UNIVERSITY OF CALGARY. Genetic Variability of Bovine Viral Diarrhea Virus in Persistently Infected Cattle. Natalie Rose Dow A THESIS

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1 UNIVERSITY OF CALGARY Genetic Variability of Bovine Viral Diarrhea Virus in Persistently Infected Cattle by Natalie Rose Dow A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE VETERINARY MEDICAL SCIENCES GRADUATE PROGRAM CALGARY, ALBERTA SEPTEMBER, 2013 Natalie Rose Dow 2013

2 Abstract Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle worldwide. The primary propagators of the virus are immunotolerant persistently infected (PI) cattle, which constantly shed large quantities of virus throughout life. The current study has characterized the viral variability in multiple body compartments of PI cattle through analysis of E2 and NS5B sequences. Phylogenetic analysis demonstrated that BVDV exists as a quasispecies distribution within PI cattle. Viral variants were not strictly compartmentalized, although the central nervous system was implicated as an important viral reservoir that may play a role in the emergence of neurovirulent strains. Additionally, vertical transmission of PI resulted in a genetic bottleneck, which is likely followed by generation of diversity by polymerase infidelity and selection processes thereafter. The identification of quasispecies within PI cattle exemplifies the role of this host in viral propagation and highlights the complex dynamics of BVDV pathogenesis, transmission, and evolution. ii

3 Acknowledgements First and foremost, I would like to express my gratitude to Dr. Frank van der Meer for his guidance and support throughout the past two years. He has been pivotal in providing valuable feedback and inspiring critical thinking. It has been a pleasure working with van der Meer lab members Adam Chernick and Sandeep Atwal, who have provided extensive technical and moral support. I would also like to thank the UCVM virology group including the labs of Dr. Guido van Marle, Dr. Markus Czub, Dr. Carla Coffin, Dr. Faizal Careem, as well as supervisory committee members Dr. Michel Levy, Dr. Karin Orsel, and Dr. Aruna Ambagala for their support and expertise in their respective fields. Special thanks to the veterinarians, dairy farmers, and slaughterhouse operators involved in this project, without whom this research would not be possible. I would also like to thank the Natural Sciences and Engineering Research Council (NSERC) and Alberta Livestock and Meat Agency (ALMA) who kindly provided funding for this project. Last but not least, I would like to thank my family, and especially Curtis Hughes, who never fail to provide unwavering encouragement and patience. iii

4 Table of Contents Abstract... ii Acknowledgements... iii Table of Contents... iv List of Tables... vi List of Figures... vii List of Abbreviations... viii Chapter 1: Introduction Etiology of bovine viral diarrhea virus: Acute and persistent infections Molecular biology of bovine viral diarrhea virus Viral quasispecies and BVDV variability within the persistently infected host Hypothesis and objectives of the current study Genes and tissue samples of interest... 9 Chapter 2: Materials and Methods Identification of persistently infected cattle Cloning and sequencing of partial E2 and NS5B genes Sequence alignment and phylogenetic analysis Statistical analysis Chapter 3: Viral Variability and Compartmentalization of BVDV in PI Cattle Rational Results Demographics of persistently infected cattle enrolled in this study iv

5 3.2.2 Phylogenetic analysis of N-terminal partial E2 and NS5B sequences Within-sample diversity Discussion Chapter 4: Viral Variability and Vertical Transmission of BVDV in Two Families of PI Cattle Rationale Results Demographics of two families of persistently infected cattle Phylogenetic analysis of N-terminal partial E2 and NS5B sequences Discussion Chapter 5: Conclusions, Limitations, and Future Work Bibliography Appendix v

6 List of Tables Table 2.1 Oligonucleotide primer sequences used for the amplification of N-terminal E2 and NS5B regions Table 3.1 Demographics of PI cattle Table 3.2 Position and frequency of E2 mutations in clusters outlined in Figure Table 3.3 Position and frequency of NS5B mutations in clusters outlined in Figure Table 3.4 Mean intra-compartment E2 and NS5B distances Table 4.1 Demographics and new ID of PI cattle Table 4.2 Description of clustered E2 and NS5B mutations in two families of PI cattle Table 4.3 Summary of fetal bottleneck and cluster-forming observations Table A1 Descriptive statistics of intrahost E2 and NS5B distances of ten PI cattle Table A2 Significance of mean intrahost E2 (white) and NS5B (grey) distances among PI cattle Table A3 Mean total E2 (white) and NS5B (grey) interhost distances between all PI cattle Table A4 Cohen s d effect size values of E2 (white) and NS5B (grey) intrahost distances among PI family Table A5 Cohen s d effect size values of E2 (white) and NS5B (grey) intrahost distances among PI family vi

7 List of Figures Figure 3.1 Unrooted neighbor joining tree displaying the phylogenetic relationship of E2 consensus sequences from ten PI cattle identified in Western Canadian dairy herds Figure 3.2 Maximum likelihood trees displaying viral variability of E2 sequences within PI cattle Figure 3.3 Maximum likelihood trees displaying viral variability of NS5B sequences within PI cattle Figure 3.4 Mean total intrahost E2 and NS5B distances Figure 4.1 Maximum likelihood trees displaying viral variability of E2 sequences among all members of PI family 1 (A) and PI family 2 (B) Figure 4.2 Maximum likelihood trees displaying viral variability of NS5B sequences among all members of PI Family 1 (A) and PI family 2 (B) Figure 4.3 Mean total E2 and NS5B distances in two families of PI cattle based on a minimum of 70 sequences per gene per animal Figure A1 Nucleotide alignment of consensus E2 sequences for all PI cattle enrolled in this study vii

8 List of Abbreviations Abbreviation 3' 5' ANOVA BBB BDV BVDV BVDV1 BVDV1a BVDV1b BVDV2 BVDV2a C CD46 cdna CNS cp CSFV DNA E1 E2 egfp E RNS FMDV GdnHCl H HCV HIV HVR1 IFN IFN-1 MLN ncp N NGS N pro NS2 NS2/3 NS3 NS4A Definition Three prime Five prime Analysis of variance Blood brain barrier Border disease virus Bovine viral diarrhea virus Bovine viral diarrhea virus genotype 1 Bovine viral diarrhea virus subtype 1a Bovine viral diarrhea virus subtype 1b Bovine viral diarrhea virus genotype 2 Bovine viral diarrhea virus subtype 2a Capsid protein Cluster of differentiation 46 Complimentary DNA Central nervous system Cytopathic Classical swine fever virus Deoxyribonucleic acid Envelope-associated protein 1 Envelope-associated protein 2 Enhanced green fluorescent protein Envelope-associated protein with ribonuclease activity Foot and mouth disease virus Guanidine hydrochloride Hemagglutinin Hepatitis C virus Human immunodeficiency virus Hypervariable region 1 Interferon Interferon Type-1 Mesenteric lymph node Noncytopathic Amino Next generation sequencing Amino protease Nonstructural protein 2 Nonstructural protein 2/3 Nonstructural protein 3 Nonstructural protein 4A viii

