Molecular Investigation of Wildlife Herpesvirus and Parapoxvirus: Benefits and Limitations of Genetic Characterization of Dsdna Viruses from Tissues

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2019-12-18 Molecular Investigation of Wildlife Herpesvirus and Parapoxvirus: Benefits and Limitations of Genetic Characterization of dsDNA Viruses from Tissues

Dalton, Chimoné Stefni

Dalton, C. S. (2019). Molecular Investigation of Wildlife Herpesvirus and Parapoxvirus: Benefits and Limitations of Genetic Characterization of dsDNA Viruses from Tissues (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/111363 doctoral thesis

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Molecular Investigation of Wildlife Herpesvirus and Parapoxvirus: Benefits and Limitations of

Genetic Characterization of dsDNA Viruses from Tissues

Chimoné Stefni Dalton

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES

CALGARY, ALBERTA

DECEMBER, 2019

Abstract

Wildlife populations can be reservoirs or victims of pathogens shared with humans and/or domestic animals. Most diseases at the wildlife-livestock interface are caused by viruses.

Herpesviridae and Parapoxviridae are families of important double-stranded DNA (dsDNA) viruses that have been implicated in diseases of wildlife, domestic animals, and humans resulting from spill-over or zoonotic transmission, yet still little is known about viruses circulating in wildlife. Wildlife health surveillance is a primary tool for the management of zoonotic diseases, the control of diseases of domestic animals, and the preservation of wildlife populations. Studies herein conduct molecular surveillance of herpesviruses (HV) and orf virus (a parapoxvirus) through diagnostic polymerase-chain reactions (PCR), sequencing, and phylogenetic analysis using tissues of various wildlife animal species in Canada.

The viral DNA polymerase (DPOL) gene is an effective target for the detection and characterization of HV present in infected animals. Previously uncharacterized HV were characterized in marten across Canada, and Reindeer gamma-HV 1 was characterized in caribou from different herds. Phylogenetic analysis suggests HV have coevolved with their wildlife host at a species level.

Detection of orf virus was most successful when targeting the viral immunodominant envelope protein gene: B2L. Orf virus was detected in muskoxen on Victoria Island in areas managed by the Northwest Territories (NT) and Nunavut (NU), and on the adjacent mainland of

NU, Canada. Orf virus was present in males and females, from calf to adulthood, indicating this virus represents a disease threat for muskoxen. Next-generation sequencing was performed directly on the DNA extracted from tissues of four clinically infected, geographically distant

ii muskoxen in our study area. Phylogenetic analysis revealed Muskox orf virus (MxOV), to be unique from known orf viruses.

This thesis documents the diversity of HV circulating in wildlife, increases our awareness of limitations when using tissues for molecular surveillance, increases our understanding of orf virus infection in muskoxen, and highlight areas of much-needed research. Methodologies herein can be adapted for the surveillance of other dsDNA viruses, while the data directly contribute to the database of HV and orf virus sequences in Canadian wildlife that provide context for new or emerging pathogens.

iii

Preface

This thesis comprises manuscripts that have been published or are considered for publication. Chimoné Dalton, as the author, designed and performed this research and wrote the manuscripts. All work was performed at the Department of Ecosystem and Public Health,

Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada under the supervision of Dr. Frank van der Meer and guidance from the thesis committee members.

Published Manuscripts

Chapter 2 - Dalton CS, van de Rakt K, Fahlman Å, Ruckstuhl K, Neuhaus P, Popko R, Kutz S,

van der Meer F. Discovery of herpesviruses in Canadian wildlife. Archives of

virology 2017 162(2):449-456.

Manuscripts Considered for Publication

Chapter 3 – Dalton CS, Kutz S, Ruckstuhl K, Abdul-Careem MF, van der Meer F. Limited

genetic variation of herpesviruses in caribou (Rangifer tarandus spp.) and marten

(Martes americana) in Canada.

Chapter 4 – Dalton CS, Tomaselli M, Rothenburger JL, Mavrot F, Di Francesco J, Leclerc LM,

Checkley S, Kutz S, Abdul-Careem MF, van der Meer F. Detection and phylogenetic

analysis of orf virus and herpesvirus from muskoxen (Ovibos moschatus) in the

Canadian Arctic

Chapter 5 – Dalton CS, Workentine ML, Kutz S, van der Meer F. Next-generation sequencing

approach to investigate genome variability of Parapoxvirus in Canadian muskoxen

(Ovibos moschatus).

Acknowledgements

I would like to express my gratitude to Dr. Frank van der Meer for his guidance and understanding throughout this work. His commitment to the success of all his students is unmistakable. He has been pivotal in providing valuable feedback and shaping my critical thinking. I am grateful for my supervisory committee members Dr. Kathreen Ruckstuhl, Dr. Susan

Kutz, and Dr. Mohammad Abdul Faizal Careem for their involvement in my thesis and expertise in their respective fields. It has been an honour working with van der Meer Lab members Dr. Adam

Chernick, Alessa Kuczewski, Monica Rincon Garcia, and William Bremner, who have provided their friendship and moral support over the years. Thank you to the countless summer students who have brought fun and fresh perspective to the lab and to research. To the members of the Kutz lab, especially Dr. Matilde Tomaselli, Dr. Fabien Mavrot, and Juliette DiFrancesco, thank you for your hard work and long-distance friendship all the way from the other Vet Med buildings.

Special thanks to the veterinarians, hunters, trappers, and community members involved in this project, without whom this research would not be possible. I would also like to thank ArcticNet and the Faculty of Veterinary Medicine, the University of Calgary - especially the department of

Ecosystem & Public Health, for their financial support for this project.

Finally, I thank my family for their words of encouragement.

Thank you Chris, my husband, for your unconditional love, patience, and genuine support.

You believed in me when I was unable to, and for that I am forever thankful.

Table of Contents

Abstract ...... ii

Preface ...... iv

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... xii

List of Figures, Illustrations, and Graphics...... xiv

List of Symbols, Abbreviations, and Nomenclature ...... xx

CHAPTER 1: INTRODUCTION ...... 1

1.1 Impact of pathogens on wildlife ...... 1

1.2 “Infection” vs. “Disease” ...... 4

1.3 Surveillance and monitoring of wildlife pathogens...... 6

1.4 Viruses and the molecular surveillance of wildlife viruses ...... 7

1.5 Evolutionary Relationships and Phylogeny ...... 9

1.6 Herpesviruses (HV) and Poxviruses in wildlife ...... 10

1.7 Summary ...... 13

PART I: MOLECULAR SURVEILLANCE OF HV IN CANADIAN WILDLIFE ANIMALS

...... 15

CHAPTER 2: DISCOVERY OF HERPESVIRUSES IN CANADIAN WILDLIFE ...... 16

2.1 Abstract ...... 16

2.2 Introduction ...... 16

2.3 Materials and methods ...... 18

2.3.1 Sample collection ...... 18

2.3.2 Sample preparation and DNA extraction ...... 18

2.3.3 PCR conditions ...... 19

2.3.4 Gel extraction and sequencing...... 20

2.3.5 Phylogenetic analysis...... 20

2.4 Results ...... 21

2.5 Discussion...... 31

2.6 Acknowledgements ...... 33

2.7 Declaration of Conflicts of interest ...... 34

2.8 Funding ...... 34

CHAPTER 3: LIMITED GENETIC VARIATION OF HERPESVIRUSES IN CARIBOU

(RANGIFER TARANDUS SPP.) AND MARTEN (MARTES AMERICANA) IN

CANADA ...... 35

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3.1 Abstract ...... 35

3.2 Introduction ...... 36

3.3 Materials and methods ...... 39

3.3.1 Sampling and DNA Extraction...... 39

3.3.2 Polymerase chain reaction (PCR) and sequencing ...... 41

3.3.3 Quality Control and Phylogenetic analysis ...... 42

3.4 Results ...... 43

3.4.1 Caribou Sample Data ...... 43

3.4.2 Marten Sample Data ...... 47

3.5 Discussion...... 52

3.6 Acknowledgements ...... 56

3.7 Funding: ...... 56

3.8 Declaration of Conflicts of interest ...... 57

3.9 Supplementary Data ...... 57

PART II: MOLECULAR SURVEILLANCE OF ORF VIRUS IN CANADIAN

MUSKOXEN...... 71

CHAPTER 4: DETECTION AND PHYLOGENETIC ANALYSIS OF ORF VIRUS AND

HERPESVIRUS FROM MUSKOXEN (OVIBOS MOSCHATUS) IN THE CANADIAN

ARCTIC ...... 72

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4.1 Abstract ...... 72

4.2 Introduction ...... 73

4.3 Materials and methods ...... 76

4.3.1 Sampling and Study Area ...... 76

4.3.2 Histology ...... 76

4.3.3 DNA Extraction and Polymerase Chain Reaction (PCR) ...... 77

4.3.4 Sequencing and phylogenetic analysis...... 78

4.4 Results ...... 79

4.5 Discussion...... 94

4.6 Acknowledgements ...... 97

4.7 Declaration of Conflicts of interest ...... 98

4.8 Supplementary Data ...... 98

CHAPTER 5: NEXT-GENERATION SEQUENCING APPROACH TO INVESTIGATE

GENOME VARIABILITY OF PARAPOXVIRUS IN MUSKOXEN (OVIBOS

MOSCHATUS) ...... 103

5.1 Abstract ...... 103

5.2 Introduction ...... 104

5.3 Materials and Methods ...... 107

5.3.1 Sampling ...... 107

5.3.2 Density Gradient Centrifugation ...... 108

5.3.3 Library Preparation and Next-Generation Sequencing ...... 109

5.3.4 Data Analysis ...... 109

5.3.5 Recombination Analysis ...... 111

5.4 Results ...... 112

5.4.1 Reads Data ...... 112

5.4.2 Assembly Data ...... 112

5.4.3 Variants Data ...... 113

5.4.4 Comparison of Individual Proteins ...... 114

5.4.5 Whole Genome & Concatenated Protein Tree Comparison...... 115

5.4.6 Recombination Analysis ...... 121

5.5 Discussion...... 126

5.6 Acknowledgements ...... 128

5.7 Declaration of Conflicts of Interest ...... 129

5.8 Funding ...... 129

5.9 Supplementary Data ...... 129

CHAPTER 6: DISCUSSION AND CONCLUSION ...... 157

Part I: Molecular surveillance of HV ...... 157

Part II: Molecular surveillance of orf virus ...... 161

BIBLIOGRAPHY ...... 164

APPENDIX A: COPYRIGHT PERMISSIONS ...... 181

List of Tables

Table 2.1 A summary of HV DNA-dependent DPOL gene amino acid sequences from this study

and the most closely related virus sequences in the GenBank database ...... 23

Table 3.1 Summary of Canadian caribou samples collected for HV detection ...... 39

Table 3.2 Summary of Canadian marten samples collected for HV detection ...... 40

Table 3.3 Summary of DNA findings from caribou samples ...... 43

Table 3.4 Summary of DNA findings from marten samples ...... 47

Supplementary Table 3.1 Summary of HV DPOL sequences included for comparison of Cervidae-

derived viruses ...... 58

Supplementary Table 3.2 Summary of mtDNA sequences within the family Cervidae included for

comparison. Sequences spanned the partial tRNA-Thr, tRNA-Pro, D-loop, and partial

tRNA-Phe region...... 59

Supplementary Table 3.3 Summary of HV DPOL sequences included for comparison of mustelid-

derived viruses ...... 61

Supplementary Table 3.4 Summary of mtDNA sequences within the family Mustelidae included

for comparison. Sequences spanned the Partial tRNA-Thr, tRNA-Pro, D-loop, tRNA-Phe

region unless otherwise indicated...... 62

Table 4.1 Primers used in this study...... 77

Table 4.2 Summary of muskoxen included in this study. UCID represents the sampling batch.. 81

Supplementary Table 4.1 GPS location of muskoxen at time of hunt...... 99

Table 5.1 Muskox samples included in this study ...... 108

Table 5.2 Taxonomic summary of NextSeq reads from muskox samples ...... 112

Table 5.3 Variants in reads from MX samples with reference to MxOV genome ...... 114

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Table 5.4 Predicted recombination sites of MxOV with reference OV strains. The major parent, or

backbone, is the parental strain contributing a larger fraction of sequence. The minor parent

is the strain likely involved in the recombination event, contributing a smaller fraction of

sequence. Statistical significance at p>0.05...... 123

Supplementary Table 5.1 Genomes included in phylogenetic and BLASTp analysis...... 130

Supplementary Table 5.2 Predicted genes of MxOV ...... 131

Supplementary Table 5.3 Blast hits to reference sequences of MxOV ORFs identified using Prokka

1.13.3. The highest scoring blast hits are shown in bold...... 137

xiii

List of Figures, Illustrations, and Graphics

Figure 1.1 The epidemiological roles of wildlife in the movement of pathogens including viruses.

...... 1

Figure 1.2 The value of wildlife. Inspired by the review from Chardonnet et al. (2002), a research

article by Ezran et al. (2017), and book by Chivian and Bernstein (2010)...... 2

Figure 1.3 Factors affecting biodiversity loss. Inspired by the articles of Aguirre and Tabor (2008),

Aguirre (2009), and Aguirre et al. (2017)...... 3

Figure 2.1 RAxML tree of a MUSCLE alignment of 51 HV DPOL amino acid sequences. Twenty-

one viral sequences from wildlife from this study are shown in blue, and accession numbers

are shown in parentheses. PCH; Porcupine caribou herd. ML analysis was carried out in

Geneious 8.1 using the RAxML plugin, with the scale bar indicating the number of amino

acid substitutions per site. Branching support is shown as bootstrap percentages for 1,000

bootstrap replicates ...... 25

Figure 2.2 MrBayes Bayesian inference phylogenetic tree of a MUSCLE alignment of 51 HV

DPOL amino acid sequences. Twenty-one viral sequences from wildlife from this study are

shown in blue, and accession numbers are shown in parentheses. PCH; Porcupine caribou

herd. Bayesian analysis was carried out in Geneious 8.1 using the MrBayes plugin, with the

scale bar indicating the number of amino acid substitutions per site. A burn-in of 1,000 was

used, with branching support shown as posterior probability percentages (See previous

page)...... 27

Figure 2.3 Alpha- and beta-HV-like marten amino acid sequences aligned with related published

sequences using MUSCLE. Gaps in the alignment are indicated with a dash...... 30

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Figure 3.1 Bayesian phylogenetic tree of gamma-HV DPOL fragments isolated from cervid hosts.

Sequences are approximately 200 bp in length. Colours indicate caribou herd: green

(Bathurst), orange (Leaf River), purple (Porcupine). Numbers at the nodes indicate posterior

probability percentage. Scale bar indicates nucleotide substitutions per site...... 45

Figure 3.2 Bayesian phylogenetic tree of the mtDNA control region of cervids. Numbers at the

nodes indicate posterior probability percentage. Colours indicate caribou herd: blue

(unknown from BC), orange (Leaf River), purple (Porcupine). Red bar belongs to the

Beringian (BEL) linage, green bar belongs to the North American (NAL) linage, black bar

(BEL/NAL) are of mixed lineages. Rangifer tarandus caribou (woodland caribou), R. t.

terranovae (Newfoundland caribou), R. t. granti (Grant’s caribou), R. t. groenlandicus

(barren-ground caribou), Odocoileus virginianus (white-tailed deer), R. t. tarandus (Eurasian

reindeer), O. h. hemionus (mule deer), Cervus canadensis nelsoni (Rocky Mountain elk),

Dama dama (fallow deer), Bos taurus (cattle), Alces americanus (moose). Scale bar indicates

nucleotide substitutions per site...... 46

Figure 3.3 Bayesian phylogenetic tree of gamma-HV DPOL fragments isolated from mustelid

hosts. Colours indicate sample origin: blue (Pemberton, BC), orange (southern Labrador).

Marten gamma-HV 1 and 2 are from marten in the Northwest territories. Numbers at the

nodes indicate posterior probability percentage. Scale bar indicates nucleotide substitutions

per site...... 49

Figure 3.4 Bayesian phylogenetic tree of alpha-HV DPOL fragments isolated from mustelid hosts.

Colours indicate sample origin; dark blue: McBride, BC, pink: Vancouver Island, BC,

purple: Whitehorse, YK. Numbers at the nodes indicate posterior probability percentage.

Scale bar indicates nucleotide substitutions per site...... 50

Figure 3.5 Bayesian phylogenetic trees of mtDNA partial tRNA-Thr, tRNA-Pro, and partial D-

loop sequences from mustelids. Pink font indicates marten sourced from Vancouver Island,

BC. Numbers at the nodes indicate posterior probability percentage. Martes americana

(American marten), M. pennanti (fisher), M. foina (stone marten), M. zibellina (sable), M.

melampus (Japanese marten), Meles meles (Eurasian badger), Neovison vison (American

mink), Enhydra lutris (sea otter), Lutra lutra (common otter). Scale bar indicates nucleotide

substitutions per site...... 51

Supplementary Figure 3.1 ML phylogenetic tree of gamma-HV DPOL fragments isolated from

cervid hosts. Colours indicate caribou herd: green (Bathurst), orange (Leaf River), purple

(Porcupine). Numbers at the nodes indicate bootstrap percentage; scale bar indicates

nucleotide substitutions per site...... 64

Supplementary Figure 3.2 ML phylogenetic tree of mtDNA control region sequences from cervid

hosts. Colours indicate caribou herd; blue: unknown from British Columbia, orange: Leaf

River, purple: Porcupine. Numbers at the nodes indicate bootstrap percentage; scale bar

indicates nucleotide substitutions per site...... 65

Supplementary Figure 3.3 ML phylogenetic tree of gamma-HV DPOL fragments isolated from

mustelid hosts. Colours indicate sample origin; blue: Pemberton, BC, orange: Labrador.

Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide

substitutions per site...... 66

Supplementary Figure 3.4 ML phylogenetic tree of alpha-HV DPOL fragments isolated from

mustelid hosts. Colours indicate sample origin; dark blue: McBride, BC, pink: Vancouver

Island, BC, purple: Whitehorse, YK. Numbers at the nodes indicate bootstrap percentage;

scale bar indicates nucleotide substitutions per site...... 67

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Supplementary Figure 3.5 ML phylogenetic trees of mtDNA tRNA-Thr, tRNA-Pro, and D-loop

sequences from marten in this study and published sequences from mustelids. Pink font:

marten sourced from Vancouver Island, BC. Numbers at the nodes indicate bootstrap support

percentage. Scale bar indicates nucleotide substitutions per site...... 68

Supplementary Figure 3.6 Bayesian phylogenetic tree of partial mtDNA D-loop and tRNA-Phe

sequences from marten in this study with published sequences from Genbank. Colours

indicate the location of sample origin; pink: Vancouver Island, BC, light blue: Pemberton,

BC, dark blue: Lac La Hache, BC, grey: 100 Mile House, BC, yellow: Labrador. Numbers

at the nodes indicate posterior probability percentage; scale bar indicates nucleotide

substitutions per site...... 69

Supplementary Figure 3.7 ML phylogenetic tree of mtDNA D-loop and tRNA-Phe sequences from

marten in this study and published sequences from Genbank. Colours indicate the location

of sample origin; pink: Vancouver Island, BC, light blue: Pemberton, BC, dark blue: Lac La

Hache, BC, grey: 100 Mile House, BC, yellow: Labrador. Numbers at the nodes indicate

bootstrap percentage; scale bar indicates nucleotide substitutions per site...... 70

Figure 4.1 Count of muskox samples with macroscopic orf-like lesions among different age

groups. Age groups are as follows: calf (<1 year old), yearling (1-2 years old), juvenile (2-3

years old), adult (4 years old)...... 87

Figure 4.2 Count of muskox samples with macroscopic orf-like lesions among different sexes. 88

Figure 4.3 Microscopic appearance of skin samples from Muskoxen. H&E stain. A) Normal haired

skin (control sample with no macroscopic abnormalities). MX116, 4X, scale 200 m; B)

Severe proliferative dermatitis of the lip with epidermal hyperplasia (short arrow),

hyperkeratosis (thin arrow) and intracorneal pustules (thick arrow) characteristic of orf virus

xvii

infection. MX444, 1.25X, scale 1 mm; C) Severe proliferative dermatitis of haired skin with

epidermal hyperplasia (short arrow), hyperkeratosis (thin arrow) and intracorneal pustules

(thick arrow) characteristic of orf virus infection. MX319, 4X, scale 200 m; D) Bacterial

cells within the orthokeratotic hyperkeratosis affecting the lip as shown by the black arrow.

