phylogenetic analysis of pegivirus strains from different parts of the world. 10th Graduate school days, 2017, Bad Salzdetfurth, Germany. Kennedy J, Baechlein C, Hoeltig D, Becher P. Presence of porcine pegivirus in domestic pigs and phylogenetic analysis of pegivirus strains from different countries in Europe and Asia. 28th Annual Meeting of the Society for Virology (GfV), 2018, Würzburg, Germany. Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Keuling O, Ciurkiewicz M, Baumgärtner W, Becher P, Baechlein C. Porcine pegivirus: genetic variability in pigs from Europe and China, insights into tissue tropism and establishment of antibody ELISA. 11th Graduate school days, 2018, Hannover, Germany. Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Ciurkiewicz M, Baumgärtner W, Becher P, Baechlein C. Characterization of persistent pegivirus infection: serology, transmission and replication in PBMCs. 29th Annual Meeting of the Society for Virology (GfV), 2019, Düsseldorf, Germany. Kennedy J, Pfankuche VM, Hoeltig D, Postel A, Ciurkiewicz M, Baumgärtner W, Becher P, Baechlein C. Insights into porcine pegivirus infection: global distribution, tissue tropism, and transmission. Keystone Symposia Conference on Positive-Strand RNA Viruses, 2019, Killarney, Ireland. Contents Table of contents __________________________________________________________ I List of abbreviations ______________________________________________________ III List of figures ____________________________________________________________ VI List of tables _____________________________________________________________VII Table of contents 1 Introduction ___________________________________________________________ 1 1.1 Genus Pegivirus ____________________________________________________ 1 1.1.1 Discovery of pegiviruses _________________________________________ 1 1.1.2 Taxonomy _____________________________________________________ 2 1.1.3 Morphology and genome organization ____________________________ 4 1.1.4 Pegivirus protein functions _______________________________________ 5 1.2 Biology of pegivirus infection in pigs and other hosts ____________________ 6 1.2.1 Prevalence and seroprevalence ___________________________________ 6 1.2.2 Transmission __________________________________________________ 10 1.2.3 Persistence ____________________________________________________ 10 1.2.4 Tissue tropism _________________________________________________ 11 1.2.5 Co-infection with other pathogens and clinical relevance ____________ 12 1.3 Aims of the study __________________________________________________ 13 2 Genetic variability of porcine pegivirus in pigs from Europe and China and insights into tissue tropism _________________________________________________ 15 I Contents 3 Dissecting antibody reactivity and possible transmission routes in porcine pegivirus infection ________________________________________________________ 35 4 Overall Discussion ____________________________________________________ 59 4.1 PPgV RNA detection in domestic pig serum samples from Europe and Asia _ _________________________________________________________________ 59 4.2 Phylogenetic analyses of PPgV ______________________________________ 60 4.3 No detection of PPgV RNA in wild boar ______________________________ 61 4.4 Persistent and transient PPgV infections ______________________________ 62 4.5 Investigation of PPgV tissue tropism _________________________________ 65 4.6 Insights into PPgV transmission routes _______________________________ 66 4.7 Antibody reactivity in Western blot and ELISA ________________________ 67 5 Summary ____________________________________________________________ 71 6 Zusammenfassung ____________________________________________________ 73 7 References ____________________________________________________________ 75 II Contents List of abbreviations °C degrees Celcius × g gravitational acceleration µg microgram µl microliter µm micrometer aa amino acid Ab antibody BPgV bat pegivirus bp base pair cDNA complementary deoxyribonucleic acid Cq cycle quantification CSFV classical swine fever virus C-terminally carboxyl-terminally E envelope protein E2t carboxyl-terminally truncated envelope protein 2 E. coli Escherichia coli ELISA enzyme-linked immunosorbent assay ELISA100 ELISA coated with 100 ng protein per well ELISA250 ELISA coated with 250 ng protein per well EPgV equine pegivirus FISH fluorescence in situ hybridization FPLC fast protein liquid chromatography GBV GB virus h hour HCV hepatitis C virus HGV hepatitis G virus HHPgV human hepegivirus HIV human immunodeficiency virus HPgV human pegivirus HRP horseradish peroxidase IgA immunoglobulin A III Contents IgG immunoglobulin G IgM immunoglobulin M IMAC immobilized metal ion chromatography IPTG Isopropyl β-d-1-thiogalactopyranoside IRES internal ribosome entry site kb kilo base kDa kilo Dalton LB lysogeny broth M molar min minute ml milliliter mM millimolar nm nanometer no. number NS non-structural protein NS3h non-structural protein 3 helicase domain nt nucleotide N-terminal amino-terminal NW New World OD optical density OW Old World ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PBS-Tw phosphate buffered saline containing 0.05% Tween20 PCR polymerase chain reaction PPgV porcine pegivirus PVDF polyvinylidene difluoride px protein x qRT-PCR quantitative reverse transcription polymerase chain reaction RdRp RNA-dependent RNA polymerase RNA ribonucleic acid RPgV rodent pegivirus IV Contents rpm revolutions per minute RT room temperature RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulfate SPgV simian pegivirus SPgVcpz simian pegivirus (chimpanzee) ssRNA single-stranded RNA TBS Tris-buffered saline TDAV Theiler’s disease-associated virus TierSchVersV Tierschutz-Versuchstierverordnung TM TaqMan TMB tetramethylbenzidine UTR untranslated region WB Western blot V Contents List of figures Chapter 1 Figure 1-1. Phylogenetic relationship of pegivirus species A-K. __________________ 3 Figure 1-2. Predicted genome organization of porcine pegivirus. _________________ 5 Chapter 2 Figure 2-1. Phylogenetic analysis of porcine pegiviruses from different countries and other mammalian pegiviruses. ______________________________________________ 21 Figure 2-2. Fluorescence in situ hybridization of porcine pegivirus (PPgV) positive and negative pigs using a PPgV specific probe; overlay phase contrast and immunofluorescence; bar = 100 µm. _________________________________________ 24 Chapter 3 Figure 3-1. Coomassie gel of NS3h protein before and after purification by IMAC. _ 45 Figure 3-2. Western blots of purified NS3h protein incubated with serum samples as first antibody (Ab). ________________________________________________________ 46 Figure 3-3. Western blot of crude E2t protein incubated with serum samples that showed NS3h-specific antibody (Ab) reactivity in Western blot and ELISA as first Ab. _________________________________________________________________________ 48 Figure 3-4. PPgV viral genome quantity in serum (RNA positive results only) during the course of infection in domestic pigs. ______________________________________ 50 VI Contents List of tables Chapter 1 Table 1-1. Pegivirus species nomenclature and their respective hosts (Smith et al., 2016). _____________________________________________________________________ 4 Chapter 2 Table 2-1. Porcine pegivirus genome detection rates and viral genome load in serum samples from individual animals and herds from different countries in Europe and Asia. ____________________________________________________________________ 19 Table 2-2. Number of pegivirus positive pigs of different age groups from Europe and China. _______________________________________________________________ 19 Table 2-3. Porcine pegivirus RNA quantities and fluorescence in situ hybridization results in blood and different tissues from two domestic pigs from Germany. _____ 22 Chapter 3 Table 3-1. Characterization of selected serum samples._________________________ 47 VII Chapter 1 1 Introduction