9 NS4B NS5A NS5B NU ORF p p7 PBMC PBS PCR PI RdRP RNA RNase TCID UTR VP1 VSV Nonstructural protein 4B Nonstructural protein 5A Nonstructural protein 5B Neurovascular unit Open reading frame Probability according to chance 7 kilodalton protein Peripheral blood mononuclear cells Phosphate buffer saline Polymerase chain reaction Persistently infected RNA-dependent RNA polymerase Ribonucleic acid Ribonuclease Tissue culture infectious dose Untranslated region Viral protein 1 Vesicular stomatitis virus Unit Abbreviation m o C bp kb m Definition Micro (10-6 ) Degree Celcius Basepair Kilobase Milli (10-3 ) ix

10 Chapter 1: Introduction 1.1 Etiology of bovine viral diarrhea virus: Acute and persistent infections Bovine viral diarrhea virus (BVDV), a pestivirus of the family Flaviviridae, is an economically significant pathogen of cattle with a worldwide distribution. Bovine viral diarrhea virus is divided into two genotypes (BVDV1 and BVDV2) and up to eleven subgenotypes with BVDV1a, 1b, and 2a predominating in North American cattle 1-3. Independent of genotype, BVDV may be additionally classified as one of two biotypes, cytopathic (cp) or noncytopathic (ncp), based on the ability of the virus to lyse cells in tissue culture 4. This phenotypic distinction is due to the fact that cpbvdv is implicated in the induction of interferon type-i and apoptosis, whereas as ncpbvdv fails to induce their generation 5. Both cp and ncpbvdv are capable of acutely infecting immunocompetent animals, although ncpbvdv widely predominates and accounts for the majority of clinical cases 6. Furthermore, ncpbvdv is capable of crossing the placentome of an acutely infected dam, which may have various outcomes depending on the stage of gestation. When ncpbvdv infection occurs approximately between day 30 and 120 of gestation a persistently infected (PI) fetus will be produced 7. Calves infected during the final trimester of gestation or post-natally will subsequently produce neutralizing antibodies capable of viral clearance, however, a PI fetus will remain immunotolerant of the persisting strain due to viral establishment before maturation of the fetal immune system 6,8,9. Viral proteins are subsequently regarded as self, which allows viral replication in all tissues and excretions without host detection 9. Persistently infected cattle are the most significant propagators of BVDV as they continuously shed the virus and produce viral titres upwards of 10 7 TCID50/ml 10. Additionally, every calf produced by a PI dam will also be PI, although the pathway and timing 1

11 of fetal infection are not clear as all tissues including the ovarian follicles, oocytes and uterine tissues are continually infected 11,12,13. This may suggest a mechanism of fetal survival in the face of early PI establishment, as acute infection of the dam in the first month of gestation results in embryonic death. Despite the significance of the PI host in the propagation of BVDV, very little is known regarding the viral populations generated by these animals. 1.2 Molecular biology of bovine viral diarrhea virus Bovine viral diarrhea virus is a positive stranded RNA virus that encodes a single 12kb open reading frame (ORF) flanked by 5 and 3 untranslated regions (UTRs). The 5 UTR harbours the internal ribosome entry site (IRES), which mediates translation by binding the 40S ribosomal subunit 14. Translation results in a polyprotein that is processed by cellular and viral proteases to yield four structural and eight nonstructural proteins in the order of N pro -C-E rns -E1-E2-p7-NS2/3- NS4A-NS4B-NS5A-NS5B 15. The virion is composed of a central capsid surrounded by an outer lipid bilayer envelope that is associated with the three glycoproteins 16. The first envelope protein, E rns, is a non-transmembrane protein that produces ribonuclease activity and is not required for cell entry. Both E1 and E2 are structural glycoproteins with N-terminal ectodomains anchored to the viral membrane via C-terminal helices 17. Disulfide linked E2 homodimers are formed quickly on the surface of the virion, while the E1-E2 heterodimers are formed later, as folding of the E1 occurs more slowly than that of the E2 18. Heterodimer formation between E1 and E2 glycoproteins is essential for infectivity, where the E1 is thought to act as a chaperone protein while the E2 possesses the receptor binding function and membrane fusion domain However, the precise mechanism of fusion is not currently understood, as the BVDV E2 unexpectedly does not contain a class II fusion protein fold 17. The N-terminal ectodomain of the 2

12 E2 encodes the dominant neutralizing epitopes and is characterized by significant genetic diversity 17,22. The first nonstructural protein, N pro, serves as an autoprotease, which cleaves itself from the polyprotein 23. Nonstructural p7 is required for production of infectious virus but not replication, although its specific role is poorly understood 24. The NS2/3 protein contains the molecular biotype determinants, as these proteins are cleaved into separate NS2 and NS3 components in cytopathic strains of BVDV 25. The NS2/3 cleaves itself and the remaining proteins from the polyprotein with NS4A serving as a cofactor 15. The replicase component is composed of NS4B, NS5A, and NS5B, where NS5B encodes the RNA dependent RNA polymerase (RdRP) 26. Bovine viral diarrhea virus binds the cellular receptor CD46 17,27. Following clathrinmediated endocytosis the RNA is uncoated and released into the cytoplasm where transcription and translation occur 16. Following translation, replication takes place where a complimentary negative RNA strand is synthesized to serve as template for the positive stranded RNA genome. The virion is assembled in the endoplasmic reticulum and is then transported to the Golgi apparatus, where mature particles are released by exocytosis Viral quasispecies and BVDV variability within the persistently infected host The BVDV PI host represents a unique model of viral evolution, as persistence is achieved by establishing immunotolerance. In contrast, persisting viral infections in immunocompetent hosts rely on mechanisms such as epitope diversity to continuously escape neutralization by host antibodies As such, population diversity is an important hallmark of RNA viruses attributed to the sloppy replication enzyme, RNA-dependent RNA polymerase (RdRP), which incorporates mutations at a rate of approximately 10-4 substitutions per nucleotide 3

13 site 29,30. The resulting viral populations exist as a swarm of closely related mutants, which are collectively referred to as quasispecies. Viral diversity is an important mechanism of pathogenesis for quasispecies, as it affords rapid adaptability to the dynamic features of the cellular environment, host immune response, and antiviral therapy. With each round of replication, mutations are spontaneously introduced and may undergo selection, as mutations inferring fitness gains are maintained while deleterious traits are removed. Additionally, neutral mutations are accumulated and may be added to the repertoire of viral diversity, over time contributing to a process called genetic drift 32. Thus, viral populations can produce a highly fine-tuned and host-specific response by adapting to the host through selection processes. That being said, quasispecies dynamics extend beyond classical gene theory and population genetics, as cooperative functioning among constituent subpopulations in the host leads to evolution of groups of interdependent variants rather than individual genomes 33,34. Evidence of this phenomenon is described in poliovirus, where reduced quasispecies diversity resulted in the loss of neurotropism 33. Thereafter, invasion of the central nervous system (CNS) was restored by addition of a mutagen, exemplifying the biological impact of quasispecies and the function of viral diversity as a mechanism of pathogenesis 33. One of the outcomes of efficient adaptability to the host is compartmentalization of viral variants, where viral populations are genetically distinct between different tissues and body compartments due to localized drivers. Shaping of tissue-specific viral subpopulations is a complex process attributed to various combinations of localized influences such as viral load, immune pressure, antiviral therapy, and modification to or distribution of cellular receptors. Compartmentalization has been reported for viruses such as human immunodeficiency virus type 1 (HIV-1), where studies have identified tissue-specific viral variants in compartments including 4