MX444, 60X, scale 20 m...... 89

Figure 4.4 Map of northern Canada showing the number of sequence-confirmed infections of orf

virus in muskoxen. White numbers indicate the total number of muskoxen sampled...... 90

Figure 4.5 Bayesian phylogenetic tree of the orf virus B2L gene fragment. Bold indicates

sequences from muskoxen and domestic goat from this study. Branch support values > 50%

are shown; NCBI GenBank accession numbers are in parentheses; BPSV: Bovine papular

stomatitis virus; PCPV: Pseudocowpoxvirus; scale bars show nucleotide substitutions per

site...... 92

Figure 4.6 Phylogenetic trees of the HV DPOL fragment. Sequences from this study are shown in

blue. Branch support values > 50% are shown; NCBI GenBank accession numbers are shown

in parentheses. Scale bars show nucleotide substitutions per site...... 93

Supplementary Figure 4.1 RAxML phylogenetic tree of the orf virus B2L gene fragment. Bold

indicates 493 bp sequences from muskoxen and domestic goat from this study. NCBI

GenBank accession numbers are in parentheses. Scale bars show nucleotide substitutions per

site...... 101

Supplementary Figure 4.2 RAxML phylogenetic tree of the HV DPOL gene fragment. Bold

indicates sequences from muskoxen in this study. Branch support values > 50% are shown;

NCBI GenBank accession numbers are in parentheses. Scale bars show nucleotide

substitutions per site...... 102

xviii

Figure 5.1 Amino acid protein trees of key parapoxvirus genes and virulence factors. Ultrafast

bootstrap support values are given at the nodes. Trees were rooted on OV-D1701 when

PCPV homologue sequences were unavailable. Scale bars indicate amino acid substitutions

per site (See previous page)...... 117

Figure 5.2 Heatmap showing percent identity of MxOV blast hits to related genomes ordered by

genomic position. Blast hits with a percent identity > 50% and coverage of > 10% were

retained...... 118

Figure 5.3 Whole-genome nucleotide comparison of MxOV and parapoxvirus reference

sequences. Ultrafast bootstrap support values are given at the nodes...... 119

Figure 5.4 Comparison of concatenated gene sequences from MxOV and reference

parapoxviruses. Ultrafast bootstrap support values are given at the nodes...... 120

Figure 5.5 MxOV genome graphic depicting significant predicted recombination events. Purple

indicates the genome of MxOV; grey shading along the genome identifies other regions or

recombination excluded by our selection criteria. Names of the most likely donor sequence

(minor parent) with approximate recombination region are given in coloured font...... 122

Figure 5.6 Phylogenetic trees of the three most significant recombinant regions of MxOV. Branch

labels are ML bootstrap support values. Scale bars indicate nucleotide substitutions per site

(See previous page)...... 125

Supplementary Figure 5.1 Heatmap ordered by percent identity of MxOV blast hits to related

reference genomes. Blast hits with a percent identity > 50% and coverage of > 10% were

retained...... 156

xix

List of Symbols, Abbreviations, and Nomenclature

Abbreviation Definition

3’ Three prime

5’ Five prime

Alpha-HV Alphaherpesvirus

B2L Immunodominant Orf virus envelope protein

Beta-HV Betaherpesvirus

BLAST Basic local alignment search tool

BLASTn Basic local alignment search tool for nucleotides

BLASTp Basic local alignment search tool for protein amino acids

BPSV Bovine papular stomatitis virus

CBP Chemokine binding protein

CCV Channel catfish herpesvirus

CE Contagious ecthyma

CHV-1 Canid herpesvirus 1

CoHV-1 Columbid herpesvirus 1

CWD Chronic wasting disease

D_MX Discovered (dead) muskox

DNA Deoxyribonucleic acid

DNAtopo DNA topoisomerase dNTP Deoxyribonucleotide triphosphate

DPOL DNA polymerase dsDNA Double-stranded DNA dUTPase deoxyuridine 5′-triphosphate nucleotidohydrolase

EBV Epstein-Barr virus

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent assay

F1L Immunodominant Orf virus envelope protein

Gamma-HV Gammaherpesvirus

GIF Granulocyte-macrophage colony-stimulating factor inhibitory factor

GPS Global positioning system

FHV-1 Felid herpesvirus 1

H&E Hematoxylin and eosin stain

HSV-1 Herpes simplex virus 1

HV Herpesvirus

IL-2 Interleukin 2

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KHV Koi herpesvirus

KSHV Kaposi’s sarcoma-associated herpesvirus

ML Maximum likelihood mRNA Messenger RNA mtDNA Mitochondrial DNA

MX Muskox nuDNA Nuclear DNA

PCH Porcupine Caribou Herd

PCPV Pseudocowpoxvirus

PCR Polymerase chain reaction

RAxML Randomized Axelerated Maximum Likelihood

RNA Ribonucleic acid

SARS Severe acute respiratory syndrome virus

VEGF Vascular endothelial growth factor vIL-10 Viral interleukin 10

VZV Varicella zoster virus

xxii

Unit Abbreviation Definition

C Degree Celsius bp Base pair g Gram g Gravitational force kbp Kilobase pair km Kilometer, one thousand meters

L Microlitre; 10-6 litre

m Micrometer; 10-6 meter mg Milligram; 10-3 gram min Minute mM Millimolar; 10-3 mol/litre mya Million years ago; time scale nm Nanometer; 10-9 meter pmol Picomole; 10-12 mol w/v Weight by volume; g of solute/100 mL of solution

xxiii

All living things need their instruction manual (even nonliving things like viruses) and that is all they need, carried in one very small suitcase.

- L.L. Larison Cudmore, The Center of Life: A Natural History of the Cell (1977)

xxiv

CHAPTER 1: INTRODUCTION

1.1 Impact of pathogens on wildlife

Emerging diseases of human or veterinary importance are a major challenge to human society (Artois et al., 2009). Wildlife are implicated as a reservoir for many pathogens, and depending on the pathogen, wildlife may play a role as maintenance, spillover, or spillback host

(Figure 1.1) (Bengis et al., 2002; Siembieda et al., 2011; Aguirre, 2017). Approximately 43% of emerging human pathogens originate in wildlife (Jones et al., 2008). Ebola virus, West Nile virus, severe acute respiratory syndrome virus (SARS), Hendra virus, and Nipah virus are among many pathogens that have jumped directly or indirectly from wildlife to humans, resulting in disease

(Funk and Piot, 2014; Marra et al., 2004; Kuehn, 2013; Egbetade et al., 2015). Historically, wildlife pathogens were studied when they were regarded as a threat to human or livestock health, or zoo and game animal survival and welfare (Daszak et al., 2000; Gortazar et al., 2006;

Cunningham et al., 2017). Classical swine fever, foot-and-mouth disease, bovine tuberculosis, and brucellosis are well-known wildlife pathogens that cause substantial economic loss due to direct mortality, trade restrictions, or farm-wide culling of domestic animals (Joseph et al., 2013).

Figure 1.1 The epidemiological roles of wildlife in the movement of pathogens including viruses.

Wildlife refers to any biological entity that lives, grows, disperses, and reproduces independently of humankind with phenotypes not selected by humans, and constitute most of the living biodiversity on earth and are valuable in many ways to humankind (Figure 1.2). However, these animals migrate without regard for jurisdictional boundaries, and the movement of migratory wildlife may straddle international borders (Young, 1997; Grant and Quinn, 2007). Importantly, their pathogens migrate with them, causing significant challenges for control. The study of wildlife disease and wildlife health plays a critical role in the conservation of biodiversity, wildlife management, and the protection of the health of domestic animals and humans (Gortazar et al.,

2014). The magnitude of wildlife disease impacts rests on our ability to properly plan, prepare, and cope (Dunbar et al., 2007).

Figure 1.2 The value of wildlife. Inspired by the review from Chardonnet et al. (2002), a research article by Ezran et al. (2017), and book by Chivian and Bernstein (2010).

Several factors have been identified as major contributors to wildlife and biodiversity loss, these are depicted in Figure 1.3. Pathogens can interact with other factors to contribute to temporary or permanent declines in animal numbers of wildlife species or lead to local and/or global extinctions (Smith et al., 2009). Recovery of wildlife populations that are damaged or lost, for example through emerging pathogens, can take decades, if at all possible (WWF, 2018).

Currently, there is a lack of knowledge regarding the diversity and abundance of pathogens in wildlife species, making it difficult to establish the relative importance of pathogens as a driver of species extinction (Smith et al., 2009).

Figure 1.3 Factors affecting biodiversity loss. Inspired by the articles of Aguirre and Tabor (2008),

Aguirre (2009), and Aguirre et al. (2017).

There are several well-known examples in which pathogens have played a role in the decimation of wildlife. Fungal pathogens such as Batrachochytrium dendrobatidis and B. salamandrivorans have caused the extinction of at least 90 species of amphibians worldwide

(Scheele et al., 2019). Bacillus anthracis, a bacterial pathogen causing anthrax, impacted 26.4% of hippopotamus populations and 3.8% of Cape buffalo in Namibia in 2017 (Cossaboom et al.,

2019). Wildlife diseases include those caused by infectious proteins (prions) such as chronic wasting disease (CWD). CWD is fatal to deer, elk, moose, and caribou, and has caused a decrease in total cervid numbers in heavily affected areas (Carlson et al., 2018). Viral pathogens such as rinderpest virus resulted in the extirpation of more than 90% of wild buffalo populations in Kenya and caused local extinctions of the tsetse fly (Daszak et al., 2000).

1.2 “Infection” vs. “Disease”

“Wildlife disease” is a widely used term that refers to infectious and non-infectious diseases affecting wildlife, and all diseases related to wildlife, i.e. infectious agents carried by healthy wildlife that have the potential to cause disease in humans or domestic animals (Ryser-

Degiorgis et al., 2015). For the purpose of this thesis, the terms infection and disease are not synonymous as it relates to wildlife pathogens. An infection results when a pathogen invades and replicates within a host. Disease results when, as a consequence of the invasion and replication of a pathogen, tissue function is impaired (NIH, 2007).

Diseases may be classified as endemic, cluster, outbreak or epidemic depending on the prevalence (the overall number of cases at one point in time), incidence (the number of new cases given a period or time), and geographic scope of a particular disease at a point in time. Endemic diseases are those constantly maintained at a baseline level in a population or geographic area

4 without external inputs (Riley, 2019). A disease cluster is defined by unusually high incidence in close proximity in space and time (Riley, 2019). Given enough clusters, a disease may be classified as an outbreak, which is when cases occur in greater numbers than expected in a population or area (Riley, 2019). Disease outbreaks are often caused by novel pathogens, the emergence of pathogens having undergone antigenic drift, or the appearance of known pathogens in new hosts

(Joseph et al., 2013; Aguirre 2017). When outbreaks spread rapidly to more populations, the disease status may be classified as an epidemic (Riley, 2019). The ability to detect and characterize infections when they occur is paramount to determining the disease status of a pathogen in a population.

Infection relies on the transmission of pathogens which depends on the dynamics of how infectious and susceptible hosts interact, both spatially and socially (Altizer et al., 2003).

Transmission is influenced by the density of a population, the frequency of interaction within and between populations, and the patterns of mixing at the individual level (Ferrari et al., 2011).

Vector-borne and sexually transmitted diseases are generally assumed to be transmitted in a frequency-dependent manner because the number of contacts is independent of population density

(Ferrari et al., 2011). On the other hand, directly transmitted diseases are typically expected to spread in a density-dependent manner because the number of encounters may increase with density and/or population size (Ferrari et al., 2011). In the context of wildlife, the dynamics of pathogen transmission are often poorly understood (Smith et al., 2009). A wide range of factors may affect contact rates and transmission in these populations; breeding male and female territoriality, movement patterns, and dispersal may be seasonal- and density-dependent (Smith et al., 2009).

Surveillance of wild populations can help elucidate the seasonal variations in social behavior and/or host susceptibility to infection (Smith et al., 2009).

1.3 Surveillance and monitoring of wildlife pathogens

Wildlife surveillance is undertaken to limit the harmful consequences of pathogens or diseases to humans, livestock, the agrifood industry, and to preserve wildlife populations and natural resources (Artois et al., 2012). Disease control in wildlife requires the establishment of proper surveillance and monitoring strategies (Gortazar et al., 2015). Surveillance and monitoring build on the steady collection, collation, and analysis of data related to animal health but differs at the aim and target population (Gortazar et al., 2015). Surveillance targets wildlife populations classified as healthy to demonstrate the absence of infection or infestation, or for the early detection of possible exotic diseases or emerging diseases (OIE, 2019). Animal health surveillance is a tool to facilitate the control of infection or infestation, to provide data for use in risk analysis, for animal or public health purposes, to substantiate the rationale for sanitary measures and for providing assurances to trading partners (OIE, 2009). Monitoring aims to detect spatial and temporal trends of known infected wildlife populations (Artois et al., 2009).

Comprehensive surveillance and monitoring of wild mammal populations generates data needed to inform conservation decisions (population size or trends, distribution, habitat preferences, and limiting factors), and enhances our capacity to detect and control infectious diseases that may emerge in human or domestic animal populations (Artois et al., 2009; Sullivan et al., 2017). Wildlife present some challenges to the practicality of health surveillance since populations are usually remote, difficult to assess, and their pathogens are not always similar to those of domestic species. Consequently, surveillance data are frequently sparse, biased or inaccurate (Artois et al., 2012). However, when wildlife samples are properly stored and analysed, they have the capacity to detect relevant health events in natural animal populations.

Surveillance and monitoring of pathogens in wildlife are similar in many respects in terms of their objectives, concepts, and methodology to those undertaken for domestic animal health surveillance and monitoring (Artois et al., 2009). However, there are substantial differences and challenges owing to the zoological, behavioural and ecological characteristics of wildlife populations (Artois et al., 2009; OIE, 2019). Consequently, definitions, methods, and procedures must often be adapted to suit the unique conditions of wildlife disease surveillance (Artois et al.,

2009).

1.4 Viruses and the molecular surveillance of wildlife viruses

Viruses comprise the most abundant group of biological entities on earth, and can infect vertebrates, invertebrates, plants, protozoa, fungi, archaea, and bacteria (Gelderblom 1996;

Krupovič and Bamford, 2010; Rosario and Breitbart, 2011; Kazlauskas et al., 2016). The number of virus particles typically is 10–100 times greater than the number of cells in marine, soil, and animal-associated environments (Koonin and Dolja, 2013). The virus world displays an enormous diversity of genome structures and sizes, replication and expression mechanisms, and virus particle structure (Koonin et al., 2015). Viral genes typically evolve much faster than genes of cellular organisms, and as a result, most of the genetic diversity on earth is probably concentrated in the virus world (Koonin et al., 2015).

An extensive literature search by Wiethoelter et al. (2015) showed that 60% of the diseases studied at a wildlife-livestock interface from 1912 to 2013 were caused by viruses (Wiethoelter et al., 2015). Many viruses infect wildlife without evidence of disease (Levinson et al., 2013; Ryser-

Degiorgis, 2013). Among 312 mammal-virus associations analyzed by Levinson et al. (2013), 72% of wildlife viruses were reported without evidence of disease in their host. Viral strategies may

7 favour the development of visible disease or asymptomatic infection, but ultimately the probability of having disease depends on the interaction between the virus and the host (Levinson et al., 2013).

There are many viruses (families: Hepadnaviridae, Polyomaviridae, Papillomaviridae,

Adenoviridae, Herpesviridae, Poxviridae, and Asfarviridae) with a double-stranded DNA

(dsDNA) genome that infect humans and animals (Modrow et al., 2013). These viruses have many ways to regulate and influence cell division of the host, sometimes leading to persistent infection and/or the development of cancers (Modrow et al., 2013).

DsDNA viruses belong to Class I viruses that replicate by transcribing their DNA into messenger RNA (mRNA), much like cells (Baltimore, 1971). Most dsDNA viruses are nuclear replicating with the exception of poxviruses which replicate in the cytoplasm of infected vertebrate host cells (Schmid et al., 2014). The genomes of dsDNA viruses infecting vertebrates can vary from as small as 5 kbp in the case of Bovine polyomavirus, to as large as 360 kbp in the case of

Canarypox virus (Van Etten et al., 2010; Campillo-Balderas et al., 2015). Plants such as algae are host to the dsDNA virus family Phycodnaviridae but currently, however to date there are no reports of dsDNA viruses that can infect flowering plants, trees, shrubs, or fungi (Yu et al., 2010; King et al., 2012; Campillo-Balderas et al., 2015).

The use of modern molecular techniques allows us to rapidly identify and document pathogens present during an infection (Real and Biek, 2007). Detection of viral infection can be accomplished using electron microscopic visualization of virus particles or inclusion bodies, visualization of lesions, cell culture infection, immunofluorescent or Enzyme-Linked

Immunosorbent Assay (ELISA) detection of antibodies or viral antigens, polymerase chain reaction (PCR) targeting viral genes (Anderson et al., 2014; Hjalgrim et al., 2007; Namvar et al.,

2005; Singh et al., 2005). Microscopy and cell culture diagnosis are frequently limited by sample

8 type and quality, special culturing conditions or timing of lesion biopsy, and cannot distinguish between similar viruses (Anderson et al., 2014). Diagnosis using ELISA may be limited by cross- reactivity of antibodies resulting in the inability to differentiate between closely related viruses

(Anderson et al., 2014).

PCR, one of the most important analytical tools of molecular biology, is a sensitive detection and specific method of genotyping viral pathogens in infected samples (Iserte et al.,

2013; Anderson et al., 2014). PCRs using degenerate primers allows for the amplification of all the possible combinations of nucleotide sequences coding for a given protein sequence, and for the detection of new viral family members (Iserte et al., 2013). Molecular techniques allow for the evaluation of gene sequences of pathogens, enabling further analysis of their evolutionary relationships. Surveillance of viruses in apparently healthy wildlife will maximize virus discovery and facilitates the development of more powerful models to predict, prevent, and control the emergence and spread of wildlife pathogens (Real and Biek, 2007; Siembieda et al., 2011; Joseph et al., 2013; Levinson et al., 2013; Cunningham et al., 2017).

1.5 Evolutionary Relationships and Phylogeny

Evolutionary relationships of pathogens are best represented in the form of a phylogenetic tree wherein the branching pattern reflects a hypothesis of how species evolved from a series of common ancestors. The process of placing entities into a phylogeny, known as phylogenetic characterization, can be performed using alignments of nucleotide or amino acid sequences of genes, motifs, other regions of interest, or complete genomes of organisms. Phylogenetic analysis is a useful tool in inferring evolutionary relationships of wildlife species or pathogens from

9 different sources, geographic locations and/or even different time periods (Iyer, 2001; Omland,

2014; Koonin et al., 2015).

In the context of virology, phylogenetic analysis is useful for understanding the diversity and evolution of viruses not only within a viral family but also among different viral families that may have a common origin (Iyer, 2001). Viral phylogenies can give us an indication of the risk of spillover transmission, and of the potential of reservoir hosts (Olival et al., 2017). Phylogenetic analysis has led to advances in molecular epidemiology by elucidating the actual transmission histories in wildlife populations and/or the identification of the most probable donor individual of a new infection (Real and Biek, 2007; Wang et al., 2015).

1.6 Herpesviruses (HV) and Poxviruses in wildlife

HV and poxviruses are examples of dsDNA viruses that have been characterized in wild animals around the world. Infections with these viruses may directly increase mortality or cause negative effects leading to mortality such as the reduced capacity to feed, avoid predators, reproduce and maintain homeostasis (Costantini et al., 2018).

Most vertebrates are infected with one or more HV and remain so for the rest of their lives.

HV pathogenesis occurs in three phases: acute (lytic) infection, latency, and reactivation. During the lytic phase, virus particles replicate rapidly in epithelial cells resulting in disease symptoms such as mucosal lesions, ulcers, enteritis, encephalitis, conjunctivitis, and other pathology in multiple organ systems depending on the HV (Griffin et al., 2010; White et al., 2012; Constable et al., 2017). Lytic viral replication is eventually controlled by the host adaptive immune response

(White et al., 2012). The lytic phase is followed by the prolonged latent phase occurring in tissues which is characterized by the absence of viral particles, passive viral replication in the host nucleus,

10 minimal expression of viral genes, and immune evasion (Gibbs, 1977; Griffin et al., 2010; White et al., 2012). All HV are capable of both lytic and latent stages of infection. Since there is no immune activation during latency, infected hosts express no overt evidence of disease (Das Neves et al., 2010).

Occasionally, HV may reactivate from latency and enter a lytic phase. Reactivation of latent HV is not well understood though it is often linked to immunodeficiency resulting in the production and shedding of new viral particles (Whitley, 1996). Furthermore, pathological conditions can arise due to the inherent oncogenic potential possessed by certain HV (Griffin et al., 2010). Most of our knowledge of HV replication is understood in the context of human HV:

Herpes simplex virus 1 (HSV-1), Varicella zoster virus (VZV), Epstein-Barr virus (EBV), and

Kaposi’s sarcoma-associated HV (KSHV).

There have been several cases of HV spilling over to wildlife causing severe morbidity or mortality. HSV-1, which is normally asymptomatic in the human host, has been reported to cause disease or death in wild black-tufted marmosets (Callithrix penincillata) (Costa et al., 2011), white-faced sakis (Pithecia pithecia pithecia) (Schrenzel et al., 2003), common marmosets

(Callithrix jacchus) (Huemer et al., 2002), white-handed gibbons (Hylobates lar) (Landolfi et al.,

2005), and orangutans (Pongo pygmaeus pygmaeus) (Kik et al., 2005). HV spillover from domestic animals to wild animals has also been reported. Ovine HV 2 (OvHV-2), which is enzootic and subclinical in domestic sheep worldwide, can cause morbidity or mortality resulting from disease known as malignant catarrhal fever (MCF) in cattle, bison (Bison bison), moose (Alces alces), roe deer (Capreolus capreolus), red deer (Cervus elaphus), elk (Cervus canadensis), white- tailed deer (Odocoileus virginianus) and Père David’s deer (E. davidianus) (Baxter et al., 1993;

Vikøren et al., 2006; O’Toole and Li, 2014). Another example, Columbid HV 1 (CoHV-1) from

11 feral pigeons (descendants of domestic homing pigeons) in Australia caused a fatal infection in a native Powerful owl (Ninox strenua), but CoHV-1 is not present in native pigeons and doves

(Phalen et al., 2017).

Parapoxviruses in the family Poxviridae cause infection in wild animals around the world.

Morbidity is associated with the development of proliferative dermatitis (lesions) on the lips, nostrils, eyes, teats, and skin of infected animals because of viral replication in epithelial cells

(Bowman et al., 1981; Haig and McInnes, 2002). The virus may be contracted in the absence of obvious lesions on the animal (Bowman et al., 1981), though typically the presence of lesions indicates high amounts of viral shedding and increased likelihood of infection. Severely affected animals may lose weight and be predisposed to secondary infections (Maclachlan and Dubovi,

2010). Morbidity is high in young animals, but mortality is usually low unless lesions prevent lambs and kids from suckling or grazing (Maclachlan and Dubovi, 2010). Parapoxvirus infection is typically cleared within a matter of weeks, with animals remaining susceptible to reinfection

(Barrett and McFadden, 2008).

Parapoxviruses have been globally reported in free-ranging and captive wild animals including camels (Oryan et al., 2017), North American white-tailed deer (Roess et al., 2010), New

Zealand red deer (Horner et al., 1987), Japanese serows (Inoshima et al., 2002), Finnish reindeer

(Tikkanen et al., 2004), European muskoxen (Falk, 1978), United Kingdom red squirrels

(Tompkins et al., 2002), and several pinniped species (Hicks and Worthy, 1987; Muller et al.,

2003; Nollens et al., 2006). Orf virus (known by many names such as scabby mouth virus, sore mouth virus, ecthyma contagiosum virus, contagious pustular dermatitis virus), pseudocowpox virus (PCPV, Milker’s nodule virus, paravaccinia virus), and bovine papular stomatitis virus

(BPSV) are three important parapoxviruses that affect wildlife, domestic animals, and pose a

12 zoonotic threat to public health. Orf virus is naturally maintained in sheep and goats, whereas

BPSV and PCPV are maintained in cattle (Mercer et al., 1997).

1.7 Summary

In this thesis, molecular surveillance techniques are employed to study HV and parapoxvirus in wild animals in Canada. A pan-HV nested PCR targeting the HV DNA polymerase

(DPOL) gene was used to discover and briefly characterize previously unknown HV in multiple species of wildlife (Chapter 2). HV of caribou and marten were studied further by targeting additional viral genes and host mitochondrial DNA (mtDNA) using PCR to determine the usefulness of virus evolution in elucidating host evolution (Chapter 3). In Chapter 4, symptomatic and asymptomatic muskox tissues were screened for HV and a novel orf virus recently reported on Victoria Island, Nunavut (NU) and Northwest Territories (NT), and the adjoining mainland

NU, using PCR of structural and non-structural parapoxvirus genes. In Chapter 5, the genome of this Muskox orf virus (MxOV) was sequenced and characterized from infected tissues displaying clinical signs and compared to global sequences of orf virus. Chapter 6 summarizes limitations, challenges, and new learnings in wildlife disease surveillance as it relates to this thesis.

1.8 Study hypotheses

The thesis comprises experiments that tested the following hypotheses:

1. HV can be characterized from tissues of various wildlife species using a pan-HV nested

PCR targeting the HV DPOL gene.

2. There is a similar topology of the phylogenetic trees of HV genes and the host mtDNA

control region which implies coevolution.

3. Infection with orf virus is associated with clinical orf-like lesions and a PCR positive

result in muskoxen.

4. Muskoxen on Victoria Island, NU and NT, and the adjacent mainland NU, Canada are

infected by one orf virus strain which is unique from known strains around the world.

1.9 Study Aims

The objectives of this study are:

1. To validate the use of PCR methods for the detection of HV in archived tissues.

2. To identify and characterize known and previously uncharacterized HV present in wildlife

animals of Canada.

3. To characterize the variability of caribou and marten HV across Canada,

4. To examine the co-evolution of HV and their natural host mtDNA.

5. To validate the use of PCR methods for the detection of orf virus in archived tissues.

6. To determine the prevalence and spread of orf virus infection in muskoxen from Victoria

Island, NU and NT and the adjoining mainland NU.

7. To directly sequence the genome of orf virus from tissues of muskoxen Victoria Island,

NU and NT and the adjoining mainland NU.

8. To compare the genomic features of MxOV between samples and to known reference orf

virus sequences.

PART I: MOLECULAR SURVEILLANCE OF HV IN CANADIAN WILDLIFE ANIMALS

CHAPTER 2: DISCOVERY OF HERPESVIRUSES IN CANADIAN WILDLIFE

2.1 Abstract

Herpesviruses (HV) have a wide range of hosts in the animal kingdom. The result of infection with HV can vary from asymptomatic to fatal diseases depending on subtype, strain, and host. To date, little is known about HV naturally circulating in wildlife species and the impact of these viruses on other species. In our study, we used genetic and comparative approaches to increase our understanding of circulating HV in Canadian wildlife. Using a nested polymerase chain reaction (PCR) targeting a conserved region of the HV DNA polymerase (DPOL) gene, we analyzed material derived from wildlife of western and northern Canada collected between

February 2009 and Sept 2014. For classification of new virus sequences, we compared our viral sequences with published sequences in GenBank to identify conserved residues and motifs that are unique to each subfamily, alongside phylogenetic analysis. All alpha-HV shared a conserved tryptophan (W856) and tyrosine (Y880), beta-HV all shared a serine (S836), and gamma-HV had a conserved glutamic acid (E835). Most of our wildlife HV sequences grouped together with HV from taxonomically related host species. From Martes americana, we detected previously uncharacterized alpha- and beta-HV.

2.2 Introduction

Herpesviridae is a family of enveloped, double-stranded DNA (dsDNA) viruses that emerged roughly 400 million years ago (mya) (McGeoch and Gatherer, 2005). Herpesviruses (HV) closely co-evolved with their natural human and animal hosts and can be classified into three subfamilies: Alpha-, Beta-, and Gammaherpesvirinae (McGeoch et al., 2006; Wang et al., 2007;

Blake, 2010). This classification is based on phylogeny, pathogenesis, and the organ or tissue in

16 which these viruses establish latency: neural ganglia, secretory glands, and lymphatic tissues, respectively (McGeoch et al., 1995; 2000; 2006; Blake, 2010). Transmission of HV may occur vertically or horizontally in a population where susceptible animals become infected through direct contact with mucosal surfaces of infected individuals (Wald and Corey, 2007). In the event of cross-species HV transmission, disease is often characterized by lesions, fever, weight loss, spontaneous abortions, and death (Lankester et al., 2015; Mlilo et al., 2015).

In northern Alberta, Canada, serological tests have confirmed the presence of alpha-HV in woodland caribou (Rangifer tarandus caribou) (Tessaro et al., 2005). Koi HV (KHV) was detected in wild carp (Cyprinus carpio) of Manitoba and Ontario through polymerase chain reaction (PCR) and sequencing (Garver et al., 2010). Phocid HV 1 (PhHV-1), identified through microscopic evidence of inclusion bodies, caused morbidity and mortality in neonatal and weaning harbor seal

(Phoca vitulina) pups of British Columbia, Canada, and Washington, USA (Himworth et al.,

2010). In Calgary, Alberta, Canada, Washington, USA, and Idaho, USA, numerous great horned owls (Bubo virginianus), peregrine falcons (Falco peregrinus), and gyrfalcons (Falco rusticolus) have died from infection with Columbid HV 1 originating from pigeons (Columba livia domestica) diagnosed through sequencing of HV genetic material (Gailbreath and Oaks, 2008; Rose et al.,

2012). Awareness of circulating viruses could lead to targeted post-mortem examinations, thereby improving wildlife disease surveillance (Brown et al., 2015).

The HV DNA polymerase (DPOL) gene can be detected by PCR, using published degenerate primers (VanDevanter, 1996). The HV DPOL, which encodes a B family polymerase, is common to all HV regardless of subfamily or strain. Although DPOL genes of HV are not identical, they are structurally related and contain highly conserved B (region III) and C (region I) motifs, which are targeted by this PCR assay (VanDevanter, 1996). Motif B comprises the

17 sequence KXXXNSXYGXXG and is believed to be a part of the polymerase active site involved in dNTP coordination and DNA synthesis (VanDevanter, 1996; Marchler-Bauer et al., 2015).

Motif C is composed of the sequence DTDS, which, together with motif A, is critical for cation coordination when the enzyme is active (Bennett and Götte, 2013). Mutations in these known conserved motifs lead to severe impairment of the viral polymerase enzyme activity (Ye and

Huang, 1993).

Our study analyzed the diversity of circulating HV in selected and opportunistically obtained samples collected between 2009 and 2014 from Canadian native wildlife. We used phylogenetic approaches to compare HV and found two previously uncharacterized marten HV sequences among our samples.

2.3 Materials and methods

2.3.1 Sample collection

Convenience sampling methods were used to collect tissue and blood samples from various

Canadian wildlife animals between February 2009 and September 2014. Animals presented for a necropsy to the University of Calgary originated from research, trappers, hunting, roadkill events, euthanasia, or wildlife management activities. Tissue samples were taken from spleen, mesenteric lymph nodes, liver, kidney, tonsil, and lung during post-mortem examinations. When possible, fresh whole blood was collected into serum and/or EDTA tubes.

2.3.2 Sample preparation and DNA extraction

Animal tissue pieces of approximately 0.3 g were finely minced with a scalpel blade and placed in a 1.5-mL microcentrifuge tube. DNA was extracted using an E.Z.N.A.® Tissue DNA

Kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer’s protocol. Prior to extraction, tissue pieces were incubated overnight at 55 °C with TL buffer to achieve optimal tissue digestion. DNA was eluted twice from a HiBind DNA mini-column, each time using 25 µL of 10 mM Tris HCl, pH 8.5, at 70 °C.

Serum was collected from coagulated whole blood by centrifugation for 10 minutes at

1,000 × g. Peripheral blood mononuclear cells were isolated from EDTA blood by density gradient centrifugation on Ficoll-Paque® PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Viral DNA was extracted using an E.Z.N.A.® Blood DNA Kit (Omega Bio-Tek Inc., Norcross,

GA, USA) according to the manufacturer’s protocol.

2.3.3 PCR conditions

Nested degenerate primers were used to amplify a highly conserved fragment between motifs B and C of the DNA-directed DPOL gene common to all HV (VanDevanter, 1996). These primers allow rapid amplification of the polymerase gene of all known HV subfamilies. For primary amplification, we used 75-150 ng of template DNA, 1.25 units of AccuTaq™ LA DPOL

(Sigma-Aldrich Co. LLC, St. Louis, MO, USA), 3.75 pmol of each nucleotide, and 5 pmol each of primers DFA, KG1, and ILK in a total reaction volume of 25 µL. Cycling conditions were as follows: initial denaturation of 5 min at 95 °C followed by 40 cycles of 95 °C for 30 s, 46 °C for

60 s, and 72 °C for 90 s, with a final extension step of 5 min at 72 °C. The second round of the nested protocol used 1.5 µL of the first-round PCR product as the template, with 5 pmol of primers

IYG and TGV. Thermocycling conditions were as described above, but with 30 seconds of annealing time followed by 60 seconds of extension at each cycle.

2.3.4 Gel extraction and sequencing

PCR products were separated using gel electrophoresis on a 1% agarose gel, and bands were extracted using an E.Z.N.A.® Gel Extraction Kit (Omega Bio-Tek Inc., Norcross, GA, USA), following the manufacturer’s protocol. DNA was eluted from the minicolumn using 30 µL of elution buffer heated to 70 °C. DNA samples were premixed with IYG primer and sequenced

(Eurofins MWG Operon LLC, Huntsville, AL, USA). Weak amplification bands were purified from the gel, and the DNA was ligated into pGEM®-T Easy Vector (Promega Corp. Madison, WI,

USA), followed by transformation using One Shot® TOP10 Escherichia coli (Invitrogen Corp.,

Carlsbad, CA, USA). LB agar plates with 1% ampicillin and spread with 1.6 mg X-gal were used for blue-white colony screening. Five white colonies were grown in LB with 1% ampicillin.

Plasmids were recovered using an E.Z.N.A.® Plasmid Mini Kit (Omega Bio-Tek Inc., Norcross,

GA, USA) following the manufacturer’s protocol and sequenced using T7 and SP6 primers

(Eurofins MWG Operon LLC, Huntsville, AL, USA).

2.3.5 Phylogenetic analysis

Chromatograms of sequences were visually inspected for quality. Poor-quality nucleotide sequences identified as having overlapping chromatogram peaks, ambiguous nucleotides, and/or less than 50 bp in length, were discarded from the analysis. Fragments generated from T7 and SP6 primers were used to construct a consensus DNA fragment for each replicate of each sample.

Primer and vector sequences were trimmed prior to alignment using Geneious v8.1.3 (Kearse et al., 2012). Only amino acid sequences that covered the full range between motif B and C on the

DPOL gene were included in further analysis. These viral sequences were searched against published sequences in GenBank using the protein Basic Local Alignment Search Tool (BLASTp)

(NCBI, 2013). Known DPOL sequences of HV were obtained from GenBank and included in amino acid alignments using the multiple sequence comparison by log-expectation (MUSCLE) algorithm (Edgar, 2004). Since this region of the viral DPOL gene is commonly used to detect

HV, amino acid sequences of wildlife and published HV sequences were aligned to examine conserved residues and motifs characteristic of each subfamily. Phylogenetic trees were constructed using the Randomized Axelerated Maximum Likelihood (RAxML) tree builder based on Maximum likelihood (ML) inference, with 100 bootstrap replicates (Stamatakis, 2014), and using the MrBayes Bayesian inference plugin available in Geneious with a burn-in of 1000. Tree files were exported and visually labelled for clarity in FigTree v1.4.2 (Morariu et al., 2008).

2.4 Results

A fragment of the HV DNA-dependent DPOL gene was successfully amplified by nested

PCR from wildlife tissue and blood samples from different wildlife species. High-quality nucleotide sequences were obtained from 21 animals of six different species, and these are summarised in Table 2.1 along with their most similar sequence found using BLASTp. Tissue samples from some Porcupine caribou herd members (Rangifer tarandus granti), fox (Vulpes vulpes), wolf (Canis lupus), cougar (Puma concolor), black bear (Ursus americanus), grizzly bear

(Ursus arctos horribilis), and raccoon (Procyon lotor) were negative using the described PCR methodology. Overall, the use of spleen tissue resulted in the largest number of successful sequencing attempts when compared to other tissue types. Fifty-one sequences were included in the MUSCLE amino acid alignment and RAxML phylogenetic tree (Figure 2.1). These same sequences were included in Bayesian phylogenetic analysis to strengthen our interpretations.

Similar topologies were seen for both phylogenetic trees, with greater branching support in the

Bayesian tree. Three major viral groups formed in both trees, corresponding to the HV subfamilies

Alpha-, Beta-, and Gammaherpesvirinae. However, Felid HV-1 (FHV-1) did not follow this topology in the RAxML analysis.

Bison (Bison bison) sample 15 was positive for American bison gamma-HV (AAL29891,

100%), and our four elk (Cervus canadensis) samples were infected with variant strains of

Ruminant rhadinovirus 2 of elk (AAO88180, 95%-100%) as summarized in Table 2.1.

Two coyote (Canis latrans) samples from Alberta, Canada, shared 100% nucleic acid and amino acid identity with Canid HV 1 (CHV-1) (accession number AAC55646) isolated from domestic Labrador retriever (Canis lupus familiaris) puppies. CHV-1 is an alpha-HV known to infect domestic and wild canines. Coyote sequences clustered within the alpha-HV subfamily with

CHV-1 in phylogenetic analysis (Figure 2.1 and 2.2).

Table 2.1 A summary of HV DNA-dependent DPOL gene amino acid sequences from this study and the most closely related virus sequences in the GenBank database

Host Organism Sample GenBank accession Most similar BLASTpa match ID number (accession no., % identity)

Bighorn sheep (Ovis 5, 13 KX062141, KX062142 Ruminant rhadinovirus 2 canadensis) (AAO88175, 98%)

Bison (Bison bison) 15 KX062140 American bison gammaherpesvirus (AAL29891, 100%)

Caribou (Rangifer tarandus 15, 21, KX062137, KX062138, Reindeer gammaherpesvirus granti) 24 KX062139 (AFV98876, 98-100%)

Coyote (Canis latrans) 12, 27 KX062143, KX062144 Canid herpesvirus 1 (AAC55646, 100%)

Elk (Cervus canadensis) 11, 13, KX062145, KX062146, Ruminant rhadinovirus 2 32, 40 KX062147, KX062148 (AA088180, 95-100%)

Marten (Martes americana) 8, 32 KX062128, KX062130 Mustelid herpesvirus 1 (AAL55728, 93%)

33, 41, KX062131, KX062132, Felid herpesvirus 1 46 KX062133 (AAC55649, 72%)

11, 49, KX062129, KX062134, Aotine herpesvirus 1 52, 65 KX062135, KX062136 (AAC55643, 52%)

a protein Basic Local Alignment Search Tool (NCBI, 2013)

Figure 2.1 RAxML tree of a MUSCLE alignment of 51 HV DPOL amino acid sequences. Twenty- one viral sequences from wildlife from this study are shown in blue, and accession numbers are shown in parentheses. PCH; Porcupine caribou herd. ML analysis was carried out in Geneious 8.1 using the RAxML plugin, with the scale bar indicating the number of amino acid substitutions per site. Branching support is shown as bootstrap percentages for 1,000 bootstrap replicates

Figure 2.2 MrBayes Bayesian inference phylogenetic tree of a MUSCLE alignment of 51 HV

DPOL amino acid sequences. Twenty-one viral sequences from wildlife from this study are shown in blue, and accession numbers are shown in parentheses. PCH; Porcupine caribou herd. Bayesian analysis was carried out in Geneious 8.1 using the MrBayes plugin, with the scale bar indicating the number of amino acid substitutions per site. A burn-in of 1,000 was used, with branching support shown as posterior probability percentages (See previous page).