14 peripheral blood, brain, spleen, lymphoid tissues, and the gut Compartmentalization serves as a strong predictor of HIV treatment efficacy, as localized suboptimal antiviral concentrations can result in viral sanctuary sites and may lead to the selection of antiviral resistant variants. This was described in a model by Kepler and Perelson, which showed that the likelihood of emergence of resistance is increased when multiple spatially distinct compartments exist where one compartment receives suboptimal drug concentration 39. In this scenario, resistant variants are generated in the viral sanctuary site and then selected in other compartments with adequate antiviral concentrations, where such variants achieve a strong selective advantage. This phenomenon is particularly concerning when a high level of trafficking of viruses occurs between compartments, as it can lead to the accumulation of resistant mutants and may lead to the spread of such variants throughout the host 39. Compartmentalization has also been evidenced in hepatitis C virus (HCV), a Flavivirus closely related to BVDV. The HCV hypervariable region 1 (HVR1) located at the N-terminal region of the E2 glycoprotein is the focus of most compartmentalization studies, where many patients harbour variants with distinct cellular tropisms between viral populations in the liver, serum, and peripheral blood mononuclear cells (PBMCs) Evidence of intrahost variants with various cellular tropisms is a challenge for treatment, as different subpopulations may have varying levels of sensitivity to antiviral therapy 40. In addition to antiviral escape, immune escape mutants have been identified in chronically infected HCV patients where emergence of escape variants is correlated with extensive viral variability that increases over the course of chronic infection 41,43. Compartmentalization of such variants may further exacerbate the host immune defense through the production of distinct epitopes distributed in different compartments and cell populations. As evidenced by these examples, intrahost quasispecies analysis can provide insight of localized evolutionary drivers 5

15 and can reveal important characteristics of viral pathogenesis that may contribute to the outcome of infection; however, this approach has yet to be applied to BVDV. The integral role of the BVDV PI host along with the unique mechanism of immunotolerant persistence raises many questions regarding viral variability within PI cattle. Bovine viral diarrhea virus strains circulating between livestock herds exhibit a high degree of variability in nucleotide and amino acid sequences, however, it is currently unclear how this viral diversity is introduced Previously, it has been proposed that acute infections are the most significant contributors to BVDV variability, with adaptive immunity driving evolution of structural proteins 45. Conversely, PI cattle were expected to maintain stable populations as immune surveillance eliminates variants that differ from the persisting strain 47. However, this assumption disregards the fact that, as compared to acute infection, BVDV infects a far wider range of tissues and produces much higher viral titers within the PI host 10,48. In addition to constant production of massive viral populations, BVDV PI cattle remain infected throughout life and therefore permit viral replication over significantly longer periods of time than acutely infected cattle 49. Collectively, these characteristics of PI cattle serve as predictors of viral variability, despite the immunotolerance of the host. Questions regarding viral variability within the PI host have been previously raised by Collins et al., who investigated diversity of E2 and NS3 sequences of two experimentally infected PI cattle 50. Although the NS3 was highly conserved within both PI hosts, the E2 displayed genetic diversity that increased over time. Temporal increases in E2 amino acid sequence diversity suggests potential for generation of antigenically variant viruses, therefore Collins et al. additionally collected antibody data from 21 experimentally infected PI calves that were housed in isolation followed birth. Four PI calves produced low levels of neutralizing 6

16 antibody, indicating generation of viral diversity to the point of antigenic variation 50. Intrahost diversity of BVDV was further investigated by Jones et al. who analyzed clones of the 5 UTR derived from the kidney, spleen, and lung from a field infected PI fetus 51. This study identified unique sequences in different organs of the PI fetus however, the quasispecies did not form phylogenetic groups based on the organ of origin. Therefore, it was concluded that BVDV variants can exists at low frequencies in different tissues and it is possible that certain viral subpopulations replicate at high rates in some tissues and lower rates in others 51. This study provided insight into genetic diversity within PI cattle, however it is isolated to a highly conserved region of the genome and phylogenetic grouping of unique variants may become evident through analysis of a more diverse region of the genome. Furthermore, analysis of viral diversity a PI fetus may not be generalizable to other PI cattle, as this represents a relatively early time point following establishment of PI and the fetus likely does not have a fully developed immune system. To fully assess compartmentalization in PI cattle, a variety of tissues in multiple PI cattle should be assessed. This study demonstrated that fetal PI cattle possess low frequency variants, however it is not clear whether these variants were transmitted to the fetus or if they arose following transmission. The level of viral diversity associated with vertical transmission of PI was assessed in a recent study by Neill et al 44. Consensus sequence diversity was compared between a PI dam and her PI calf where 12 of 21 nucleotide substitutions in the ORF were nonsynonymous 44. Evidence of viral variability following vertical transmission from PI dam to PI calf was unexpected and no explanation was offered for this finding. Vertical transmission from PI dam to fetus remains a poorly understood process, as it is not clear when fetal infection occurs. Infection may occur much earlier than congenital transmission from an acutely infected 7

17 dam, as BVDV antigen has been detected in ovarian follicles, oocytes, and uterine tissues of PI dams 13. While little is known regarding the clinical implications of BVDV viral diversity, it is possible that it plays a role in virulence as evidenced in classical swine fever virus (CSFV), another member of the pestivirus genus 52. Recently, next generation 454 sequencing correlated highly virulent strains with high E2 and NS5B quasispecies diversity as compared to isolates from pigs infected with low and moderately virulent strains 52. Thus, previous studies and knowledge of BVDV PI suggest that PI cattle produce genetic variability, however the complexity and distribution of viral diversity within these animals is not clear. Persistently infected cattle represent the most significant propagators of BVDV, therefore it is important to understand the dynamics of the viral populations produced by these animals. Despite the absence of an adaptive immune response viral adaptation may occur in response to other factors such as interaction with the cellular receptor, evasion of the innate immune system, and replicative capacity. Additionally, vertical transmission of PI is an important aspect of BVDV epidemiology, as it continues the cycle of viral propagation within the herd. Viral populations between PI dam and offspring have not been described at the quasispecies level, however this knowledge would provide insight into the consequences of long term BVDV infection in a herd over multiple generations of PI. Knowledge of genetic diversity generated by PI cattle may have important implications in elucidating mechanisms of pathogenesis, transmission, and evolution of BVDV. 8