Marten (Martes americana) samples were collected from trappers in the Sahtú Settlement

Region, Northwest Territories, Canada. Amino acid and nucleic acid sequences from martens 8 and 32 were identical and shared 93% identity with Mustelid HV 1 (AAL55728), a gamma-HV found in sea otters (Enhydra lutris) and other members of the family Mustelidae such as badgers

(Meles meles) and pine martens (Martes martes) of Europe.

Bighorn sheep (Ovis canadensis) sample 5 and sample 13, native to Sheep River Provincial

Park, Alberta, Canada, shared 98% amino acid sequence identity with the gamma-HV Ruminant rhadinovirus 2 (AAO88175) from free-range bighorn sheep in Washington, USA (Li et al., 2005).

Marten samples 33, 41, and 46 shared 72% identity with FHV-1 (AAC55649), an alpha-

HV which infects domestic cats. The amino acid sequence identified from marten 46 differed from those from martens 33 and 41 by one residue at position 38; the nucleotide sequence CTT in marten

46 was changed to CCT, thereby encoding a proline instead of leucine at this position. Martens 33,

41, and 46 composed a single phyletic group within the subfamily Betaherpesvirinae (Figure 2.1 and 2.2).

Marten samples 11, 49, 52, and 65 were infected with an uncharacterized beta-HV.

Sequences from these marten sequences are identical in amino acid sequence and have no close relatives in GenBank (Table 2.1). These martens aligned in a single phyletic group within the subfamily Betaherpesvirinae; however, other sequences in this subfamily are distant (Figure 2.1 and 2.2).

Spleen samples from animals 15, 21, and 24 of the Porcupine caribou herd (PCH) native to northwestern Canada, produced amino acid sequences that were 98-100% identical to those of

Reindeer gamma-HV 1 (AFV98876) characterized in Norway from reindeer (Rangifer tarandus tarandus) (Das Neves et al., 2013). Sample PCH 21 differed from the Norwegian virus by an

28 amino acid mutation from isoleucine to valine, sharing 98.3% identity. PCH sample 24 was more variable in the terminal region of the fragment, differing from those from other herd members and

Reindeer gamma-HV 1.

Starting at the first residue after the TGV primer corresponding to position 824 of the polymerase, all sequences shared a proline (P829), cysteine (C830), alanine (A834), threonine

(T838), glycine (G841), serine (S842), methionine (M844), and leucine (L845). Variation in the amino acid sequence occurs more frequently in the terminal end of the fragment between motif B

(region III) approaching motif C (region I) of the DPOL polypeptide.

The sequences from martens 11, 33, 41, 46, 49, 52, and 65 were aligned with most similar published sequences in GenBank using MUSCLE to highlight amino acid sequence differences

(Figure 2.3). Published alpha-HV sequences share a conserved aromatic hydrophobic tryptophan

(W856) residue and a tyrosine (Y880) at a position of five residues from the end of the sequence.

The previously undescribed sequences from martens 33, 41, and 46 in this region are most similar to those of alpha-HV since they align with the alpha-HV sequences in ML and Bayesian phylogenetic trees and share subfamily-specific conserved residues.

Viral sequences from the spleens of martens 11, 49, 52, and 65 are most similar to beta-

HV since they share the subfamily-specific conserved serine (S836) residue at position 13. These beta-like marten sequences also cluster with other beta-HV sequences in ML and Bayesian phylogenetic trees, although the relationship is distant in both.

Published gamma-HV sequences all share a glutamic acid (E835) residue at position 12.

Our previously undescribed viral sequences identified in marten 8 and 32, bighorn sheep 5 and 13, elk 11, 13 and 49, and PCH 21 and 24 belong to the subfamily Gammaherpesvirinae based on conserved residues and clustering in both phylogenetic trees.

Figure 2.3 Alpha- and beta-HV-like marten amino acid sequences aligned with related published sequences using MUSCLE. Gaps in the alignment are indicated with a dash.

2.5 Discussion

The Porcupine caribou herd spans Alaska, USA, and the northwest Yukon, Canada

(COSEWIC, 2011). Evidence from this study suggests that Reindeer gamma-HV 1 and at least one other closely related virus is circulating in the Porcupine caribou herd. Our Canadian caribou HV sequences share an ancestor with the Norway strain, which is not surprising, as North American caribou originally came from Eurasia (Yannic et al., 2014).

Coyote samples in our study were infected with CHV-1, which is known to cause morbidity and mortality in wild and domestic Canidae pups. Coyotes are small canids native to North

America that were historically confined to open plains and arid regions. With the expansion of urban developments, it is possible that CHV-1 was transmitted from domestic dogs to wild coyotes, and as such, CHV-1 was carried into the wild-coyote population. Wild coyotes are known to interact with wolves and foxes, both of which also have large territorial ranges (Levi and

Wilmers, 2012). Since coyotes, wolves, and foxes are susceptible to CHV-1, a single spillover event from domestic dogs could propagate disease in many animal populations.

As expected, our bighorn sheep, bison, and elk samples were infected with HV characterized in previous studies from members of the same species. It is possible that related strains of ruminant rhadinovirus 2 are endemic in bison and elk, while American bison gamma-

HV is endemic in bison.

Martens are mid-sized carnivorous mammals that are distributed across the forests of

Canada and Alaska (Broquet et al., 2006). They are integrated into various habitats in urban and rural communities and hunt on the ground or in trees. These animals are solitary and territorial, with some dispersing over 80 km (Broquet et al., 2006). We identified different marten HV that segregated into all three HV subfamilies. Marten gamma-HV sequences are closely related to

31 mustelid HV, which is as expected since martens are of the Mustelidae animal family. In some cases, HV in martens are distantly related to other HV in the alpha and beta subfamilies, likely because such viral sequences have not yet been characterized in closely related animals.

The fragment between the B and C motif contains several amino acid residues that are conserved within particular HV subfamilies, as confirmed in this study. It has been shown in studies with Human HV 1 that a glycine-to-serine mutation at position 841 in the polymerase results in altered drug sensitivity (Larder et al., 1987). It is possible that mutations in other conserved residues common to all HV could also directly impact the function of the polymerase or the virus itself. The influence of subfamily-specific conserved residues identified in this study

(alpha: W856, Y880; beta: S836; gamma: E835) on the function of the polymerase is also unknown but warrants investigation. The terminal region approaching motif C shows greater variability, suggesting that this region may not be critical for polymerase function. Future studies could determine the role of this region in infectivity or pathogenesis.

Convenience sampling allowed us to access tissues from numerous wildlife species, including more-scarce tissues from cougar, wolf, fox, raccoon, and bear. However, many of these tissues tested negative when using the described methods. The high frequency of negative PCR results could be affected by the handling, storage, or age of tissues prior to testing for HV. Plumb et al. (1972) showed that the infectivity and detectability of Channel catfish HV (CCV) in recently deceased channel catfish (Ictalurus punctatus) was greatly influenced by the storage temperature and duration of storage (Plumb et al., 1973). After 48 hours at 22 °C, CCV in dead fish was no longer infectious in cell culture. CCV could be detected up to 14 days when dead fish were stored on ice, up to 162 days when stored at –20 °C, and up to 210 days when the fish were frozen at –

80 °C. Some of our wildlife samples were sourced from carcasses that were found dead, or from

32 remote regions where access to a freezer was not always possible. The ability to detect HV using

PCR could be affected by the time of death and the status of tissue and viral degradation. Biopsies of available tissues were sometimes taken in non-sterile, wilderness settings, potentially introducing PCR inhibitors that might have contributed to the negative PCR results. Uniform sampling from different tissues of different animals, followed by immediate PCR analysis or proper tissue storage may reveal HV present in species that tested negative in our study.

It is important that we understand the relationships between viruses and animals in a wild or domestic setting so that we can examine the overall health status of a population. In this study, we determined the nucleotide and predicted amino acid sequences of previously undescribed HV in Canadian wildlife. Observing the conserved motifs of the HV DPOL gene and additional conserved subfamily-specific residues allowed us to determine the relationship of these unknown

HV to known viruses. Amplifying larger DNA fragments of the HV genome will allow stronger evolutionary comparisons of HV genes and may give insights regarding the relatedness of host species harboring these co-evolving viruses. Finally, samples from other wild species and uniform sampling are needed for phylogenetic resolution when comparing previously undescribed viruses to known viruses.

2.6 Acknowledgements

We would like to thank the members of the Canadian Cooperative Wildlife Health Centre and the pathology department at the University of Calgary Spy Hill Campus for their help and expertise in animal sample collection during necropsy. Caribou samples were collected through

CARMA project; some marten samples were collected by the youth of the Sahtú region as part of

33 an NSERC PromoScience outreach program. Finally, Alasdair Veitch, Ale Massolo, and Cynthia

Kashivakura participated largely in this project.

2.7 Declaration of Conflicts of interest

Compliance with ethical standards

The authors declare that they have no conflicts of interest.

2.8 Funding

This study was funded by the University Research Grants Committee (URGC) Seed Grant

Program, University of Calgary, Alberta, Canada.

CHAPTER 3: LIMITED GENETIC VARIATION OF HERPESVIRUSES IN CARIBOU

(RANGIFER TARANDUS SPP.) AND MARTEN (MARTES AMERICANA) IN CANADA

3.1 Abstract

Herpesviruses (HV) are ubiquitous in people and have been identified in animals around the world, including wildlife. Research on HV in wildlife is often focused on those that cause overt disease, whereas little is known about the broader diversity of HV in wild animals. Previous studies on human HV have used the genetic variation in viral sequences to infer the phylogeographic history of humans. Herein we co-investigate the genetic variation of HV DNA polymerase (DPOL) gene as a marker of virus evolution, and the control region of host mitochondrial DNA (mtDNA) as a marker of host evolution in tissue samples of wild caribou and marten across Canada.

We sequenced Reindeer gamma-HV 1 from 32 caribou from 3 distinct herds. Sequences grouped together with no nucleotide variation and belonged to a separate phylogenetic clade than other cervid-derived gamma-HV. Control region sequences from caribou in this study grouped together with others from Rangifer tarandus in a monophyletic clade in the family Cervidae. No distinct grouping was seen by caribou subspecies, rather grouping supported genetic separation based on ancestral caribou lineages. Cervidae host mtDNA and endemic HV phylogenies shared similar branching patterns.

We sequenced HV from 19 marten belonging to different provinces and territories in

Canada. Marten gamma-HV 3 was identified in five marten from Pemberton, BC, while Marten gamma-HV 4 was identified in three marten from southern Labrador, NL. These gamma-HV grouped into independent clades sharing a common ancestor with Fisher HV from Martes pennanti. Marten alpha-HV 1 was sequenced in five marten from Vancouver Island, BC and

Marten alpha-HV 2 was sequenced in three marten each from McBride, BC, and Whitehorse, YK.

Marten mtDNA sequences grouped in a clade separate from other mustelid sequences with little sequence variation and sharing a recent common ancestor with Martes pennanti.

The further molecular investigation into wildlife HV and especially the generation of more complete sequence data could develop gamma-HV as a valuable tool in the study of evolutionary history.

3.2 Introduction

Investigating the evolutionary history of mammals generally relies on fossil record data, and nuclear (nuDNA) or mitochondrial (mtDNA) genetics. Interpretations of host evolution from nuDNA can be complicated by large host genomes, allelic heterozygosity, multiple copies of certain loci, and very slow mutation rates (Rubinoff and Holland, 2005). Often, mtDNA is used to determine relatedness at the species or genus level due to its faster mutation rate than nuDNA, however, mtDNA does not always reflect nuclear inheritance patterns (Rubinoff and Holland,

2005). Since various sources of data each have their advantages, there is a growing preference for integrating multiple molecular data sources to infer the evolutionary history of a species (Rubinoff and Holland, 2005).

Viruses have a higher mutation rate, smaller genomes, and a much shorter generation time, meaning they evolve faster than nuDNA or mtDNA of mammals (Hungnes et al., 2000; Sanjuán et al., 2010). Molecular studies of viruses allow us to understand the dissemination and evolution of viruses within a specific population or geographical region (Hungnes et al., 2000, Lam et al.,

2010). DNA viruses with narrow host ranges tend to coevolve with their natural host (Madinda et al., 2016). In a study by Kolb et al. (2013), 31 genome sequences of Human HV 1 (HHV-1; Herpes simplex virus 1, HSV-1), a virus known to frequently infect humans, were compared using

36 phylogenetic methods. The study found viral isolates grouped together by geographic origin of patients. They concluded that herpesvirus (HV), which have coevolved with their natural host, could serve as an indicator of host evolutionary history (Kolb et al., 2013).

Herpesviridae is a family of large double-stranded DNA (dsDNA) viruses that naturally infect mammals, birds, and reptiles leading to life-long infection (Davison et al., 2009; Barton et al., 2011). HV can be transmitted horizontally through direct contact with mucosal surfaces and bodily fluids, or vertically in utero (Maclachlan and Dubovi, 2010; Grinde, 2013). In a natural, immunocompetent host, primary HV infection is followed by viral transition into latency – a period during which the virus expresses latency-associated transcripts without overt disease or virus particle production (Barton et al., 2011; Smith and Whitley, 2017). There are three subfamilies within Herpesviridae: Alpha-, Beta-, and Gammaherpesvirinae which are classified based on biologic properties including pathogenesis and cell tropism wherein these viruses establish latency

(Burrell et al., 2017; Smith and Whitley, 2017). Gamma-HV have a tropism for lymphatic tissues and establish long-term latency in T- or B-cells, whereas alpha-HV are neurotropic with latency occurring in neural ganglia, and beta-HV mainly target cells in secretory glands (Ackermann,

2006; Louten, 2016; Smith and Whitley, 2017).

HV have been detected in populations of caribou (Rangifer tarandus; also known as reindeer) across North America and Scandinavia through molecular and serological assays (Jordan et al., 2003; Tessaro et al., 2005; Evans et al., 2012; Rimstad et al., 1992; Dalton et al., 2017;

Bondo et al., 2018; Carlsson et al., 2019). Caribou numbers are declining in many natural habitats due to climate change, anthropogenic developments, hunting pressure, and other factors including infectious agents (COSEWIC, 2016). The decline of caribou threatens the food security and

37 resources of subsistence hunters in the Arctic, including indigenous communities who have relied on caribou for millennia (Burch, 1972; COSEWIC, 2011).

Dalton et al. (2017) identified several HV in marten (Martes americana) from the

Northwest Territories, Canada. Marten are small, solitary carnivores that reside in boreal forests across Canada and parts of the USA. These valued furbearers live within home ranges of up to 45 km2 for males and 27 km2 for females, depending on the presence of undesirable bog and shrub forest habitat (Smith and Schaefer, 2002). Due to habitat loss and in some instances, excessive trapping, marten populations have been extirpated from many natural areas in North America

(Jackson 1961; Helgen and Reid, 2016). Marten, like caribou, are grouped into subspecies, however marten subspecies are not widely accepted (Hicks and Carr, 1997; Dawson and Cook,

2012).

Conservation of species, especially with declining populations, is informed through the understanding of genetic diversity and relatedness. Using endemic gamma-HV as a marker for evolution offers a unique additional data source for consideration of host evolutionary history. In this study, we use molecular methods to investigate whether genetic variation in endemic gamma-

HV is associated with the taxonomy of the infected animal host. We determine the relative prevalence of gamma-HV infection in sampled marten and caribou of Canada, then characterize the genetic variation of gamma-HV DNA polymerase (DPOL) gene, and of the mtDNA control region of the hosts. Using phylogenetic analyses, we separately infer the evolutionary history of caribou and marten, and of their gamma-HV.

3.3 Materials and methods

3.3.1 Sampling and DNA Extraction

Convenience sampling was used to collect tissues from caribou and marten across Canada.

Caribou samples were sourced from frozen archives initially collected in 2009-2014 as part of other research initiatives. When available, Peyer’s Patches (lymphoid nodules) were sampled.

Samples obtained from caribou included in this study are summarized in Table 3.1.

Table 3.1 Summary of Canadian caribou samples collected for HV detection

Ecotype Province Herd Subspecies Number of Tissues* (Designatable or caribou (n) Unit) Territory

Barren-ground YK/NT Porcupine R.t. granti 16 Spleen (10), PP (7) (DU3)

Barren-ground NT Bathurst R.t. 29 LN (54), PP (28) (DU3) groenlandicus

Central Mountain BC Unspecified R.t. caribou 3 Spleen (3), LN (3), (DU8) PP (3)

Central Mountain AB Unspecified R.t. caribou 2 Spleen (2), LN (2) (DU8)

Eastern Migratory QC Leaf River R.t. caribou 16 PP (16) Woodland (DU4)

Eastern Migratory QC/NL George R.t. caribou 24 PP (24) Woodland (DU4) River

* Tissue abbreviations: LN, lymph node; PP, Peyer’s Patches

Marten tissues were collected through cooperation with provincial hunter and trapper organizations during the hunting seasons of 2016-2017 (Table 3.2). Marten commercially trapped for their pelts were skinned by the trapper, then carcasses were frozen, packaged, and shipped to

39 the University of Calgary AB, Canada for processing and dissection. Organs and tissues of the lymphatic system were resected and used in this study.

DNA was extracted from tissue samples using the E.Z.N.A® Tissue DNA extraction kit

(Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer’s protocol. Prior to extraction, tissue pieces were homogenized and incubated overnight at 55 ºC with OB Protease solution and TL Buffer included in the kit. Elution was performed twice with heated buffer.

Table 3.2 Summary of Canadian marten samples collected for HV detection

Province or Location Number of Spleen Tonsil Lymph Territory marten (n) node

YK Whitehorse 8 8 8 8

NT Norman Wells 25 25 - 22

BC Vancouver Island 5 5 5 5

Dawson Creek 9 9 9 9

Pemberton 5 5 5 5

Lac La Hache 3 3 3 3

McBride 5 5 5 5

100 Mile House 5 5 5 5

AB Birch Hills 3 3 3 3

SK multiple 21 21 - 21

MB multiple 30 30 30 30

ON multiple 28 28 28 28

QC multiple 32 32 32 32

NL Labrador South 30 30 29 29

3.3.2 Polymerase chain reaction (PCR) and sequencing

PCR was used for the detection and amplification of the HV DPOL gene. HV DPOL amplification involved a nested PCR approach using primers described by VanDevanter et al.

(1996). The first round of nested PCR contained of 75-150 ng template DNA, 1 unit of TaKaRa

Ex Taq™ DNA Polymerase (Takara Bio USA Inc., Mountain View, CA, USA), 3.75 pmol of each nucleotide, 2.5 µl of 10x Takara Ex Taq DNA Polymerase buffer, and 5 pmol of each first-round primer (DFA, KG1, ILK) in a total reaction volume of 25 µl. Cycling consisted of an initial denaturation of 5 min at 95°C followed by 40 cycles of 95°C for 30 sec, annealing at 45°C for 60 sec, extension at 72°C for 90 sec, and a final extension step of 5 min at 72°C. The second round of the nested PCR used 1.5 µl of the first-round product as the template, with 5 pmol of each second-round primer (TGV, IYG) with extension shortened to 60 sec. A Felid HV 1 (FHV-1) containing vaccine (Nobivac® Tricat Trio; Merck Animal Health Canada, Kirkland, QC, CAN) was used as a HV positive control whereas nuclease-free water served as a PCR negative control.

The control region of mtDNA was amplified using PCR as a marker of host evolution. The mtDNA control region including the tRNA-Pro and tRNA-Phe genes which bound the mtDNA D- loop region was targeted using primers L15926 and H00651 described by Kocher et al. (1989), cycling for 35 cycles.

PCR products were analyzed using agarose gel electrophoresis and purified using the

E.Z.N.A® Gel Extraction Kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer’s protocol, eluting twice from the Hi-Bind™ column with 25 µl hot (70°C) elution buffer. Gel purified PCR products of mtDNA were cloned into competent Escherichia coli (Top

10) using the pGEM®-T Vector I system (Promega Corp., Madison, WI, USA). Plasmids were purified using the E.Z.N.A® Plasmid Mini Kit I (Omega Bio-Tek Inc., Norcross, GA, USA).

Plasmids with mtDNA and HV PCR products were sent for bidirectional Sanger sequencing at the

Centre for Advanced Genetics (Toronto, ON, CA) with T7 and SP6, and TGVseq and IYGseq primers (VanDevanter et al., 1996), respectively.

3.3.3 Quality Control and Phylogenetic analysis

Nucleotide sequences were trimmed in Geneious 10 (Kearse et al., 2012) to remove low- quality bases identified as having overlapping chromatogram peaks, ambiguous nucleotides, and/or sequences less than 50 bp in length were discarded from the analysis. Remaining sequences were searched against published entries in GenBank using the Basic Local Alignment Search Tool

(BLAST; NCBI 2013) to confirm the identity prior to alignment. Gene fragments from this study were aligned with sequences retrieved from NCBI’s GenBank using the multiple sequence comparison by the log-expectation (MUSCLE) algorithm which offers a rapid, progressive alignment with established accuracy (Edgar, 2004). Sequences included in alignments and phylogenetic trees are summarized in supplementary tables 3.1-3.4.

Model testing with jModelTest2 identified the HKY85 (Hasegawa-Kishino-Yano, 85) as the best-fit nucleotide substitution model for our phylogenetic analysis. Maximum Likelihood

(ML) and Bayesian phylogenies were inferred for HV DPOL gene and the mtDNA control region.

ML analysis was performed using the RAxML (Stamatakis et al., 2014) software with bootstrapping 1000 replicates. Bayesian analysis was performed using the MrBayes software

(Huelsenbeck and Ronquist, 2001) installed in Geneious. Analyses were run until effective sample sizes (>200) were reached. Phylogenetic trees of HV and mtDNA genes were visually compared for similarities in branching patterns.

3.4 Results

3.4.1 Caribou Sample Data

Of the 90 caribou samples included in this study, 40 were PCR-positive for HV DNA.

These belonged to the Bathurst, Porcupine, and Leaf River herds. No HVes were detected in caribou samples from British Columbia, Alberta, or the George River herds (Table 3.3). Of the

PCR-positive HV samples, 80% were successfully sequenced. Fifty-four out of 90 caribou samples were successful in the amplification of the mtDNA control region, while only 14 produced quality sequences for comparison (Table 3.3). The lack of mtDNA sequences obtained from Bathurst caribou is attributed to a failed Sanger sequencing run for unknown reasons.

Table 3.3 Summary of DNA findings from caribou samples

Herd HV DPOL mtDNA D-Loop region (/total)

PCR-positive Sequence obtained PCR-positive Sequence obtained

Porcupine 10/16 9/10 14/16 6/14

Bathurst 25/29 19/25 24/29 0/24

BC 0/3 - 3/3 3/3

AB 0/2 - 2/2 0/2

Leaf River 5/16 4/5 6/16 5/6

George River 0/24 - 5/24 0/5

BLASTn results of caribou HV sequences matched with 100% identity to Reindeer gamma-HV 1 and were grouped in a monophyletic clade apart from other published HV isolated from Cervidae hosts in Bayesian and ML trees (Figure 3.1, Supplementary Figure 3.1, respectively).

Caribou mtDNA control region sequences from this study differed by an average of 1.50%

(16/1066 nucleotides), and 1.69% (18/1066 nucleotides) when compared to other published caribou and reindeer sequences included in alignments. Sequences of caribou and other cervids produced an alignment of 1270 bp (data not shown). Phylogenetic analysis of the mtDNA control region of Cervidae grouped caribou together in a monophyletic clade as expected in Bayesian

(Figure 3.2) and ML (Supplementary Figure 3.2) trees. Caribou mtDNA sequences share a more recent ancestor with mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) compared to elk (Cervus canadensis nelsoni) (Figure 3.2).

Figure 3.1 Bayesian phylogenetic tree of gamma-HV DPOL fragments isolated from cervid hosts.

Sequences are approximately 200 bp in length. Colours indicate caribou herd: green (Bathurst), orange (Leaf River), purple (Porcupine). Numbers at the nodes indicate posterior probability percentage. Scale bar indicates nucleotide substitutions per site.

Figure 3.2 Bayesian phylogenetic tree of the mtDNA control region of cervids. Numbers at the nodes indicate posterior probability percentage. Colours indicate caribou herd: blue (unknown from BC), orange (Leaf River), purple (Porcupine). Red bar belongs to the Beringian (BEL) linage, green bar belongs to the North American (NAL) linage, black bar (BEL/NAL) are of mixed lineages. Rangifer tarandus caribou (woodland caribou), R. t. terranovae (Newfoundland caribou), R. t. granti (Grant’s caribou), R. t. groenlandicus (barren-ground caribou), Odocoileus virginianus (white-tailed deer), R. t. tarandus (Eurasian reindeer), O. h. hemionus (mule deer),

Cervus canadensis nelsoni (Rocky Mountain elk), Dama dama (fallow deer), Bos taurus (cattle),

Alces americanus (moose). Scale bar indicates nucleotide substitutions per site.

3.4.2 Marten Sample Data

Of the 209 sampled marten, a total of 27 were PCR-positive for HV, and 51 were PCR- positive for the mtDNA control region (Table 3.4). Nineteen HV and 10 marten mtDNA PCR fragments were successfully sequenced.

Table 3.4 Summary of DNA findings from marten samples

HV DPOL mtDNA D-loop region (/total) Province or Location Territory Sequence Sequence PCR-positive PCR-positive obtained obtained

YK Whitehorse 3/8 3/3 5/8 0/8

NT Norman Wells 3/25 0/3 0/25 -

Vancouver BC 5/5 5/5 5/5 4/5 Island

Dawson Creek 0/9 - 0/9 -

Pemberton 5/5 5/5 5/5 3/5

Lac La Hache 1/3 0/1 2/3 1/2

McBride 3/5 3/3 1/5 0/1

100 Mile 0/5 - 5/5 1/5 House

AB Birch Hills 0/3 - 1/3 0/3

SK multiple 3/21 0/3 3/21 1/3

MB multiple 0/30 - 1/30 -

ON multiple 0/28 - 3/28 0/3

QC multiple 1/32 0/1 10/32 0/32

Southern NL 3/30 3/3 10/30 0/10 Labrador

HV sequences from Pemberton marten shared 93.9% nucleotide identity to Marten gamma-HV 2

(KX063130), suggesting the presence of an uncharacterized virus proposed as Marten gamma-HV

3. Virus sequences from southern Labrador marten were 98.2-98.8% identical in nucleotide sequence to Marten gamma-HV 2, and represent an uncharacterized virus proposed as Marten gamma-HV 4. Sequences of Marten gamma-HV were grouped together in Bayesian (Figure 3.3), and ML (Supplementary Figure 3.3) trees with identical branching patterns. Marten gamma-HV sequences share a recent common ancestor with Fisher HV (HM579931) isolated from Martes pannanti.

BLASTn results of Vancouver Island marten matched with 100% identity to Marten alpha-

HV 1 (KX062131), with no other closely related hits in Genbank. Marten from McBride, BC, and

Whitehorse, YK, shared only 98.9% identity with Marten alpha-HV 1 from the Northwest

Territories, suggesting infection by a previously uncharacterized virus herein named Marten alpha-HV 2. Sequences of Marten alpha-HV 2 were grouped together in Bayesian (Figure 3.4), and ML (Supplementary Figure 3.4) analyses with identical branching patterns.

Vancouver Island marten sequences of the mtDNA control region differed among each other by an average of 2.92% (26/890 nucleotides), whereas they differed an average of 7.64%

(68/890 nucleotides) when aligned with a previously reported marten sequence from Ontario,

Canada (HM106324).

Figure 3.3 Bayesian phylogenetic tree of gamma-HV DPOL fragments isolated from mustelid hosts. Colours indicate sample origin: blue (Pemberton, BC), orange (southern Labrador). Marten gamma-HV 1 and 2 are from marten in the Northwest territories. Numbers at the nodes indicate posterior probability percentage. Scale bar indicates nucleotide substitutions per site.

Figure 3.4 Bayesian phylogenetic tree of alpha-HV DPOL fragments isolated from mustelid hosts.

Colours indicate sample origin; dark blue: McBride, BC, pink: Vancouver Island, BC, purple:

Whitehorse, YK. Numbers at the nodes indicate posterior probability percentage. Scale bar indicates nucleotide substitutions per site.

Figure 3.5 Bayesian phylogenetic trees of mtDNA partial tRNA-Thr, tRNA-Pro, and partial D- loop sequences from mustelids. Pink font indicates marten sourced from Vancouver Island, BC.

Numbers at the nodes indicate posterior probability percentage. Martes americana (American marten), M. pennanti (fisher), M. foina (stone marten), M. zibellina (sable), M. melampus

(Japanese marten), Meles meles (Eurasian badger), Neovison vison (American mink), Enhydra lutris (sea otter), Lutra lutra (common otter). Scale bar indicates nucleotide substitutions per site.

Mustelid mtDNA sequences of the partial tRNA-Thr, t-RNA-Pro, and 5’ end of the D-loop region produced an alignment of 647 bp. Phylogenetic analyses showed similar branching patterns between Bayesian trees (Figure 3.5) and ML trees (Supplementary Figure 3.5), with sequences from Vancouver Island marten grouped together with 100% support within the M. americana clade. These mtDNA sequences share a recent common ancestor with M. pennanti (HM106327) and M. foina (HM106325).

Sequence fragments of the 3’ end of the mtDNA D-loop, and partial tRNA-Phe produced an alignment of 131 bp, however, the sequence from Neovison vison and Enhydra lutris did not span the full length of the alignment and were removed. Phylogenetic trees did not show strong branch support (Supplementary Figures 3.6-3.7) and were therefore excluded from further interpretation.

3.5 Discussion

Mammalian gamma-HV tend to exhibit a narrow natural host range often limited to one animal species suggesting these viruses have diverged with their host during speciation (Davison et al., 2009; Barton et al., 2011; Wertheim et al., 2014; Escalera-Zamudio et al., 2016). If HV closely co-evolve with caribou beyond the species level, we would expect to see distinct clades within the HV phylogeny representing subspecies or designatable units much like mtDNA phylogenies. Only one type of HV, Reindeer gamma-HV 1, was detected in caribou but no variation was found among caribou in this study even when compared to that of reindeer in

Norway. Several HV were identified in marten, but we did not find the same HV species present in multiple populations for comparison. Data from this study suggests that HV have coevolved

52 with their wildlife host at a species level but perhaps not at a finer population level. There may yet be discernible differences once full genomes are compared.

Based on fossil evidence, ancestral Eurasian caribou crossed into North America during the Pleistocene era by way of the Bering Strait land bridge connecting Siberia and Alaska (Flagstad and Roed, 2003; Evans et al., 2012). The species can be separated into a northern lineage and southern lineage, which arose from geographic isolation in two or three glacial refugia (Yannic et al. 2014). The northern (Beringian) lineage range near the Arctic circle from Eurasia to northwestern America, Greenland, Svalbard, and the Canadian archipelagos, whereas the southern

(North American) lineage is distributed from the island of Newfoundland, across the boreal forests and into the mountains of Canada (Yannic et al., 2014). Phylogenetic analysis of the mtDNA control region of Porcupine and Leaf River caribou in this study show similar separation into

Beringian (BEL) and North American (NAL) lineages, and those from British Columbia belonging to DU8 forming a distinct clade of BEL and NAL origin as previously seen (McDevitt et al. 2009).

However, no strong genetic differentiation is evident at the subspecies or herd level. This is likely due to the relatively recent post-glacial spread of caribou in Canada and the absence of geographic barriers (McFarlane et al. 2016).

Ancestral marten colonized North America approximately 1 mya, after which they were separated into glacial refugia in the East and West (Stone and Cook, 2002; Hughes, 2012). Easterly marten became M. americana and spread westward, recolonizing boreal forests from

Newfoundland to the Rocky Mountains and Alaska (Stone et al., 2002; Helgen and Reid, 2016).

In marten, several subfamilies of HV were identified but within each viral species, no nucleotide variation was evident. Marten from Vancouver Island in southern British Columbia shared the

53 same alpha-HV as marten from Norman Wells, Northwest Territories approximately 1,800 km north.

The Martes genus is paraphyletic with respect to Gulo gulo as is shown by phylogenetic analysis of the mtDNA, nuclear, and concatenated nuclear-mtDNA sequences (Stone and Cook,

2002; Koepfli et al., 2008; Sato et al., 2012). However, Bayesian and ML phylogenetic trees of the non-coding mtDNA control region sequences demonstrate Martes as a monophyletic genus.

The mtDNA control region is attractive to evolutionary biologists for fine-scale comparative studies since it is one of the fastest evolving segments in the animal mitochondrial genome (Koh et al., 2000; Matson and Baker, 2001). Previous studies comparing M. americana and M. caurina

– a subspecies of M. americana – do not show clear trends in sequence variation of mtDNA cytb and 304 bp of the control region among marten from geographically distant sampling areas in

North America (Stone et al., 2002; Dawson et al., 2017). This finding was supported by phylogenetic analysis of longer mtDNA control region sequences from marten in this study.

Tissues such as spleen and lymph node were selectively sampled for the detection of gamma-HV which are known to be lymphotropic during latency. However, two alpha-HV were detected in the lymphatic tissues of marten. Most well-studied alpha-HV establish latency in neurons, however Equine HV 1 can also establish latency in lymphoid cells of horses (Abdelgawad et al., 2016). During systemic infection of Leporid HV 4 in rabbits, viral DNA can be found in lymph nodes, spleen, and other lymphatic tissues (Jin et al., 2008). No health status was known for sampled marten to evaluate the presence or absence of disease associated with infection.

All caribou samples were obtained from archived material gathered over many years and used in several different studies. This format of opportunistic sampling is limited in sample availability, and consequently, some herds and ecotypes were underrepresented. Marten samples

54 were collected from skinned carcasses provided by trappers and harvesters active in the fur industry. Trappers in Alberta suffered a poor trapping season, therefore, sampling was underrepresented in this province. In some cases, marten carcasses were damaged by scavengers prior to shipment thereby rendering samples unusable.

It is likely that PCR inhibitors from degraded, decomposed, or highly fatty samples influenced the amplification of DNA targets in this study. During processing, all frozen tissues were briefly thawed. Freeze-thawing of tissues contributes to sample degradation (Ji et al., 2017).

Archived tissues of caribou and carcasses of marten were at various stages of decomposition at the time of receipt and may have experienced freeze-thaw events on numerous occasions during the study, transport, storage, or collection. Sample degradation can lead to breaks in DNA molecules and increase the likelihood of PCR inhibitors (Hofreiter et al., 2001; McCord et al., 2011). PCR inhibitors can interact directly with amplification primers or the polymerase, thereby reducing the affinity of the PCR target (McCord et al., 2011).

Phylogenetic trees of mtDNA were limited in the availability of comparable sequences that are geographically relevant. Many sequences in Genbank were omitted from analyses due to the lack of supporting publication, vague sequence qualifiers, or isolation from non-natural hosts. In the case of host mtDNA, sequence targets vary greatly between studies and are often focused on coding genes, making scarce the availability of the non-coding control region.

Longer gene sequences or complete HV genomes may yet provide finer resolution with which to study subgroups of caribou and marten. Insights from host-virus coevolution could offer an additional resource for understanding the genetic relationship between animal groups that are historically associated.

3.6 Acknowledgements

This study would not have been possible without the cooperation of hunters and trappers across Canada; Richard Popko from the Northwest Territories, Jackie Yaklin and Brian Melanson of the Yukon Trappers Association, Rob Andrushuk, Dean Berezanski, Barry Jahn, Murray Imrie, and Gord Hannibal of the Manitoba Trappers Association, Don Gordon from the Saskatchewan

Trappers Association, Tim Killey, Jerry Baker, Carl Gitscheff, Stuart Maitland, Bryan Monroe, and Paul Blackwell of the British Columbia Trappers Association, Clark Shecapio of the Cree

Trappers Association, Mark Deans of the Northwestern Fur Trappers Association, and Shawn Reid and William Larkham Jr. of the Newfoundland and Labrador Trappers Association. For caribou samples we thank Martin Kienzler of the Government of Yukon, the CircumArctic Rangifer

Monitoring and Assessment Network (CARMA) with funding through International Polar Year from the Government of Canada, Brett Elkin and Dean Brown of the University of Calgary, Joëlle

Taillon of the Government of Quebec, Steeve Côté of the Université Laval in Québec, Bryan

MacBeth of Parks Canada, Laura Finnegan of the fRI Research Caribou Program, and Helen

Schwantje of the Government of British Columbia. Thank you to Marnie Zimmer and Trent

Bollinger of the Canadian Cooperative Wildlife Health Centre and the Department of Pathology,

Faculty of Veterinary Medicine, the University of Calgary for assisting with specimen collection and storage.

3.7 Funding:

This work was supported by the University of Calgary Faculty of Veterinary Medicine

[Grant number 10006449].

3.8 Declaration of Conflicts of interest

None declared.

3.9 Supplementary Data

See next page

Supplementary Table 3.1 Summary of HV DPOL sequences included for comparison of Cervidae-derived viruses

Species Host Host Origin Genbank Accession Reference

Reindeer gammaherpesvirus 1 Rt. caribou (Leaf River herd) Canada MN087690 - MN087693 This study

Rt. granti (Porcupine herd) Canada MN087681 - MN087689 This study

Rt. groenlandicus (Bathurst herd) Canada MN087662 - MN087680 This study

Rt. tarandus Norway JX036282 Ihlebæk, 2010

Fallow deer lymphotropic Dama dama North Carolina, USA DQ083951 Li et al., 2005 herpesvirus

Type 2 ruminant rhadinovirus Odocoileus hemionus Washington, USA AY237363 Li et al., 2005 hemionus

Type 2 ruminant rhadinovirus Cervus elaphus California, USA AY237365 Li et al., 2005

Elk gammaherpesvirus 1 Cervus canadensis Canada KX062145 Dalton et al., 2017 nelsoni

Type 2 ruminant rhadinovirus Cervus elaphus Baotou, Inner Mongolia KY462774 Zhu et al., 2018

Type 2 ruminant rhadinovirus Odocoileus hemionus Washington, USA AY237362 Li et al., 2005 columbianus

Gammaherpesvirus of milu Elaphurus davidianus Yancheng, Jiangsu, China KY621347 Zhu et al., 2018

Sambar gammaherpesvirus Rusa unicolor Tunchang, Hainan, China KY612408 Zhu et al., 2018

*Bovine gammaherpesvirus 4 Bos taurus Colorado, USA AF031811 Rovnak et al., 1998

*Sequence used as outgroup

Supplementary Table 3.2 Summary of mtDNA sequences within the family Cervidae included for comparison. Sequences spanned the partial tRNA-Thr, tRNA-Pro, D-loop, and partial tRNA-Phe region.