18 1.4 Hypothesis and objectives of the current study The goal of the current study is to describe the extent and distribution of BVDV variability within and between ten PI cattle from Western Canadian dairy herds. It is hypothesized that BVDV is diverse within PI cattle and that viral variants are genetically distinct between different body compartments due to localized drivers in different tissues and body compartments. Furthermore, the sample set includes two families of PI cattle- one family of three generations (PI dam, heifer, and fetus) and a second family consisting of a PI dam and fetus pair. Hence, the current study will also describe how viral diversity is reflected in progeny PI cattle following vertical transmission from PI dam to fetus. This research will be hypothesis generating, as it aims to describe the presence and frequency of mutations in attempt to identify viral reservoirs within the PI host. Additionally, the current study aims to assess viral populations between the PI dam and calf, which may generate new hypotheses regarding early establishment of PI and the dynamics of BVDV evolution following transmission. 1.5 Genes and tissues samples of interest To appropriately assess BVDV variability within the PI host, partial N-terminal E2 and NS5B genes have been selected as representative structural and nonstructural protein-coding genes respectively. Analysis of the N-terminal region of the E2 glycoprotein is appropriate for genetic variability studies because it has been identified as the most diverse region of the BVDV genome 45,50. This region has been used extensively to characterize quasispecies distributions of HCV, where it has been labeled a hypervariable region (HVR1) 42, Variability of the N- terminal region in particular will be assessed, as it encodes the ectodomain and is expected to 9

19 reflect adaptation to the cellular environment 17. Furthermore, this region possesses dominant neutralizing epitopes and plays a vital role in cell fusion and binding, although the specific determinants of cell tropism have yet to be identified 3,17. Therefore, knowledge of the E2 function and previous studies of compartmentalization justify this envelope glycoprotein as a strong candidate for BVDV viral variability studies. In contrast to the E2, the nonstructural gene NS5B encoding the RdRP, is highly conserved among pestiviruses 57,58. The replication enzyme occupies a smaller sequence space than structural proteins, as single amino acid substitutions can have a major impact on enzyme fidelity by dramatically changing mutation rates and subsequent quasispecies diversity and pathogenesis 59,60. Replication of RNA viruses relies on achieving an adequate mutation rate to afford adaptability through viral diversity, but must not surpass the error threshold, where an abundance of deleterious mutations results in a profusion of nonfunctional genomes, also known as lethal mutagenesis 29,30. While RdRPs have stringent amino acid constraints, enzyme fidelity may be modified to adjust mutation rates in the face of environmental challenges. This has been evidenced in both poliovirus and HCV, where increased enzyme fidelity confers resistance to the mutagenic properties of ribavirin 60,61. Alternatively, beneficial implications of increased mutation rates have been suggested in bacteriophage populations, as a broader scope of viral genomes can enhance the rate of adaptation 62. In addition to the two genes of interest, seven samples from different body compartments were selected to provide an overview of genetic variability in multiple compartments of PI cattle. Viral populations in the ileum and colon were selected, as the gut has been implicated as a viral reservoir of HIV 36. Milk samples were selected, as this represents viral particles that had to successfully pass through the selective membranes of the alveolus to be excreted. Lymphoid 10

20 tissues often display lesions in PI cattle that develop mucosal disease; therefore tonsil and mesenteric lymph node (MLN) were included in the study. Finally serum was investigated to analyze circulating virus while the obex served as a representative of the CNS. This metagenomics approach provides an unprecedented overview of viral variability in the PI host and sheds light on the extent and distribution of viral variability in various body compartments. Additionally, investigation of intrahost evolution of BVDV within the PI host may be extrapolated to predict potential consequences for other members of the herd. 11

21 Chapter 2: Materials and Methods 2.1 Identification of persistently infected cattle Ten BVDV PI cattle were identified in Western Canadian dairy herds. Bovine viral diarrhea virus was identified on farms by a combination of bulk milk and serum PCR and antibody testing of suspected BVDV-positive herds. Infection was initially detected on farms 1 and 3 by bulk milk testing (See Table 3.1. for description of farm and PI identification numbers). An E.Z.N.A. Viral RNA Kit (Omega Bio- Tek, Norcross, GA, USA) was used to extract RNA from bulk milk according to the manufacturer s instructions. Polymerase chain reaction amplification of the 5 UTR region of the BVDV genome was carried out using a BluePrint TM One-Step RT-PCR kit (TAKARA Bio Inc, Otsu, Shiga, Japan) and primers 5 UTR-for and 5 UTR-rev listed in Table 2.1. A 25µl reaction mixture was prepared consisting of 12.5µl of 2X One Step BluePrint TM Buffer, 10µl RNase free water, 1µl One Step BluePrint TM RT Enzyme Mix, 0.5µl each of forward and reverse primers, and 0.5µl template RNA. Cycling conditions were as follows: 50 o C for 30 minutes, 94 o C for 2 minutes, 35 rounds of 98 o C for 10 seconds, 52 o C for 30s, and 72 o C for 1 minute, followed by a final extension of 72 o C for ten minutes. Following positive bulk milk testing, serum samples were collected from the entire herd to identify PI cattle. Total RNA was extracted from serum using a Mag-Bind Viral DNA/RNA Kit (Omega bio-tek, Norcross, GA, USA) on the MagMAX TM Express-96 Deep Well Magnetic Particle Processor (Applied Biosystems, Burlington ON, Canada). Amplification of BVDV RNA was performed using a VetMAX TM -Gold BVDV Detection Kit (Applied Biosystems, Burlington ON, Canada), in which cdna production and amplification are performed in one continuous process. All protocols were performed according to the manufacturer s instructions on the 12

22 CFX96 real time system (Bio-Rad, Mississauga, ON, Canada). Cattle positive for BVDV were retested three weeks later to confirm PI. Identification of PI cattle on farm 2, 4, and 5 was carried out by targeted testing of serum from recently purchased animals or poor-doing suspected BVDV-positive cattle. Total RNA was extracted and tested with VetMAX TM -Gold BVDV Detection Kit as described above. Additionally, antibody titers were measured according to the manufacturer s instructions using HerdChek BVDV Antibody Test Kit (IDEXX Switzerland AG, Bern, Switzerland) from a subset of ten cattle in herds with suspected BVDV infection. Ear notches from positive cattle were retested at necropsy using a SNAP BVDV Test (IDEXX, Westbrook, ME, USA) according to the manufacturer s instructions. All PI animals were euthanized and colon, ileum, milk, MLN, obex, serum, and tonsil samples were collected as part of a larger sampling process, which also included heart, lung, liver, kidney, spleen, duodenum, jejunum, caecum, gall bladder, ovary/testicle, udder, esophagus, skin, bile, urine, feces, saliva, nasal swab, and PBMCs. All samples were stored at 4 o C overnight, aliquoted the following day, and stored at -80 o C until further processing. 2.2 Cloning and sequencing of partial E2 and NS5B genes Viral RNA was extracted from tissue and excretion samples using an E.Z.N.A. Viral RNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer s instructions. Prior to RNA extraction, tissue samples of approximately 1cm 2 were soaked in 200µl PBS for 30 minutes and then homogenized using a pestle in a 1.5ml Eppendorf tube. Tubes were vortexed, centrifuged and supernatant was removed and used for subsequent steps of the extraction. Total RNA was used as template for PCR amplification of the E2 and NS5B regions of the genome, 13