Species Common Name (DU) Herd/Location Accession Reference

Rt. granti Grant’s Caribou (DU3) Porcupine/YK and NT, Canada MN087694- This study MN087699

Rt. caribou Woodland Caribou (DU4) Leaf River/QC, Canada MN087700- This study MN087704

(DU4) George River/QC, Canada AF096421 Dueck, 1998

(DU8) BC, Canada MN087705- This study MN087707

(DU8) Jasper National Park/AB, Canada AF096418 Dueck, 1998

(DU6) Lake Superior/ON, Canada AF096414 Dueck, 1998

(DU7) Wolf Lake/YK, Canada AF096444 Dueck, 1998

Rt. terranovae Newfoundland (DU5) NL, Canada MH266685 Wilkerson et al., 2018 Caribou

Rt. groenlandicus Barren-ground (DU3) Bathurst/NT, Canada AF096412 Dueck, 1998 Caribou

(DU3) South Baffin/NU, Canada AF096433 Dueck, 1998

Rt. tarandus Reindeer Japan NC_007703 NCBI Reference sequence

Cervus elaphus nelsoni Rocky Mountain Banff, AB, Canada AF016964 Polziehn and Strobeck Elk 1998

Dama Fallow Deer - AM419027 Hassanin et al., 2012

Odocoileus hemionus Mule Deer AB, Canada CM014077 Russell et al., 2019

Odocoileus virginianus White-tailed Deer Canada AF016978 Polziehn and Strobeck 1998

Alces americanus American Moose Canada AF016951 Polziehn and Strobeck 1998

*Bos taurus Cattle - AF492351 Hiendleder et al., 2008

*Sequence used as outgroup

Supplementary Table 3.3 Summary of HV DPOL sequences included for comparison of mustelid-derived viruses

Species Host Location of Host Genbank Accession Reference

Marten alphaherpesvirus M. americana Northwest Territories, Canada KX062131 Dalton et al., 2017

M. americana Vancouver Island, BC, Canada MN068398-MN068402 This study

Marten alphaherpesvirus 2 M. americana McBride, BC, Canada MN068392-MN068394 This study

M. americana Whitehorse, Yukon, Canada MN068395-MN068397 This study

*Mustelid alphaherpesvirus 1 Meles meles France MF042164 Kent et al., 2018

Marten gammaherpesvirus 1 M. americana Northwest Territories, Canada KX062128 Dalton et al., 2017

Marten gammaherpesvirus 2 M. americana Northwest Territories, Canada KX062130 Dalton et al., 2017

Marten gammaherpesvirus 3 M. americana Pemberton, BC, Canada MN068384-MN068388 This study

Marten gammaherpesvirus 4 M. americana Labrador South, NL, Canada MN068389-MN068391 This study

Fisher herpesvirus M. pennanti Quebec, Canada HM579931 Gagnon et al., 2011

Mustelid gammaherpesvirus 1 Meles meles Cornwall, England, UK AF376034 Banks et al., 2002

*Mustelid herpesvirus 2 Enhydra lutris Alaska, USA GU979535 Tseng et al., 2012

*Sequence used as outgroup

Supplementary Table 3.4 Summary of mtDNA sequences within the family Mustelidae included for comparison. Sequences spanned the Partial tRNA-Thr, tRNA-Pro, D-loop, tRNA-Phe region unless otherwise indicated.

Species Common Name Location Genbank Accession Reference

M. americana American Marten 100-Mile House, BC, Canada MN062191a This study

Lac la Hache, BC, Canada MN062192 a This study

Pemberton, BC, Canada MN062193-MN062195 a This study

SK, Canada MN062196 a This study

Vancouver Island, BC, MN062197 a, MN062198 b, MN062199 a, This study Canada MN062200 b, MN062201 a, MN062202 b, MN062203 a, MN062204 b

ON, Canada HM106324 Yu et al., 2011

M. foina Stone Marten China HM106325 Yu et al., 2011

M. martes European pine marten Norway HM026057 Ruiz-González et al., 2013

M. melampus Japanese Marten Japan NC_009678 Yonezawa et al., 2007

M. pennanti Fisher ON, Canada HM106327 Yu et al., 2011

M. zibellina Sable Heilongjiang, China NC_011579 Xu et al., 2012

Lutra lutra Common Otter Japan LC049955 Waku et al., 2016

Meles meles Eurasian Badger Sweden NC_011125 Arnason et al., 2007

*Enhydra lutris Sea Otter Japan NC_009692 b Yonezawa et al., 2007

Neovison vison American Mink China KM488625 b Sun et al., 2016

*Sequence used as outgroup a tRNA-Phe, partial D-loop only b Partial tRNA-Thr, tRNA-Pro, D-loop only

Supplementary Figure 3.1 ML phylogenetic tree of gamma-HV DPOL fragments isolated from cervid hosts. Colours indicate caribou herd: green (Bathurst), orange (Leaf River), purple

(Porcupine). Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.2 ML phylogenetic tree of mtDNA control region sequences from cervid hosts. Colours indicate caribou herd; blue: unknown from British Columbia, orange: Leaf River, purple: Porcupine. Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.3 ML phylogenetic tree of gamma-HV DPOL fragments isolated from mustelid hosts. Colours indicate sample origin; blue: Pemberton, BC, orange: Labrador. Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.4 ML phylogenetic tree of alpha-HV DPOL fragments isolated from mustelid hosts. Colours indicate sample origin; dark blue: McBride, BC, pink: Vancouver Island,

BC, purple: Whitehorse, YK. Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.5 ML phylogenetic trees of mtDNA tRNA-Thr, tRNA-Pro, and D-loop sequences from marten in this study and published sequences from mustelids. Pink font: marten sourced from Vancouver Island, BC. Numbers at the nodes indicate bootstrap support percentage.

Scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.6 Bayesian phylogenetic tree of partial mtDNA D-loop and tRNA-Phe sequences from marten in this study with published sequences from Genbank. Colours indicate the location of sample origin; pink: Vancouver Island, BC, light blue: Pemberton, BC, dark blue: Lac

La Hache, BC, grey: 100 Mile House, BC, yellow: Labrador. Numbers at the nodes indicate posterior probability percentage; scale bar indicates nucleotide substitutions per site.

Supplementary Figure 3.7 ML phylogenetic tree of mtDNA D-loop and tRNA-Phe sequences from marten in this study and published sequences from Genbank. Colours indicate the location of sample origin; pink: Vancouver Island, BC, light blue: Pemberton, BC, dark blue: Lac La Hache,

BC, grey: 100 Mile House, BC, yellow: Labrador. Numbers at the nodes indicate bootstrap percentage; scale bar indicates nucleotide substitutions per site.

PART II: MOLECULAR SURVEILLANCE OF ORF VIRUS IN CANADIAN MUSKOXEN

CHAPTER 4: DETECTION AND PHYLOGENETIC ANALYSIS OF ORF VIRUS AND

HERPESVIRUS FROM MUSKOXEN (OVIBOS MOSCHATUS) IN THE CANADIAN

ARCTIC

4.1 Abstract

Orf virus (genus Parapoxvirus) has been associated with clinical lesions in muskoxen on

Victoria Island, Nunavut (NU), Canada, where muskox populations are experiencing declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding which may lead to animal morbidity or mortality through secondary infections and anorexia. Our research objective was to characterize the variation of orf virus in muskoxen native to the Canadian

Arctic. Skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen that were sport-hunted, subsistence hunted, community harvested, or discovered dead on Victoria Island, NU and Northwest Territories (NT), and mainland NU. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Lesions were examined and tested for genetic evidence of orf virus and herpesvirus (HV) infection using Polymerase Chain Reaction

(PCR) targeting key viral genes. A total of 25 of 26 muskoxen with clinical lesions sampled from

2015-2017 tested positive for orf virus. Twenty out of 34 muskoxen without orf-like lesions tested positive for orf virus which may be attributed to contamination or subclinical infection. Amplified genetic products were sequenced and characterized using Bayesian and maximum likelihood phylogenetic analysis. Thirteen B2L (envelope glycoprotein) and one VIR (viral interferon resistance) gene sequence fragments were generated from orf-like lesion samples. Our results indicate that a single, unique Canadian strain of orf virus is infecting muskoxen from Victoria

Island (Nunavut and Northwest Territories), and mainland Nunavut. Muskox rhadinovirus 1, a HV endemic to muskoxen, was detected in 29 muskoxen. In all cases examined there was no

72 histological evidence of HV-specific disease. Continuous monitoring of orf virus in muskoxen may help elucidate the epidemiology of the disease that is associated with orf virus infection and its potential contribution to muskox decline in Canada.

4.2 Introduction

The muskox (Ovibos moschatus) is a unique cold-adapted ungulate that naturally resides in eastern Greenland and in the Canadian Arctic circle (Lent, 1999). These herbivores share an intricate history with the First Peoples of North America, contributing to the cultural identity and food security of Arctic Indigenous people (Lent, 1999). Tomaselli et al. (2018) reported that during times of caribou scarcity, indigenous peoples of Cambridge Bay, Victoria Island, Nunavut (NU), selectively hunt muskox until caribou populations recover. In the Arctic environment, muskoxen aide in the dispersal of seeds by grazing on vegetation, and they also provide a food source for scavengers, wolves, and grizzly bears (Elder, 2005).

Fossil evidence suggests that the ancestors of the muskox crossed the Bering land bridge to North America about 90,000 years ago, during the Pleistocene era, and survived in ice-free refugia in northern Arctic islands and Greenland (Lent, 1999). With the retreat of the glacial ice, muskoxen spread through northern Canada and Greenland, then westward into Alaska (Lent,

1999). Muskox populations have historically fluctuated in numbers and have been virtually extirpated from many regions due to die-off events and severe hunting pressures (Gunn and

Forchhammer, 2008). Since the 1930s, muskoxen have been reintroduced from Canadian wild herds to west Greenland, Alaska, Siberia, and Eurasia paralleling Ovibos’s historical circumpolar distribution (Thulin et al., 2011). On Victoria Island in the Canadian Arctic Archipelago, native

73 wild muskox populations have declined since 2005 due to numerous factors which include natural events, climate change, and infectious agents (Kutz et al., 2017).

Pathogens that have been associated with muskoxen in the Canadian Arctic include bacteria (Tomaselli et al., 2016; Kutz et al., 2015), macroparasites (Kafle et al., 2015), and viruses

(Tomaselli et al., 2016). Despite these associations, the role of infectious disease in muskoxen population dynamics remains unknown.

Orf virus, from the genus Parapoxvirus, is a large double-stranded DNA (dsDNA) virus with an envelope shaped as an ovoid particle (Delhon et al., 2004). Orf virus is the causative agent of contagious ecthyma (CE) common in sheep and goats worldwide. Transmission of orf virus occurs through direct contact with an infected animal or contaminated fomites (Friederichs et al.,

2014, Leavell et al., 1968). The virus enters through damaged skin or mucosal surfaces, resulting in proliferative and necrotizing dermatitis on the nose, lips, udder, and interdigital spaces of the feet (Tomaselli et al., 2016; Afema et al., 2017). Severe lesions with ulcerations are prone to secondary infections which can lead to mortality (Zhao et al., 2010). Animals with painful lesions around the nose and lips tend to avoid feeding which leads to poor body condition, whereas lesions around the nipples may lead to calf abandonment due to pain while nursing (Spyrou and Valiakos,

2015). Foot lesions can cause lameness, leaving infected animals weak, immobile, and vulnerable to predators (Kutz et al., 2017). Orf virus can transmit to humans such as hunters and community members that come into direct contact with contaminated skin or meat though this results in mild, self-limiting localized lesions (Friederichs et al., 2014).

In Alaska, orf virus infection is frequent in Dall’s sheep (Ovis dalli), mountain goat

(Oreamnos americanus), and muskox populations (Tryland et al., 2018; Dieterich et al., 1981).

Wild muskoxen in Norway occasionally exhibit CE, notably during an outbreak in 2004 caused

74 by an orf virus strain circulating within cohabiting animals (Vikøren et al., 2008). Orf virus in

Canada has been reported only in Rocky Mountain bighorn sheep (Ovis canadensis) in western

North America (Samuel et al., 1975), and recently in a male sport-hunted muskox near Cambridge

Bay on Victoria Island, NU (Tomaselli et al., 2016). There has been a marked increase in the number of muskoxen observed with orf-like lesions on Victoria Island (Kutz et al., 2017), however genetic confirmation as well as the impact of orf virus on the overall health of these animals in the

Canadian Arctic remains unclear.

Herpesviruses (HV), from the family Herpesviridae, are known to cause lifelong and often asymptomatic infection in natural hosts (Engels and Ackermann, 1996). During phases of active

HV infection, lesions can develop on the mucosa (Engels and Ackermann, 1996), which may be inapparent of indistinguishable from other infections in a wildlife setting. When HV infect susceptible unintended hosts, disease can be fatal (Engels and Ackermann, 1996). Muskox rhadinovirus 1 is a HV that has been reported in muskoxen from Norway, Greenland, and mainland Canada, however no illness has been associated with this virus in muskoxen to date

(Handeland et al., 2018; Li et al., 2003; Vikøren et al., 2013)

In this study, we investigate the genetic variance of orf virus in muskoxen from Victoria

Island, NU and Northwest Territories (NT), and in the Kitikmeot region of mainland Nunavut in the Canadian Arctic. We also test for evidence of HV infection which could contribute to the presence of lesions. This study represents the first large scale report of orf virus in wild, free- ranging Canadian muskoxen diagnosed through macroscopic, microscopic, and genetic evidence of infection.

4.3 Materials and methods

4.3.1 Sampling and Study Area

Tissue samples were opportunistically collected from muskoxen in 2015-2017 from

Victoria Island, NU and NT, and the Kitikmeot region of NU in the Canadian Arctic including areas surrounding Kugluktuk and Umingmaktok on the adjacent mainland, NU. Veterinarians performed field autopsies on discovered dead muskoxen and collected tissue samples from the nose, mouth, and feet. Commercially harvested, hunter-harvested or sport-hunted muskoxen were sampled using kits filled by hunters. Sample kits included the lower left hind leg, which was examined by our research team at the University of Calgary, Calgary, AB, during which case and control samples were taken. Case samples were skin samples collected from the lower legs with lesions consistent with orf virus infection, whereas control samples were taken mainly from the interdigital area and showed no macroscopic abnormalities. Tissue processing and analysis as outlined herein were performed at the Faculty of Veterinary Medicine of the University of Calgary,

Calgary, AB, Canada under tissue protocol VSACC AC16-0173.

4.3.2 Histology

Samples of case and control tissues (approximately half when available) were fixed in 10% neutral buffered formalin for at least 48 h, then processed by routine methods. Fixed tissues were embedded in paraffin, sectioned at 4 μm, mounted on a glass slide, then stained with hematoxylin and eosin (H&E) at the Diagnostic Services Unit of the Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada. A veterinary pathologist (JLR) certified by the American

College of Veterinary Pathologists screened all tissues using light microscopy and classified samples based on the presence or absence of lesions consistent with orf virus infection.

4.3.3 DNA Extraction and Polymerase Chain Reaction (PCR)

Total DNA was extracted from 40 mg of minced tissue using the E.Z.N.A.® Tissue DNA

Kit (Omega Bio-Tek Inc., Norcross, GA, USA), and quantified. Virus infection was detected using published parapoxvirus-specific primers targeting: the major viral envelope protein B2L gene resulting in 595 bp product (Inoshima et al., 2000), the DNA polymerase gene (DPOL) resulting in 536 bp product (Bracht et al., 2006), and the virus interferon resistance (VIR) gene resulting in

550 bp product (Guo et al., 2004). TREMVAC-FP® Avian encephalomyelitis – Fowlpox virus vaccine (Intervet Canada Corp., Kirkland, QC, Canada), containing Avipoxvirus, was used as a parapoxvirus PCR negative control for non-specific poxvirus amplification. Nuclease-free water

(Sigma-Aldrich Canada Co., Oakville, ON, Canada) served as a PCR contaminant control.

Muskoxen were also assayed for the presence of HV known to cause lesions on mucosal surfaces and skin. The PCR assay for HV used published pan-HV primers targeting the viral DPOL gene (VanDevanter et al., 1996), commonly targeted for diagnostic purposes. Reactions of 25 L contained 75-125 ng of sample DNA, 10 pmol of each primer, dNTP mixture, and 1 unit of

TaKaRa Ex Taq® DNA Polymerase (Takara Bio USA Inc., Mountain View, CA, USA) with the appropriate buffer. Positive PCR products were excised from 1% agarose gels and purified using the E.Z.N.A.® Gel Extraction kit (Omega Bio-tek Inc., Norcross, GA, USA). Primer sequences used in this study are summarized in Table 4.1.

Table 4.1 Primers used in this study.

Name Sequence (5’-3’) Gene target Reference

DFA GAY TTY GCN AGY YTN TAY DNA polymerase VanDevanter et al., CC 1996

KG1 GTC TTG CTC ACC AGN TCN DNA polymerase VanDevanter et al., ACN CCY TT 1996

ILK TCC TGG ACA AGC AGC ARN DNA polymerase VanDevanter et al., YSG CNM TNA A 1996

TGV TGT AAC TCG GTG TAY GGN DNA polymerase VanDevanter et al., TTY ACN GGN GT 1996

IYG CAC AGA GTC CGT RTC NCC DNA polymerase VanDevanter et al., RTA DAT 1996

Orf virus

PPP-1 GTC GTC CAC GAT GAG CAG B2L envelope protein Inoshima et al., 2000 CT

PPP-4 TAC GTG GGA AGC GCC TCG B2L envelope protein Inoshima et al., 2000 CT

VIR-F TTA GAA GCT GAT GCC GCA G interferon-resistance Guo et al., 2004

VIR-R ACA ATG GCC TGC GAG TG interferon-resistance Guo et al., 2004

PPV/DNApol-F GCG AGC ACC TGC ATC AAG DNA polymerase Bracht et al., 2006

PPV/DNApol-R CTG TTI CGG AAG CCC ATG DNA polymerase Bracht et al., 2006 AG

4.3.4 Sequencing and phylogenetic analysis

Amplified products were submitted for Sanger sequencing to The Centre for Applied

Genetics (TCAG) in Toronto, ON, Canada, then tested in the National Center for Biotechnology

Information (NCBI) nucleotide Basic Local Alignment Search Tool (BLASTn) to confirm identity. Nucleotides were aligned using MUltiple Sequence Comparison by Log-Expectation

(MUSCLE) using Geneious R10.2.3 (https://www.geneious.com). Multiple sequence alignments were produced with fragments fully spanning a 493 bp stretch of the B2L gene. These sequences include 2 from muskoxen in this study (MX319 and MX347) alongside: all published North

American orf virus strains including our first muskox orf case in 2014 (Tomaselli et al., 2016), the

B2L sequence from an orf virus infected domestic Canadian goat (Capra hircus) sample provided

78 by Prairie Diagnostic Services Inc. (PDS; Saskatoon, SK, Canada), representative orf virus strains from around the world including vaccine strains, and distantly related parapoxviruses including

Pseudocowpoxvirus (PCPV; NC013804), and Bovine popular stomatitis virus (BPSV;

NC005337).

Nucleotide alignments were analyzed using jModelTest 2.10 to identify the best fitting evolutionary model. Phylogenetic analysis was performed using maximum likelihood (ML) and

Bayesian inference. The Randomized Axelerated Maximum Likelihood (RAxML) plugin software in Geneious was used to construct ML with bootstrap values calculated from 1,000 replicates starting with a random tree and searching for the best scoring ML tree. Bayesian trees were constructed using the MrBayes software in Geneious with a burn-in length of 1,000.

4.4 Results

This study includes samples from 60 muskoxen collected over ten sampling events from

2015-2017, as indicated by the University of Calgary Identification Number (UCID) belonging to each batch of samples taken at the same time period (Table 4.2). GPS data was available for 28 muskoxen and is given in Supplementary Table 4.1. Twenty-six muskoxen that were evaluated displayed macroscopic lesions on the nose, lips or skin consistent with orf virus infection (43.3%)

(Table 4.2). Macroscopic lesions were present in calves (<1 year old), juveniles (2-3 years old), and adult muskoxen (4 years old) (Figure 4.1), and both in males and females (Figure 4.2). A total of 40 skin samples were examined by histology, and among these, 12 had microscopic lesions consistent with orf virus infection (30.0%) (Table 4.2).

Lesions were characterized by epidermal hyperplasia of varying severity with scalloping of the stratum basale and rete peg formation (Figure 4.3). Multifocal keratinocytes were swollen

79 with single, clear vacuoles. There was orthokeratotic hyperkeratosis creating thick surface keratin crusts that contained intrakeratin pustules (aggregates of viable and degenerative neutrophils) and bacterial cells. Some samples had multifocal epidermal ulcerations. Depending on the specimen, there were variable amounts of dermal inflammation that included clusters of lymphocytes, plasma cells and less frequently, neutrophils. There were also 4 samples (10.0%) that were deemed equivocal with only hyperkeratosis with or without mild epidermal hyperplasia. There was no histological evidence of multinucleated cells and/or nuclear inclusions to suggest clinical HV infection.

A total of 46 muskoxen produced a positive PCR fragment when tested for the presence of the orf virus B2L gene (Table 4.2). Twenty-five out of 26 lesions samples tested PCR positive for the B2L gene, and 20 control muskox samples produced faintly positive PCR bands (Table 4.2).

Only 12 muskoxen were PCR-positive when targeting the VIR gene, and 20 were positive for the orf virus DPOL gene (data not shown). Fowlpox vaccine and nuclease-free water negative controls were consistently negative in our PCR assays.

Of the PCR positive samples, 13 B2L and 1 VIR sequence fragments were produced using amplification primers. Sequences were inspected and trimmed to remove low quality and ambiguous nucleotide bases thereby producing high-quality B2L sequence fragments from 11 muskoxen. Muskox orf virus B2L sequences from this study ranged 279-486 bp in length and were pairwise identical (GenBank accession numbers: MN267811-MN267821).

Orf virus B2L sequences from muskoxen in Canada were identical to the sequence derived from a muskox hunted on Cambridge Bay, Victoria Island, NU, Canada in 2014 (KT719371). Our

Canadian sequences differed from Minnesota muskox orf virus by 1 nucleotide and were 2 nucleotides different from Alaskan orf virus in caribou (Rangifer tarandus), mountain goat

(Oreamnos americanus), and Alaskan muskox 147 (KJ944493). The orf virus sequence from an infected Canadian goat provided by Prairie Diagnostic Services (MN316586) was most similar to sequences from goats in Arkansas, USA in 2004 (KJ137714), Mississippi, USA in 2006

(KJ137711), and New Mexico, USA in 2006 (KJ137710) with 99.8 % identity, differing in only one nucleotide position.

Table 4.2 Summary of muskoxen included in this study. UCID represents the sampling batch.

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

305 Kugluktuk, NU Jun 2014 MX100 M Yearling Skin - + -

305 Kugluktuk, NU Jun 2014 MX101 M Yearling Skin - - n/a

305 Kugluktuk, NU Jun 2014 MX102 F Adult Skin + + **

Skin - + -

305 Kugluktuk, NU Jun 2014 MX103 F Adult Skin - - n/a

305 Kugluktuk, NU Jun 2014 MX104 F Adult Skin - - n/a

305 Kugluktuk, NU Jun 2014 MX106 F Adult Skin - + -

305 Kugluktuk, NU Jun 2014 MX109 M Adult Skin + + **

Skin - - **

305 Kugluktuk, NU Jun 2014 MX110 M Adult Skin + + -

Skin - + n/a

305 Kugluktuk, NU Jun 2014 MX115 F Adult Skin - + -

305 Kugluktuk, NU Jun 2014 MX116 F Adult Skin - + -

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

305 Kugluktuk, NU Jun 2014 MX118 F Adult Skin - + -

305 Kugluktuk, NU Jun 2014 MX119 M Yearling Skin - - n/a

331 Cambridge Bay, Vic. Nov MX182 - - Footso + + n/a Isl., NU 2014 le

Skin - + -

331 Cambridge Bay, Vic. Nov MX195 - - Skin - + - Isl., NU 2014

331 Cambridge Bay, Vic. Nov MX202 - - Skin + + + Isl., NU 2014 Skin - - +

331 Cambridge Bay, Vic. Nov MX204 - - Skin + + n/a Isl., NU 2014 Skin - - n/a

331 Cambridge Bay, Vic. Nov MX206 - - Skin + + + Isl., NU 2014 Skin - - n/a

351 West of Kugluktuk, Mar MX210 F Adult Skin - + - NU 2015

351 South of Kugluktuk, Mar MX217 F Adult Skin + - + NU 2015 Skin - - n/a

351 East of Kugluktuk, Mar MX220 F Adult Skin - + - NU 2015

351 East of Kugluktuk, Feb MX222 F Adult Skin - - n/a NU 2015

351 East of Kugluktuk, Feb MX236 M Adult Skin - + - NU 2015

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

351 East of Kugluktuk, Feb MX238 F Adult Footso + + ** NU 2015 le

Skin - - n/a

351 Lady Franklin Point, Jan 2015 MX211 F Adult Skin + + + Vic. Isl., NU Skin - + -

351 Lady Franklin Point, Jan 2015 MX212 M Adult Footso + + n/a Vic. Isl., NU le

Skin - - n/a

351 Lady Franklin Point, Jan 2015 MX213 M Adult Skin - + - Vic. Isl., NU

358 Long Point, Jun 2015 D_MX01a M Juvenile Nose + + n/a Cambridge Bay, Vic. Isl., NU

358 Cambridge Bay, Vic. Jun 2015 D_MX04 a M Adult Nose + + n/a Isl., NU

358 Gravel Pit Road, Jun 2015 D_MX06 a F Adult Lip + + n/a Cambridge Bay, Vic. Isl., NU

358 DEW Line Road, Jun 2015 D_MX08 a M Juvenile Lip + + n/a Cambridge Bay, Vic. Isl., NU

358 Cape Colborne, Jul 2015 D_MX14 a M Adult Lip + + n/a Cambridge Bay, Vic. Isl., NU

358 North Mount Pelly, Jul 2015 D_MX15 a F Adult Lip + + n/a Cambridge Bay, Vict. Isl., NU

358 West Arm, Jul 2015 D_MX16 a M Adult Lip + + n/a Cambridge Bay, Vic. Isl., NU

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

397 Ferguson Lake, Mar MX274 M Adult Skin - + - Cambridge Bay, Vic. 2016 Isl., NU

397 Ferguson Lake, Mar MX280 M Adult Skin - + - Cambridge Bay, Vic. 2016 Isl., NU

397 North of Apr MX360 M Adult Skin - + - Umingmaktok, NU 2016

397 North of Apr MX364 M Adult Skin - + - Umingmaktok, NU 2016

397 North of Apr MX365 M Adult Skin - + - Umingmaktok, NU 2016

397 North of Apr MX367 M Adult Skin - + - Umingmaktok, NU 2016

398 Augustus Hills, Mar MX278 F Adult Nose + + + Cambridge Bay, Vic. 2016 Isl., NU Skin - - n/a

399 Ferguson Lake, Apr MX366 F Adult Nose + + + Cambridge Bay, Vic. 2016 Isl., NU Skin - - n/a

402A East of Cambridge Fall MX229 - - Skin - - n/a Bay, Vic. Isl., NU 2015

402A Cambridge Bay, Vic. Fall MX318 - - Skin - + - Isl., NU 2015

402A Cambridge Bay, Vic. Oct MX319 M Adult Skin + + + Isl., NU 2015 Skin - + -

402A Cambridge Bay, Vic. Dec MX343 F Juvenile Skin - + - Isl., NU 2015

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

402A Cambridge Bay, Vic. Dec MX344 M Juvenile Skin - - n/a Isl., NU 2015

402A Cambridge Bay, Vic. Dec MX345 F Juvenile Skin - - n/a Isl., NU 2015

402A Cambridge Bay, Vic. Dec MX346 F Adult Skin + + n/a Isl., NU 2015 Skin - - n/a

402A Anderson Bay, Dec MX347 F Adult Skin + + + Cambridge Bay, Vic. 2015 Isl., NU Skin - - n/a

402A Anderson Bay, Dec MX348 F Adult Skin - - - Cambridge Bay, Vic. 2015 Isl., NU

402A Cambridge Bay, Vic. Apr MX349 - - Skin - - n/a Isl., NU 2016

402A Cambridge Bay, Vic. Apr MX351 - - Skin - - n/a Isl., NU 2016

402A Kugluktuk, NU Apr MX386 - - Skin - + - 2016

402A Kugluktuk, NU Apr MX387 - - Skin - - n/a 2016

402A Kugluktuk, NU Apr MX388 - - Skin - - n/a 2016

402A Kugluktuk, NU Apr MX389 - - Skin - + - 2016

417 Cambridge Bay, Vic. May VI16-001 M Adult Nose + + + Isl., NU 2016

442 Albert Islands, Apr MX441 M Calf Nose + + + Ulukhaktok, Vic. 2017 Isl., NT Lip - + **

UCID Location Date of Animal Sex and age Tissue Macro- B2L Histo- Death ID Type scopic PCR logical lesions* lesions*

442 Kuuk River, Apr MX444 F Calf Lip + + + Ulukhaktok, Vic. 2017 Isl., NT Lip - + +

442 Albert Islands, Apr MX445 M Calf Nose - - n/a Ulukhaktok, Vic. 2017 Isl., NT

** Lesions consistent with CE/CPD

*Equivocal histology – skin with only hyperkeratosis and/or mild epidermal hyperplasia a = D_MX; Discovered (dead) muskox n/a – not available for histological examination

Figure 4.1 Count of muskox samples with macroscopic orf-like lesions among different age groups. Age groups are as follows: calf (<1 year old), yearling (1-2 years old), juvenile (2-3 years old), adult (4 years old).

Figure 4.2 Count of muskox samples with macroscopic orf-like lesions among different sexes.

Figure 4.3 Microscopic appearance of skin samples from Muskoxen. H&E stain. A) Normal haired skin (control sample with no macroscopic abnormalities). MX116, 4X, scale 200 m; B) Severe proliferative dermatitis of the lip with epidermal hyperplasia (short arrow), hyperkeratosis (thin arrow) and intracorneal pustules (thick arrow) characteristic of orf virus infection. MX444, 1.25X, scale 1 mm; C) Severe proliferative dermatitis of haired skin with epidermal hyperplasia (short arrow), hyperkeratosis (thin arrow) and intracorneal pustules (thick arrow) characteristic of orf virus infection. MX319, 4X, scale 200 m; D) Bacterial cells within the orthokeratotic hyperkeratosis affecting the lip as shown by the black arrow. MX444, 60X, scale 20 m.

Figure 4.4 Map of northern Canada showing the number of sequence-confirmed infections of orf virus in muskoxen. White numbers indicate the total number of muskoxen sampled.

Nucleotide alignments of the orf virus B2L gene fragment supported the general time- reversible (GTR) model of evolution, with gamma distribution. Bayesian (Figure 4.5) and ML

(Supplementary Figure 4.1) trees of the orf virus B2L gene fragment depict similar branching patterns. RAxML bootstrap values were poor overall and could not confidently distinguish most evolutionary branches.

Orf virus DPOL fragments were successfully sequenced from 7 muskoxen (Accession:

MN316550- MN316556). These sequences were pairwise identical, and 99.8% identical to the highest scoring BLASTn hit: Orf virus reference strain OV-IA82 (AY386263), from sheep.

Twenty-nine muskoxen were PCR positive when tested for the HV DPOL gene and produced nucleotide sequence fragments ranging from 154-216 bp in length. Sequences were pairwise identical and matched with 100 % identity to Muskox rhadinovirus 1 (KX025089).

Nucleotide alignments of the HV DPOL gene fragment supported the Hasegawa-Kishino-Yano

(HKY) model of evolution, with gamma distribution. Twenty-three HV DPOL sequences were deposited in Genbank (Accession: MN316557-MN316585), and one representative sequence was included in phylogenetic analysis. Bayesian (Figure 4.6) and RAxML trees (Supplementary Figure

4.2) grouped our muskox HV DPOL sequences with those of Muskox rhadinovirus 1 characterized in captive muskoxen from Saskatchewan in 2003 (Accession: AY212111; Li et al., 2003), as well as in wild muskoxen from Greenland in 2014 (Accession: KX025101; Handeland et al., 2018).

Figure 4.5 Bayesian phylogenetic tree of the orf virus B2L gene fragment. Bold indicates sequences from muskoxen and domestic goat from this study. Branch support values > 50% are shown; NCBI GenBank accession numbers are in parentheses; BPSV: Bovine papular stomatitis virus; PCPV: Pseudocowpoxvirus; scale bars show nucleotide substitutions per site.

Figure 4.6 Phylogenetic trees of the HV DPOL fragment. Sequences from this study are shown in blue. Branch support values > 50% are shown; NCBI GenBank accession numbers are shown in parentheses. Scale bars show nucleotide substitutions per site.

4.5 Discussion

This study represents the first large scale report of orf virus in wild, free-ranging Canadian muskoxen diagnosed through macroscopic, microscopic, and genetic evidence of infection. We characterized orf virus in Canadian muskoxen sampled in multiple geographic locations over three years, with sampling success driven directly by the participation of community members and hunters. There was no genetic variability in the B2L sequences generated from infected muskoxen, however, these sequences were different from the orf virus identified in wild Alaskan ruminants.

There is no known overlap in the range of Canadian muskoxen with the range of Alaskan muskox populations (Gunn and Forchhammer, 2008), and from our sequence data, there is no evidence to suggest the clinical cases in Canadian muskoxen were linked by disease outbreak or spillover of orf virus to, or from, Alaskan ruminants.

In this study we also present the orf virus B2L sequence from an infected domestic

Canadian goat, thereby providing more context for Canadian strains. PCR targeting the orf virus

DPOL and VIR genes were not able to consistently detect infection, as previously experienced by

Kottaridi et al. (2006).

Outbreaks of CE have been documented in captive muskoxen of Alaska, and free-ranging muskoxen of Norway (Dieterich et al., 1981; Vikøren et al., 2008). Recently, orf virus infection has been characterized in free-ranging ruminants including muskoxen in Alaska (Tryland et al.,

2018). Among the orf virus-positive Alaskan ruminants, PCR sequences of the orf virus B2L gene amplified in muskox, caribou, and mountain goat showed homology despite being sourced from different geographic locations across many years (Tryland et al., 2018). Likewise, the sequences sourced from Canadian muskoxen in different geographic areas, various sampling batches, and over several years are homologous.

Muskoxen from Victoria Island and mainland Canada were positive of Muskox rhadinovirus 1 with no sequence variation in the HV DPOL gene fragment. Li et al. (2003) found identical Muskox rhadinovirus 1 sequences in 7 randomly selected HV PCR-positive muskoxen from Saskatchewan, Canada (captive), and Sachs harbour, Banks Island, NT (wild). These sequences are identical to Muskox rhadinovirus 1 later characterized in wild muskoxen from

Greenland which also found no sequence variation (Handeland et al., 2018).

This lack of sequence variability in two different dsDNA viruses suggests there may be limited evolutionary pressure on these viruses. It is well reported that modern-day muskoxen have low genetic variability (Van Coeverden de Groot, 2001; Hansen et al., 2018; Cuyler et al., 2019) which frequently calls into question the effectiveness of their immune system. Desforges et al.

(2018) showed greater cytotoxic sensitivity of target cells toward muskox and caribou natural killer cells compared to polar bear, suggesting these ungulates are immune-competent. It is possible that the lack of genetic variability in viral sequences is a result of viral DNA polymerase proof-reading activity, as has been demonstrated for Vaccinia virus (Gammon et al., 2009), ensuring a low mutation rate in the gene sequences investigated herein.

Ideally, tissues for histology should have been fixed in formalin immediately after collection and stored at room temperature. However, since samples in this study were opportunistically available from material harvested for consumption, sampling strategies in the field become limited by time and resources, such as sterilize instruments. Control samples were not available for all cases of macroscopic lesions, and samples were sometimes too small for use in both histology and DNA extraction. Taking this into consideration, the most feasible approach was to ensure that different sampling batches were kept separate.

It is probable that control samples were contaminated with material from case samples within the same sampling batch. Virus particles from lesions or the associated scabs could have been transferred to the hair or skin of an uninfected muskox, either by the reuse of dirty instruments or by contact with contaminated equipment and clothing. However, subsampling, DNA extraction, and PCR amplification were performed aseptically in separate batches with necessary negative controls to avoid cross-contamination between different batches. Despite possible contamination, data from this study confirm the presence of orf virus in muskoxen collected from different years and geographical locations suggesting that infection is widespread in the Canadian arctic.

Orf virus outbreaks in sheep and goats are typically characterized by clinical disease present in all age groups, though adult animals are typically less severely affected (Spickler, 2015).

Muskoxen of all ages in our study showed clinical lesions and genetic evidence of orf virus infection. Sampling in this study was biased towards adult muskoxen since these animals are typically targeted by subsistence hunters for meat and by sport hunters as trophies. Additionally, our samples are biased toward muskoxen from Victoria Island since the density of muskoxen is much higher than on mainland Nunavut (Leclerc, 2015).

Our data indicate that orf virus is present in muskoxen from the Canadian mainland, as well as island populations. Island muskoxen are separated from mainland muskoxen by water gaps for most of the year (Van Coeverden de Groot, 2001). These gaps freeze over in the winter which facilitates the movement of animals, however, muskox movement between mainland Nunavut and

Victoria Island has not been regularly observed (Van Coeverden de Groot, 2001; Dumond, 2006).

Studies comparing the genetic diversity of mainland and island muskoxen suggest historical isolation between these populations due to the natural water gap barrier (MacPhee et al., 2005).

Therefore, it is unlikely that the movement of muskoxen across water gaps separating mainland and island populations is the main contributor to the presence of orf virus in both.

Muskoxen cohabit with caribou throughout most of their range however unlike muskoxen, tundra caribou are migratory and traverse great distances in large herds (COSEWIC, 2004).

Caribou are naturally susceptible to orf virus (Tryland et al., 2018), and moose (Alces alces), known to overlap with the southern limits of muskox range, have been shown to be experimentally susceptible to sheep orf virus (Zarnke et al., 1983). It is possible that Orf virus transmission is facilitated by infected cohabiting animals.

It is also possible that orf virus is endemic in Canadian muskoxen whereby muskoxen are a reservoir for the virus as is the case in sheep. Muskoxen are known to rub their faces on prominent objects in their surroundings such as rocks (Flood et al., 1989). Sharing of a contaminated rock could facilitate the indirect transmission of orf virus to susceptible muskoxen. More research is needed to elucidate the mode of virus transmission in muskoxen.

Samples in this study were collected over three years, limiting the use of our data in epidemiological analysis. Multi-year surveillance of orf virus in muskoxen across the Canadian north is needed to determine whether orf virus infection in muskoxen is the result of recent outbreak event caused by the introduction of the virus from domestic or other wildlife sources, or if there is an ongoing infection and sustained transmission with no genetic variability.

4.6 Acknowledgements

Thank you to the communities, hunters, and Hunters & Trappers Organization of

Kugluktuk, Cambridge Bay, and Ulukhaktok. Funding from ArcticNet, NSERC PromoScience, and support from Nunavut Arctic College and Irving Maritime Ship Building, Polar Knowledge

Canada, and Canadian North Outfitters aided this project. Sam Sharpe, James Wang and the staff at the Clinical Skills Building of the University of Calgary were essential in facilitating this research. Diagnostic Services Unit Histology Technicians Susan Calder-Lodge and Jennifer Larios prepared the histology slides.

4.7 Declaration of Conflicts of interest

None declared.

4.8 Supplementary Data

See next page

Supplementary Table 4.1 GPS location of muskoxen at time of hunt.

UCID Muskox ID GPS location (latitude, longitude)

351 MX210 68.128611, -116.530278

351 MX211 68.619510, -112.685930

351 MX212 68.619510, -112.685930

351 MX213 68.619510, -112.685930

351 MX217 67.387720, -115.230490

351 MX220 67.890000, -113.813333

351 MX222 67.758890, -114.708470

351 MX236 67.637010, -114.521510

351 MX238 67.769730, -114.757770

397 MX274 69.377667, -105.524167

398 MX278 69.132980, -105.471880

397 MX280 69.377667, -105.524167

402 MX319 69.050000, -104.250000

402 MX343 68.530000, -104.300000

402 MX344 68.530000, -104.300000

402 MX345 68.530000, -104.300000

402 MX346 68.530000, -104.300000

402 MX347 68.530000, -104.300000

402 MX348 68.530000, -104.300000

397 MX360 68.285117, -108.110783

397 MX364 68.318183, -107.908917

397 MX365 68.428283, -108.055867

399 MX366 69.583333, -104.366667

397 MX367 68.467517, -107.940767

442 MX441 70.553880, -115.865000

442 MX444 70.617000, -112.832000

442 MX445 70.553880, -115.865000

100

Supplementary Figure 4.1 RAxML phylogenetic tree of the orf virus B2L gene fragment. Bold indicates 493 bp sequences from muskoxen and domestic goat from this study. NCBI GenBank accession numbers are in parentheses. Scale bars show nucleotide substitutions per site.

101

Supplementary Figure 4.2 RAxML phylogenetic tree of the HV DPOL gene fragment. Bold indicates sequences from muskoxen in this study. Branch support values > 50% are shown; NCBI

GenBank accession numbers are in parentheses. Scale bars show nucleotide substitutions per site.