23 which was carried out using a BluePrint TM One-Step RT-PCR kit (TAKARA Bio Inc, Otsu, Shiga, Japan) on the T100 TM Thermal Cycler (Bio-Rad, Mississauga, ON, Canada) using primers listed in Table 2.1 and a 25µl reaction mixture as described above. Cycling conditions for the E2 fragment included 50 o C for 30 minutes, 94 o C for 2 minutes, 35 rounds of 94 o C for 30 seconds, 56 o C for 1 minute, and 72 o C for 1 minute, followed by a final extension of 72 o C for ten minutes. Cycling conditions for the NS5B fragment were 50 o C for 30 minutes, 94 o C for 2 minutes, 35 rounds of 94 o C for 20 seconds, 50.5 o C for 30 seconds, and 72 o C for 30 seconds, and a final extension of 72 o C for 15 minutes. Amplified PCR fragments were run on 1.5% agarose gels and bands of expected size (606bp for E2 and 1162bp for NS5B) were isolated and purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) and then ligated into pgem -T Easy vectors (Promega, Madison, WI, USA) according to he manufacturer s instructions. Ampicillin resistant recombinant colonies were selected by blue/white selection method. Plasmid DNA was isolated and purified using an E.Z.N.A. Plasmid Mini Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer s instructions and were sent to Eurofins MWG Operon (Huntsville, AL, USA) to be sequenced using T7 and SP6 primers. A minimum of ten clones per tissue were sequenced to obtain an overview of viral variability within the PI host. Table 2.1 Oligonucleotide primer sequences used for the amplification of N-terminal E2 and NS5B regions. Primer Sequence (5-3 ) Position * Reference 5 UTR-for CATGCCCATAGTAGGAC UTR-rev CCATGTGCCATGTACAG E2-for TAAGACCARATTGGTGGCCTTATGAGAC E2-rev GGGCAWACCATYTGGAAGGCYGG NS5B-for AAGATCCACCCTTATGARGC NS5B-rev AAGAAGCCATCATCMCCACA * On the NADL genome sequence 14

24 2.3 Sequence alignment and phylogenetic analysis Vectors and primers were trimmed prior to assembly of sequence contigs using Geneious software (Biomatters Ltd, Auckland, New Zealand). Nucleotide alignments were constructed in Geneious using multiple sequence comparison by log-expectation (MUSCLE) and exported into MEGA (version 5 66 ) for mean total distance calculation and phylogenetic tree construction. Unrooted phylogenetic trees were constructed by maximum likelihood method using a Kimura-2 parameter model with a gamma distribution using five discrete gamma categories, a cut off of 95%, and 1000 bootstrap replicates. This nucleotide substitution model treats transitions and transversions with different substitution rates and assumes that rate variation over sites is drawn from a continuous distribution 66. Viral clones in the maximum likelihood trees were coloured based on the tissue from which they were sequences and visually assessed for clustering of clones from the same compartment. Mutations shared between compartmentalized clusters were then identified by reference to the nucleotide alignment. 2.4 Statistical analysis Significance differences between mean total distances was performed using a one-way ANOVA and post hoc Tukey analysis using SPSS software (version 21, IBM, NY, USA). Effect size was calculated using Cohen s d formula d = M 1 - M 2 / s pooled. See appendix for example calculation. 15

25 Chapter 3: Viral Variability and Compartmentalization of Bovine Viral Diarrhea Virus in Persistently Infected Cattle 3.1 Rational Bovine viral diarrhea virus PI hosts are immunotolerant lifelong viral shedders that represent the most significant propagators of the virus 49. Despite the significance of the PI host as a viral reservoir within the herd, little is known regarding the viral populations produced by these animals. Many RNA viruses acquire significant viral variability through large population sizes, rapid generations time, and high mutation rates 29,67,68 ; however the immunotolerant nature of the PI host presents a unique model of viral evolution where viral variability has not been thoroughly characterized. As such, the principle aim of the present study is to provide a detailed description of viral variability within and between different PI cattle. It is hypothesized that with the absence of an adaptive immune response, viral diversity will be directed by variations in the cellular environment and the local influence of the innate immunity, thus resulting in compartmentalization of viral variants. The current chapter investigates viral compartmentalization of partial E2 and NS5B sequences obtained from the colon, ileum, milk, MLN, obex, tonsil, and serum of ten PI cattle identified in Western Canadian dairy herds. Presence and frequency of mutations from various organs of origin will be analyzed to evaluate the role of different body compartments as viral reservoirs. 3.2 Results Demographics of persistently infected cattle enrolled in this study A total of ten PI cattle were identified across five Western Canadian dairy herds, as described in Table 3.1. Persistently infected fetuses in approximately the fourth and eighth 16

26 month of gestation on farm 1 and 5 respectively were included in the sample set. A minimum of ten E2 and NS5B sequences were obtained from each compartment or excretion of interest (colon, ileum, milk, MLN, obex, tonsil, and serum) with the exception of NS5B clones derived from the colon, ileum, and obex of PI 6 and 7, which were not positive by PCR amplification. Table 3.1 Demographics of PI cattle. PI ID Farm ID BVDV Genotype No of E2 Clones No of NS5B Clones 1 1 1a a a * 1 1a b b b a b ** 5 1b * Fetus in approximately the fourth month of gestation ** Fetus in approximately the eighth month of gestation Phylogenetic analysis of N-terminal partial E2 and NS5B sequences Degenerate primers were used to amplify the first 123 amino acids of the E2 gene. The phylogenetic analysis of E2 consensus sequences (Figure 3.1) showed that viruses from different farms clearly segregated into different strains, while PI cattle from the same farm produced identical consensus sequences. The consensus sequence is defined as the representative sequence in which the nucleotide at each position is the one that occurs most frequently in a population 68. Alignment of E2 consensus sequences is available in the appendix. 17

27 PI PI 2 PI 3 PI 4 NADL (M31182) PI 8 Oregon (AF091605) PI Osloss (M96687) PI 6 PI 7 Farm 1 Farm 2 Farm 3 PI 9 Farm 4 84 PI 10 Farm Figure 3.1 Unrooted neighbour joining tree displaying the phylogenetic relationship of E2 consensus sequences from ten PI cattle identified in Western Canadian dairy herds. Cattle from the same farm formed monophyletic groups while cattle from different farms segregated into different strains. Reference strains for BVDV1a (Oregon and NADL) and BVDV1b (Osloss) are included along with Genbank reference numbers. Maximum likelihood trees were constructed to provide an in depth analysis of the E2 and NS5B variability within PI cattle, shown in Figure 3.2 and 3.3. All viral variants are colourcoded according to the body compartment of origin from which the virus was cloned and sequenced. Maximum likelihood trees display an accumulation of intrahost viral variability where very few variants were identical and the majority of clones existed as a spectrum of mutants. Although many variants were interspersed, tissue-specific clustering was observed in E2 maximum likelihood trees for PI 1, 2, 8, and 9. Similarly, NS5B sequences produced tissuespecific clustering for PI 1, 3, 4, 8, and 9. Clustering variants from the same compartment were designated a Cluster ID and mutations shared within clusters are listed in Table 3.2 (E2) and 3.3 (NS5B). 18

28 Cluster PI 1 PI PI 3 90 Cluster PI 4 PI 5 75 PI Cluster PI 7 PI 8 PI 9 Cluster 8.2 Cluster PI 10 Colon Ileum Milk MLN Obex Serum Tonsil Figure 3.2 Maximum likelihood trees displaying viral variability of E2 sequences within PI cattle. Four PI animals produced evidence of compartmentalized clustering in the obex and/or tonsil, as indicated by black boxes. Specific mutations shared by these clusters are described in Table

29 Table 3.2 Position and frequency of E2 mutations in clusters outlined in Figure 3.2. Compartmentalized variants were found in tissues of the obex and/or tonsil in four of ten PI cattle in this study. Cluster 1.1 contained the highest frequency of shared mutations, six of which being nonsynonymous. No mutations were shared among multiple PI hosts from different farms, although both PI 1 and 9 had mutations at position 170 in obex clusters (bold and underlined). Compartment Total # clones in cluster Nucleotide position and change * aa change Mutation frequency in cluster Cluster 1.1 Obex T:C Wà R C:T Pà L A:G & 183 A:C Rà G A:C Rà S A:G Rà G C:T Silent A:G Kà R 5 Cluster 2.1 Tonsil T:C Wà R 9 Cluster 8.1 Obex T:C Ià T T:C Fà S G:A Eà K 7 Cluster 8.2 Tonsil T:C Là P A:G Kà E G:A Silent G:A Eà K T:C Silent 3 Cluster 9.1 Obex C:T Tà I A:G & 170 T:C Ià A T:C Ià T 9 * Position on the NADL genome sequence 20

30 PI Cluster PI PI Cluster 1.1 Cluster 1.2 Cluster PI 5 84 PI 6 85 PI PI 8 87 PI 9 95 PI Cluster Cluster 9.1 Cluster PI Colon Ileum Milk MLN Obex Serum Tonsil Figure 3.3 Maximum likelihood trees displaying viral variability of NS5B sequences within PI cattle. Five PI animals produced evidence of compartmentalized clustering, as indicated by black boxes. Specific mutations shared by these clusters are described in Table

31 Table 3.3 Position and frequency of NS5B mutations in clusters outlined in Figure 3.3. Compartmentalized variants were found in tissues of the serum, obex or tonsil in five of ten PI cattle in this study. Two silent mutations (bold and underlined) were shared among PI hosts from different farms. As opposed to E2 compartments, the majority of NS5B mutations were synonymous. Compartment Total # clones in cluster Nucleotide position and change * aa change Cluster 1.1 Obex T:C Silent C:T Silent T:C Silent A:G Silent G:A Silent C:T Silent 2 Cluster 1.2 Obex G:A Silent 4 Cluster 3.1 Serum A:G Kà R G:A Silent A:G Kà R C:T Silent A:G Silent C:T Silent 4 Cluster 4.1 Tonsil C:A Dà E A:G Silent 5 Cluster 8.1 Obex G:A Ià V A:G Hà R G:A Silent C:T Silent 10 Cluster 8.2 Tonsil A:G Kà R T:C Silent G:A Silent T:C Silent C:T Silent 2 Cluster 9.1 Obex G:A Silent T:C Silent C:T Silent A:G Kà V A:G Sà G 2 * Position on the NADL genome sequence Mutation frequency in cluster 22

32 3.2.3 Within-sample diversity Mean total intrahost distances were calculated to quantify the diversity illustrated in Figure 3.2 and 3.3. This calculation represents the average of all pairwise distances between the sequences within a group 66. For example, an average distance of 0.01 infers that all sequences within the group are 99% identical. The sample group was specified as either the entire host (Figure 3.4) or as a specific body compartment within the host (Table 3.4). Average intrahost diversities of NS5B sequences were significantly more conserved than those of E2, although both genes followed a similar pattern across all PI cattle. Mean interhost distances are additionally reported in the Appendix fgg Distance ± SE E2 NS5B 0 PI 1 PI 2 PI 3 PI 4 PI 5 PI 6 PI 7 PI 8 PI 9 PI 10 Farm 1 BVDV1a Farm 2 BVDV1b Farm 3 BVDV1b Farm 4 BVDV1a Farm 5 BVDV1b Figure 3.4 Mean total intrahost E2 and NS5B distances. Intrahost E2 distances were significantly higher than NS5B distances for all PI cattle. Significance matrix is available in the appendix. Mean intrahost distances were broken down for each compartment for both E2 and NS5B genes. The heat map in Table 3.4 shows that E2 sequences typically had the greatest diversity in 23

33 the obex, particularly evidenced in PI 1, 2, and 3 from farm 1. However, this genetic variability was not reflected in the fetal PI 4 from the same farm. Another highly diverse viral E2 sequence subset was identified in the milk of PI 9, although this finding was not reproduced in milk samples from other PI cattle in this study. The same pattern was not observed in NS5B sequences, as the obex was not the most diverse compartment nor did the milk of PI 9 produce highly diverse variants. Farm 1 displayed a similar scenario between E2 and NS5B sequences, as both genes from PI 1, 2, and 3, were highly variable, although this finding was not reflected in PI 4 from the same farm. 24

34 Table 3.4 Mean intra-compartment E2 and NS5B distances. Graded colour scale illustrates relative extent of mean within-sample variations of E2 and NS5B sequences. Nucleotide sequences were more highly conserved among NS5B sequences than E2 and patterns across different tissues varied between farms. The greatest diversity among both genes was observed in PI 1, 2, and 3 of farm 1. Typically obex-derived tissues produced the most E2 diversity, while NS5B sequence diversity varied between all PI hosts. PI ID E2 Colon Ileum Milk NA * NA * NA * NA * NA * NA * MLN Obex Tonsil Serum NS5B Colon NS ** NS ** Ileum NS ** NS ** Milk NA * NA * NA * NA * NA * NA * MLN Obex NS ** NS ** Tonsil Serum *N Farm 1 BVDV1a * Not applicable: non-lactating ** Not successful: PCR negative Farm 2 BVDV1b Farm 3 BVDV1b Farm 4 BVDV1a Farm 5 BVDV1b 3.3 Discussion Sequence variability of cloned E2 and NS5B genomic regions was assessed to describe the level of viral diversity within and between PI cattle and, furthermore, identify the role of different body compartments as viral reservoirs within the host. Analysis of E2 consensus sequences from all cattle indicated that PI cattle from the same farm produced identical 25

35 consensus sequences, which is in support of previous work by Hamers et al. 69 and Paton et al. 70 who suggest that PI cattle establish herd specificity by stabilizing BVDV strains. Despite this finding, clonal analysis is merited, as changes in quasispecies fitness are not necessarily accompanied by changes to the consensus sequence 68,71. An in depth analysis of clones derived from the colon, ileum, MLN, milk, obex, serum, and tonsil revealed that few variants were identical to the consensus sequence while the majority existed as a mutant spectrum surrounding the consensus. This collection of closely related genotypes is indicative of a quasispecies population. Patterns of intrahost quasispecies distributions varied between all ten PI cattle, although sequence diversity of the N-terminal E2 ectodomain-encoding region was significantly more diverse than NS5B RdRP-encoding sequences among all PI cattle. This finding was not unexpected, as the E2 is known to be the most diverse region of the BVDV genome between different strains. Mutations are likely tolerated in this region, as it must adapt to the cellular environment and is additionally driven by the host immune response and tissue tropism requirements. Historically, the E2 glycoprotein of pestiviruses has accumulated mutations to accommodate a diversified host range, as BVDV, border disease virus (BDV), and classical swine fever virus (CSFV) are respectively adapted to cattle, sheep, and swine 20,72. The NS5B on the other hand is less tolerant of amino acid changes, as reflected by a low proportion of nonsynonymous changes. The RdRP is an integral factor in quasispecies dynamics, as enzyme fidelity affects population diversity and subsequent cooperative interactions of viral subpopulations contributing to pathogenesis 33,59,60. Within most PI cattle, viral clones were highly interspersed, suggesting a random distribution of genotypes and equilibrium of viral particles in body compartments. However, Figures 3.2 and 3.3 displayed clustering of variants derived from the obex (PI 1, 8, 9), tonsil (PI 26

36 2, 4, 8), and serum (PI 3) implicating these tissues as viral reservoirs within these animals. The greatest extent of compartmentalization was observed in the obex, where PI 1, 8, and 9 all produced clustering variants of both E2 and NS5B sequences. Identification of clustering in both genomic regions implies that tissue-specific drivers existed in the obex at levels of both glycoprotein structure and enzymatic function. Not only were obex-derived populations compartmentalized within a subset of animals, but they also typically exhibited greater E2 genetic diversity than other tissues. While the mechanism of compartmentalization of diverse variants within the obex has yet to be defined, several explanations could account for this observation. The CNS represents an immunologically specialized site, as it can become locally immune reactive due to the immunemodulating function of the neurovascular unit (NU) 73. Tissue-specific mutations could subsequently occur in response to unique immune activity that is not observed in any other compartment. In conjunction with this hypothesis, another major driver could be the selective expression of the cellular receptor CD46, a regulator of complement activation that is present on all nucleated cells 27. It has been documented that CD46 is in abundance at the blood-brainbarrier (BBB), which is additionally a property exploited by the measles virus in entry of the CNS 74. Cellular receptor density has been implicated in the production of larger viral populations and subsequent increases in pathogenicity, as shown with Coxsackie B Virus in mice 75. It is possible that the same scenario occurs with BVDV, as increased receptor density locally expands the genomic repertoire. In turn, more substantial quasispecies populations facilitate increasingly complex interactions, thus altering selective gains and variant survival potential 30. This has been evidenced in vitro with foot and mouth disease virus (FMDV), where a double substitution in a conserved region of VP1 became dominant when passaged in large numbers, but 27

37 was not observed in a reduced population size 76. Similarly, poliovirus requires a minimum critical threshold of viral diversity to facilitate migration to the CNS 33. As such, compartmentalization in the CNS may be the product of increased viral diversity, which permits highly specific adaptation to the CNS. Although host-specific adaptation occurs, it is evident that similar evolutionary drivers are present in different cattle, as NS5B clones gave rise to shared mutations in PI cattle from different farms at positions 1110 and Although there were no identical obex-derived E2 mutations in PI cattle from different farms, PI 1 and 8 both had clusters harbouring amino acid changes of codon 57. While the significance of these particular mutations is currently unclear, it is evident that the cellular environment of the obex induced selection of mutation of this amino acid. It should be noted that despite the evidence for the CNS as an important viral reservoir, the clinical relevance of compartmentalization of diverse viral variants within the obex has yet to be defined, as it is unclear whether or not these variants affected the progression of disease or if they were able to circulate outside the CNS. Persistent infection with BVDV can result in presentation of neurological clinical signs and hypomyelination of the CNS at necropsy 12. It is possible that compartmentalization in the CNS plays a role in these features, although they were not present in PI cattle from the current study. Apart from the obex, high levels of viral diversity were also identified in the milk-derived E2 sequences from PI 9, however common mutations did not accumulate to result in compartmentalization. Nevertheless, this finding is concerning, as viral variants excreted in the milk could result in transmission to other members of the herd. Novel mutations in the E2 may affect the antigenicity and cell tropism of the virus, and could have unpredictably consequences even within a vaccinated herd. 28

38 This study provides evidence that despite the stability of consensus sequences, quasispecies distributions may vary between hosts, as supported by farm 1, where PI 1, 2, and 3 display high viral diversity, which was not reflected in PI 4. This phenomenon may be explained by the fact that PI 4 was a fetus in the fourth month of gestation, which represents an early time point following establishment of PI. Evaluation of quasispecies of this fetus may provide insight into vertical transmission of PI by revealing founding variants and determinants that facilitate infection. Interestingly, PI 2 is the dam of PI 3 and PI 3 is the dam of PI 4, resulting in a family of three PI generations. A second family exists in this study, as PI 9 was in the eighth month of gestation with PI 10 at the time of sampling. These two families are the focus of Chapter 4. 29

39 Chapter 4: Viral Variability and Vertical Transmission of Bovine Viral Diarrhea Virus in Two Families of Persistently Infected Cattle 4.1 Rationale Persistently infected cattle constantly shed large quantities of virus, resulting in horizontal transmission and rapid seroconversion of the herd 49. Additionally, vertical transmission results in the generation of new PI cattle, as every calf produced by a PI dam will also be PI 11. The previous chapter evidenced that, although PI cattle from the same herd produce identical consensus sequences, viral populations exist as a diverse cloud of mutants within the PI host. Currently, it is unclear as to how BVDV quasispecies populations develop following vertical transmission. Viral vertical transmission is often accompanied by a significant transmission bottleneck, as multiple barriers must be surpassed to allow establishment of the pathogen 77. Thereafter, bottleneck events in addition to population size and duration of viral replication within the host serve as determinants of fitness and virulence of viral populations 77. As such, description of quasispecies between multiple generations of PI cattle has potential to provide insight into PI vertical transmission events and reveal mutations that facilitate transmission and establishment of infection in the fetal host. The current sample size includes two families of PI cattle- one with three generations (PI dam, heifer, and fetus) and another consisting of a PI dam and fetus pair. This chapter evaluates quasispecies populations within these families through analysis of partial N-terminal E2 and NS5B coding regions. The goal of this work is to describe the effect of vertical transmission on quasispecies populations and, furthermore, test the transmission bottleneck hypothesis, gain insight into founder viral variants, and identify potential mutational facilitators of BVDV establishment in a new host. 30

40 4.2 Results Demographics of two families of persistently infected cattle To gain insight into viral variability within multiple generations of PI cattle N-terminal partial E2 and NS5B sequences from two PI families were analyzed. Identification numbers were reassigned to PI cattle to clarify the generation in which they represent within the family. Family 1 consists of three generations (F1-G1 is the dam of F1-G2, who was in the fourth month of gestation with F1-G3) and PI family 2 includes a PI dam-fetus pair (F2-G1 and F2-G2 respectively). Table 4.1 Demographics and new ID of PI cattle. The F within the identification code refers to the family ID and the G refers to the generation in which the PI represents within the PI family. ie. F1-G1 represents the first PI generation from family 1. Previous PI ID New PI ID Family BVDV Genotype Approximate Age 2 F1-G1 1 1a 5 years 3 F1-G2 1 1a 3 years 4 F1-G3 1 1a 4mo fetus 9 F2-G1 2 1b 3 years 10 F2-G2 2 1b 8mo fetus Phylogenetic analysis of N-terminal partial E2 and NS5B sequences Consensus sequences among members of the same family were identical, as described in Chapter 3 (Figure 3.1, appendix). Similarly, maximum likelihood trees display a range of diversity extending past the consensus sequence (Figure 4.1 and 4.2). Clusters of viral variants were designated a Cluster ID and specific mutations are listed in Table 4.2. Within family 1 over 83% of all fetal E2 sequences were grouped into one of two clusters (Cluster A and B, as denoted in Figure 4.1 A). Both clusters additionally gave rise to sub- 31

41 branches, which represented the accumulation of mutations that were exclusive to the fetus and not observed in other PI family members. It should also be noted that mutations in Cluster A existed at low frequencies in older PI generations (F1-G1 and F1-G2) and mutations in Cluster B were not observed in the first PI generation within that family (F1-G1). The patterns of variation between members of family 2 differed in that progenitor and progeny E2 variants were more highly interspersed and were not grouped into distinct clusters (Figure 4.1 B). However, the fetus in family 2 gave rise to four novel nonsynonymous mutations that were not observed in the progenitor PI, F2-G1 (listed in Table 4.2). 32

42 (A) Cluster B Cluster A A.1 B.1 F1-G1 F1-G2 F1-G3 (B) Cluster C Cluster D F2-G1 F2-G2 Cluster E Cluster F Figure 4.1 Maximum likelihood trees displaying viral variability of E2 sequences among all members of PI family 1 (A) and PI family 2 (B). Within family 1 (A), 83.3% of all fetal variants were grouped into one of two clusters and gave rise to sub-clusters exclusive to the fetus. Fetal variants within family 2 (B) did not form distinct clusters, however, four clusters produced novel mutations as listed in Table

43 Conservation of NS5B sequences was greater than that of E2, as emphasized by the average intrahost nucleotide variation in Figure 4.3. Within family 1, fetal clones were distributed around the consensus sequence, however, 39% of fetal variants were grouped in Cluster H, which was defined by one nonsynonymous mutation that was not observed in progenitor PI F1-G1. The family 1 fetus F1-G3 did not give rise to any novel NS5B mutations. The most diverse NS5B cluster of variants in family 1 (Cluster G) was defined by three silent mutations. This cluster was composed primarily of variants from F1-G2, although one fetal clone also acquired these three mutations as well. As opposed to family 1, fetal variants from family 2 formed distinct clusters that were primarily exclusive to the fetus. Cluster I displayed the accumulation of three silent mutations, two of which were only found in the fetus. Cluster J and K respectively displayed two and one novel silent mutations in the fetus. 34

44 A) Cluster G Cluster H F1-G1 F1-G2 F1-G3 (B) Cluster J F2-G1 F2-G2 I.2 I.1 K.1 Cluster I Cluster K Figure 4.2 Maximum likelihood trees displaying viral variability of NS5B sequences among all members of PI family 1 (A) and PI family 2 (B). Family 1 NS5B sequences formed two major clusters composed of variants from F1-G2 and F1-G3. Fetal variants from family 2 formed three major clusters, all defined by silent mutations. 35

45 Table 4.2 Descriptions of clustered E2 and NS5B mutations in two families of PI cattle. Clusters of E2 and NS5B sequences are displayed in Figure 4.1 and 4.2. Underlined mutations indicate mutations that were observed only in the fetus. Nucleotide position and change * aa change E2 Family 1 Cluster A 366 C:T Silent Cluster A C:T & 209 A:G Silent & Hà R Cluster B 237 G:A Silent Cluster B G:A & 59 C:T Silent & Pà L Family 2 Cluster C 80 C:T & 87 G:T & 166 C:T Tà I & Rà H and Silent Cluster D 166 C:T Silent Cluster E 221 T:C & 345 A:G Là S & Kà N Cluster F 342 A:G Silent NS5B Family 1 Cluster G 285 G:A & 813 C:T & 1230 G:A All silent Cluster H 1146 C:A Dà E Family 2 Cluster I 1302 T:C Silent Cluster I T:C & 1143 A:T Both Silent Cluster I T:C & 1143 A:T & 1188 A:G All Silent Cluster J 1290 G:A & 1329 C:T Both Silent Cluster K 1107 C:T Silent Cluster K C:T& 465 C:T Both Silent * Position on the NADL genome sequence Observed variability in the maximum likelihood trees of Figure 4.1 and 4.2 were quantified by calculating mean total pair-wise distances, which describe the average nucleotide variation within the host (Figure 4.3). Average intrahost nucleotide diversity for NS5B sequences was significantly more conserved than E2 diversity. However, patterns between PI family members were similar between both genes. The average intrahost nucleotide variation of the 4- month fetus (F1-G3) was significantly lower than other PI family members and the diversity of F1-G2 was significantly higher than both family members as well. Cohen s d was calculated for 36

46 both genes to assess the effect size of comparisons between all family members. The mean intrahost diversity for each PI is the product of thousands of pairwaise distances, as approximately 80 clones were sequenced for both genes from all PI cattle. Therefore, such large sample sizes may artificially produce statistical significance but do not necessarily reflect a biological signifance. Evaluation of Cohen s d is a method of analyzing the effect size of a comparison independent of statistical significance, where the effect of the PI generation on genetic diversity is the parameter of question. Among E2 sequences from family 1 the greatest effect size existed between F1-G2 and F1-G3, where a measurement of 0.6 indicated a moderate effect. Therefore, the effect of the PI generation (the condition of being a dam versus a fourth month fetus) moderately affects the genetic diversity of the E2 glycoprotein. Moreover, the effect size of fetal F1-G3 NS5B sequences compared to both progenitor PI cattle was large, with a value of 1.2 in both cases. Within family 2, identical trends were observed between E2 and NS5B sequences, where the fetus was significantly less diverse than the PI dam, although the effect size was small at 0.2. Calculations of Cohen s d are reported in the appendix for all PI cattle. 37

47 !"#$%&'())*))+,) ) * * * !"# $%&'# F1-G1 F1-G2 F1-G3 F2-G1 F2-G2 Figure 4.3 Mean total E2 and NS5B distances in two families of PI cattle based on a minimum of 70 sequences per gene per animal. All E2 sequences were significantly more diverse than NS5B sequences, although both genomic regions followed similar patterns between PI cattle. Sequences isolated from fetal PI cattle were significantly more conserved than other PI cattle in their respective families. 4.3 Discussion The previous chapter demonstrated that BVDV exists as a quasispecies distribution within the PI host, however it is currently unclear as to how BVDV diversity is shaped following vertical transmission from a PI dam to fetus. The current study evaluated E2 and NS5B sequence variability among members of two PI families to elucidate the role of vertical transmission in generation of BVDV diversity among PI cattle. Within both families, the viral variability of E2 and NS5B sequences derived from the PI fetus was significantly reduced compared to the PI dam, which is indicative of a transmission bottleneck. This phenomenon is not uncommon among viral infections and it is in support of Pfeiffer and Kirkegaard s tough transit model 78. Such model proposes that natural physical barriers and the immune system limit passage of the majority of viral variants and a limited 38