102

CHAPTER 5: NEXT-GENERATION SEQUENCING APPROACH TO INVESTIGATE

GENOME VARIABILITY OF PARAPOXVIRUS IN MUSKOXEN (OVIBOS

MOSCHATUS)

5.1 Abstract

Orf virus, a double-stranded DNA (dsDNA) virus of the genus parapoxvirus, was first isolated from a muskox from Victoria Island, Nunavut (NU), Canada in 2016. Sequencing of the viral envelope gene B2L suggested that this isolate was a previously uncharacterized strain. We used deep sequencing on DNA extracted from orf virus-positive tissues from wild muskoxen from locations on Victoria Island and the adjacent mainland to characterize the strain further. Orf virus sequence reads derived from four samples were nearly identical. These data were pooled to generate a consensus sequence of Muskox orf virus (MxOV). MxOV presented herein comprises a large contiguous sequence (contig) of 131,759 bp and a smaller right terminal contig of 3,552 bp contig, containing all coding sequences identified as Parapoxvirus. MxOV open reading frames were predicted using Prodigal 2.6 and annotated using Prokka 1.13.3 in addition to the manual annotation of three genes confirmed using BLASTn. Based on nucleotide percent identity and genomic layout, MxOV is closely related to the reference genome of an orf virus isolated from a sheep in New Zealand (OV-NZ2). Phylogenetic analysis using whole-genome amino acid sequences identifies the closest known relative of MxOV as reference strain OV-IA82 identified in a sheep from Iowa, USA. Individual gene comparisons reveal that MxOV shares genetic characteristics with reference strains from both sheep and goat origin. Recombination analysis using Bootscan, MAXCHI, GENECONV, CHIMAERA, SISCAN, and RDP algorithms within the RDP4 software predicted recombination events in two virulence factors, and a large 3,000 bp segment of the MxOV genome. The genome sequence of MxOV reported here is derived directly

103 from clinical field samples and is considered representative of the naturally occurring orf virus strain in muskoxen from Victoria Island (Nunavut and Northwest Territories) in the Canadian

North. Our analyses demonstrate little MxOV sequence variability among infected muskoxen from geographically distant regions on Victoria Island.

5.2 Introduction

Orf virus is the causative agent of contagious ecthyma, an acute skin disease common in sheep (Ovis spp.) and goats (Capra spp.) worldwide (Haig et al., 2006). The virus belongs to the genus Parapoxvirus in the subfamily Chordopoxvirinae and family Poxviridae (Mercer et al.,

2006). Orf virus causes non-systemic proliferative lesions in the skin, udders, and/or in the mucosa typically around the nose and lips (Greig, 1956; Haig et al., 2006). Painful lesions can lead to lethargy and anorexia due to the avoidance of feeding (Delhon et al., 2003). The lesions progress to pustules which develop into scabs that harbour orf virus particles (Haig et al., 2006).

Transmission of the virus occurs when susceptible hosts encounter infested scab material or come in direct contact with infected animals (Delhon et al., 2003; Günther et al., 2017). Orf virus is a highly contagious zoonotic pathogen capable of causing disease in humans, especially those who handle sheep, goats, and other ruminants (Hosamani et al., 2009). Viral lesions offer an entry point for secondary infection which is the main cause of orf virus-associated mortality in animals

(Demiraslan et al., 2017). Infected immune-competent animals typically clear the virus within a matter of weeks, however, no long-term immunity to re-infection is induced (Delhon et al., 2003).

Vikøren et al. (2008) reported the first known outbreak of orf virus infection in free-ranging muskoxen (Ovibos moschatus) of Norway. Using polymerase chain reaction (PCR) targeting the orf virus B2L gene (ORFV011) coding for a viral envelope protein, they were able to show that

104 the outbreak in muskoxen was caused by an orf virus strain circulating among cattle (Bos taurus), reindeer (Rangifer tarandus tarandus), and sheep in Norway (Vikøren et al., 2008).

Recently, Tryland et al. (2018) reported orf virus infection in archived tissues from muskoxen, mountain goats (Oreamnos americanus), caribou (Rangier tarandus granti), Sitka black-tailed deer (Odocoileus hemionus sitkensis), and Dall’s sheep (Ovis dalli dalli) sampled between 2002-2012 in Alaska, USA. Sequences of several orf virus genes from these animals were nearly identical apart from those originating from Dall’s sheep, demonstrating that wild ruminants can carry and be affected by orf virus (Tryland et al., 2018).

Clinical signs consistent with orf infection (scabs around the nose, mouth, and eyes) in muskoxen have been reported sporadically by local knowledge holders and sport hunters on

Victoria and Banks Islands in the Canadian Arctic Archipelago (Tomaselli et al., 2018). In 2016, orf virus was first isolated from a clinically affected wild muskox from Victoria Island, NU,

Canada, confirming this virus as a cause of morbidity (Tomaselli et al., 2016). Muskoxen are endemic to the Canadian Arctic Archipelago and represent approximately 65% of the global muskox population (Cuyler et al., 2019).

Molecular characterization of the orf virus B2L gene from the isolate from the Victoria

Island muskox suggested infection by a previously uncharacterized orf virus strain (Tomaselli et al., 2016). However, information from only one gene may bias the interpretation of virus diversity and does not provide much information about the genetic characteristics of this virus. Chi et al.

(2013) showed that clustering of orf virus isolates from goats in Northern Fujian, China, differed greatly when the phylogenetic analysis was based on the immunodominant envelope proteins: B2L and F1L. Comparisons of the full genomes of orf virus isolates from Fujian revealed similarities

105 in genome structure, but differences in the terminal regions and gene content among the isolates

(Chi et al., 2015).

Genome sequences also allowed for a distinction between orf viruses of sheep or goat origin (Chi et al., 2015). Sequencing the genomes of orf viruses allows for the comparison of genome sizes, conserved and unique genes, and their arrangement within the orf virus genome

(Gubser et al., 2004). Orf virus genomes have an average G+C content of ~64% but show a distinctive pattern of deviation in G+C content at terminal regions, unlike the uniform G+C content seen in all other genera of Chordopoxvirinae (Mercer et al., 2006). The genome of orf virus has a central core region (ORFV009 to ORFV111) which contains homologues of conserved poxvirus genes responsible for the replication, structure, and maturation of virus particles (Delhon et al.,

2003). The flanking terminal regions of the orf virus genome are more variable and code for virulence factors that play a role in immune evasion and host specificity (Delhon et al., 2003;

Fleming et al., 2015). Virulence factors such as virus interleukin 10 (vIL-10) and vascular endothelial growth factor (VEGF) have been captured from their specific host and can be highly variable among strains of the same orf virus (Fleming et al., 2015).

Recombination, the exchange of genetic information between two nucleotide sequences, reshuffles existing variation and even creates new variants. One main assumption of most phylogenetic methods is that there is only one phylogeny underlying the evolution of the sequences under study (Posada and Crandall, 2001). Recombination violates this assumption by generating mosaic genes, where different regions have different phylogenetic histories (Posada and Crandall,

2001). For the recombination of poxviruses to take place, one cell must be infected by two or more strains with homologous genomic regions (Paszkowski et al., 2016). The study of recombination events allows us to better understand the dynamics of genomes, and better interpret hypotheses

106 based on the estimation of phylogenetic trees. By ignoring the presence of recombination, a phylogenetic analysis may be severely compromised. (Posada and Crandall, 2001).

Performing deep sequencing directly on clinical samples offers an opportunity to investigate the most representative viral genome at play without adaptations caused by in vitro or in vivo methods (Günther et al., 2017). In this study, we sequence DNA extracted directly from clinical samples to generate the genome of orf virus infecting Canadian muskoxen. Phylogenetic and recombination analysis is performed to gain a better understanding of evolutionary relationships between orf virus strains, and the diversity present on Victoria Island and the adjacent mainland of Nunavut in the Canadian Arctic.

5.3 Materials and Methods

5.3.1 Sampling

Tissues with clinical orf virus-like lesions were collected from muskoxen that were sampled as part of a broader community-based wildlife health surveillance program (collaboration among communities, Governments of Nunavut and NWT, and Kutz Lab; University of Calgary

Animal Care and Use Protocol AC13-0121, AC17-0010). These animals were either hunted for subsistence or as guided hunts, or discovered dead on Victoria Island (NU, and NT) and the adjacent mainland Nunavut between 2015 and 2017. Samples from four clinically infected muskoxen were selected for sequencing as they were geographically distant from one another within the sampling area. Briefly, tissues were homogenized, and DNA was extracted using the

E.Z.N.A.® Tissue DNA Kit (Omega Bio-tek Inc., Norcross, GA, US). Primers targeting the B2L gene as previously described (Inoshima et al., 2000) were used to confirm the presence of orf virus.

Details of clinical samples included in this study are summarized in Table 5.1.

107

Table 5.1 Muskox samples included in this study

Sample Location Tissue Date Collected

MX08 Cambridge Bay, Victoria Island, NU Skin lesion June 2015

MX238 Kugluktuk, NU Footsole lesion February 2015

MX347 Cambridge Bay, Victoria Island, NU Skin lesion December 2015

MX444 Ulukhaktok, Victoria Island, NT Lip lesion August 2017

5.3.2 Density Gradient Centrifugation

Approximately 0.3 g of each lesion tissue was homogenized in 4 mL of sterile PBS using a gentleMACS™ Dissociator (Miltenyi Biotec, Auburn, CA, USA). The homogenate was filtered through a 0.45 m Polypropylene VWR® syringe filter (VWR International, Edmonton, AB,

Canada) to enrich for Orf virus particles (140-170 nm wide, and 220-300 nm long) through depletion of intact host cells, bacterial cells, and contaminants > 450 nm in size. A discontinuous iodixanol gradient was prepared by diluting OptiPrep™ Density Gradient Medium (60% w/v solution of iodixanol in water) with sterile PBS to produce 25%, 20%, 15%, and 10% w/v iodixanol solutions. A 30% w/v iodixanol solution was prepared by combining 350 L of homogenized biopsy with 350 L of OptiPrep™ Density Gradient Medium. The gradient was carefully generated by sequentially layering 700 L of the 30% (w/v) iodixanol solution, followed by 650

L each of 25, 20, 15, and 10% (w/v) iodixanol solutions. The gradient was centrifuged at 10 C in an Optima MAX-XP Ultracentrifuge (Beckman Coulter Inc., Brea, CA, USA) using an MLS-

50 rotor for 4 hours at 168,000 x g, with slow acceleration for 3 mins, and slow deceleration for

10 mins. Twelve fractions of 250 L, and a final fraction of 300 L were collected. DNA was

108 extracted from each fraction and PCR as previously described (Tomaselli et al., 2016) was used to detect the Orf virus B2L gene to confirm the presence of orf virus.

5.3.3 Library Preparation and Next-Generation Sequencing

DNA from Orf virus PCR-positive fractions were pooled for each sample and submitted to the University Core DNA Services (University of Calgary, AB, Canada) for library preparation.

Libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®

(New England Biolabs Ltd., Whitby, ON, CAN) following the manufacturer’s protocol and paired- end sequenced using a NextSeq 500/550 Mid Output v2 kit (300 cycles) (Illumina Inc., San Diego,

CA, USA).

5.3.4 Data Analysis

Raw sequence files were trimmed with fastp 0.19.4 (Chen et al., 2018) using default parameters. Read-based taxonomic assignments were generated with Kaiju 1.6.3 (Menzel et al.,

2016) with NCBI non-redundant protein sequences, using “greedy” mode allowing for 5 substitutions and a minimum match score of 60. Kaiju, using a protein-based alignment, is more sensitive for viral sequences but less sensitive for non-coding regions of Eukaryota and will, therefore, miss intergenic reads originating from the host genome. To identify host sequences (note that no reference genome is currently available for Ovibos moschatus), reads were also classified with Centrifuge 1.0.4 (Kim et al., 2016) against NCBI non-redundant nucleotide sequences (nt) with default parameters. Reads that matched the subfamily Caprinae were removed from each sample using SeqKit v0.8.0 (Shen et al., 2016). The remaining reads were co-assembled with

Megahit 1.1.3 (Li et al., 2015) using default parameters.

109

To identify orf virus contiguous sequences (contigs) from the assembly, open reading frames (ORFs) were identified with Prodigal 2.6.3 (Hyatt et al., 2010) and the taxonomy of the coding sequences was identified using Kaiju as above. A 131,759 and a 3,552 bp contig were identified that contained all coding sequences identified as Parapoxvirus. These contigs were concatenated into what is referred as the Muskox orf virus (MxOV genome) and used for downstream analysis. The MxOV genome was annotated using Prokka 1.13.3 (Seemann, 2014).

Several known orf virus genes such as dUTPase (deoxyuridine 5′-triphosphate nucleotidohydrolase) and VEGF-like protein were not identified in the original annotation. To identify these the MxOV DNA sequence was searched against a database of parapoxvirus genomes with BLASTn. Hits to dUTPase and VEGF were identified on the ends of the large MxOV contig.

These regions had not been identified as potential genes by Prodigal, reasons for which are not clear. These annotations were added based on the blast hits. Coverage of MxOV in each individual sample was calculated by mapping the trimmed reads to the MxOV contig using BBMap 37.90

(Bushnell, 2017).

MxOV proteins were searched against a database of Parapoxvirus proteins using BLASTp

(2.7.1) and the best scoring hit (bitscore) was retained. Whole genomes, individual protein, and concatenated protein sequences of MxOV and other orf virus strains were aligned with MAFFT

7.407 (Katoh et al., 2002) and a phylogenetic tree constructed with IQ-TREE 1.6.8 (Nguyen et al.,

2015). Unless otherwise stated, trees were rooted on Pseudocowpoxvirus (PCPV). Ten genes were compared in individual protein trees with reference strains: B2L (an immunodominant envelope protein), F1L (an immunodominant envelope protein), DPOL (involved in replication), DNA topoisomerase (DNAtopo; involved in relaxation of supercoiled DNA), virus interferon resistance

(VIR; inhibits host interferon response, immune evasion), viral interleukin 10 (vIL-10; inhibits

110 maturation and function of antigen-presenting cells, immune evasion), granulocyte-macrophage colony-stimulating factor inhibitory factor (GIF; inhibits host IL-2, immune evasion), virus chemokine binding protein (CBP; competitive binding of chemokine receptors, immune evasion), viral vascular endothelial growth factor (VEGF; essential for contagious pustular symptoms and vascular permeability), and dUTPase (prevents excessive dUTP incorporation, decreases mutation frequency, maintains genetic stability, may be involved in between-species infections). For concatenated protein phylogenetic trees, sequences were concatenated in the order of B2L, CBP,

DNA pol, DNA topo, dUTPase, F1L, GIF, VEGF, vIL10, then VIR.

Bootstrap values were generated using IQ-TREE’s ultrafast bootstrap method (Hoang et al., 2018). The ultrafast bootstrap (UFBoot) feature (-bb option) was published in (Minh et al.,

2013), and produces support values that are more unbiased when compared to normal bootstrap supports; 95 % UFBoot support corresponds roughly to a probability of 95 % that a clade is true, whereas regular bootstrap values are considered true when values are > 80 %.

To evaluate variability in the MxOV sequence across samples, variants were identified with FreeBayes 1.2.0 (Garrison and Marth, 2012), filtered for quality of greater than 100, and annotated with SnpEff 4.3.1t (Cingolani et al., 2012).

All software was installed using Bioconda (Grüning et al., 2018) and analyses were run as

Snakemake (Köster and Rahmann, 2012) workflows. Genomes included in this study for analyses are summarized in Supplementary Table 5.1.

5.3.5 Recombination Analysis

An alignment consisting of twelve complete orf virus genomes was constructed as previously described and subjected to an exploratory recombination analysis using the full suite of

111 options including Bootscan (Martin et al. 2005), MAXCHI (Maynard 1992), GENECONV

(Padidam et al., 1999), CHIMAERA (Posada and Crandall 2001) and the SISCAN method (Gibbs et al., 2000) within the RDP4 software package (v4.97) with default settings (Martin et al., 2015), to identify potential recombination events. Events detectable by more than 3 methods, with a p- value < 0.05 were considered significant. Recombinant and non-recombinant regions of the genome were used to generate separate phylogenetic trees using the FastTree (Price et al., 2010) maximum likelihood (ML) algorithm in the RDP4 software.

5.4 Results

5.4.1 Reads Data

NextSeq data of field samples from muskoxen produced 36 – 40 million reads per sample

(Table 5.2). Our samples contained 0.26 - 0.40 % viral reads, with most reads remaining unclassified

Table 5.2 Taxonomic summary of NextSeq reads from muskox samples

Sample Total Archaea Bacteria Eukaryota Unclassified Viruses

MX08 36,684,136 40,270 10,264,607 7,338,562 18,892,469 148,228

MX238 38,110,841 39,980 11,043,826 7,408,225 19,518,121 100,689

MX347 36,052,048 42,193 9,990,130 7,547,864 18,329,421 142,440

MX444 40,312,141 36,201 10,057,404 7,191,462 22,885,891 141,183

5.4.2 Assembly Data

Reads were assembled into 14,338 contigs ranging in size from 1,000 to 164,158 bp in length, with an average contig size of 1,617 bp. Reads separated by sample produced nearly

112 identical contigs but with regions of low coverage due to relatively few viral reads in each sample.

Therefore, viral reads from all samples were grouped together to generate longer contigs. MxOV is represented as two major contigs: one of 131,795 bp with a GC content of 64.14 %, and a smaller right terminal contig of 3,552 bp containing 59.46 % GC with coverage > 50 across in three of four muskox samples. Although most raw viral reads from MX238 matched with identity to

Parapoxvirus, these reads showed poor coverage across the MxOV genome for reasons that are unclear. Data from MX238 were excluded from subsequent analysis.

Annotation Data

The genome of MxOV contains 131 predicted genes (Supplementary Table 5.2) annotated based on the highest bitscore (Supplementary Table 5.3), which is comparable to the number of genes in reference orf virus genomes (Chi et al., 2015; Fleming et al., 2015). Most proteins were annotated on the major contig except for hypothetical proteins MxOV129 to MxOV131.

5.4.3 Variants Data

Sequences from MX08 and MX347 showed the same two variant nucleotides when compared to the consensus MxOV genome (Table 5.3). The impact of 26345G>T and 111515C>T are moderate in that they both result in a missense mutation, however, these genes encode for hypothetical proteins with unknown function.

The only variant of MX444 occurs between genes of Mx0V107 (encoding an envelope glycoprotein) and MxOV108 (encoding a hypothetical protein) (Table 5.3). This intergenic region of MxOV is 149 bp long whereas the region is 32 bp long in Orf virus NP, SJ1, NZ2, HN3/12,

113

NA1/11, and YX strains, and 28 bp long in GO, SA00, and D1701. OV-182 does not contain an intergenic region between the equivalent hypothetical protein and envelope glycoprotein.

Table 5.3 Variants in reads from MX samples with reference to MxOV genome

Sample Position nta aab Type Impact MxOV Product Gene

MX08 26345 G→T R755S Missense Moderate 024 hypothetical protein

MX08 42335 T→C G61G Synonymous Low 041 late transcription factor VLTF-1

MX08 111515 C→T P307L Missense Moderate 111 hypothetical protein

MX347 26345 G→T R755S Missense Moderate 024 hypothetical protein

MX347 42335 T→C G61G Synonymous Low 041 late transcription factor VLTF-1

MX347 111515 C→T P307L Missense Moderate 111 hypothetical protein

MX444 108394 C→T N/A Intergenic Modifier 107-108 ant – nucleotide variant baa – amino acid variant

5.4.4 Comparison of Individual Proteins

DPOL, GIF, and B2L gene sequences of MxOV are closely related to OV-IA82 (Figure

5.1). No PCPV homologues were available for vIL-10 and dUTPase sequences, therefore trees were rooted on OV-D1701. The vIL-10 and dUTPase proteins of MxOV are closest to those from

OV-NZ2 (Figure 5.1). MxOV VEGF is only distantly related to reference sequences, the closest

114 in amino acid similarity being OV-SA00 from a goat in San Antonio, Texas, USA with a shared amino acid identity of 90.3% (Figure 5.1; Supplementary Table 5.3). Sequences of MxOV genes

CBP, DNA Topo, VIR, and F1L are closely associated with reference orf virus strains from sheep in China (OV-HN3/12 and OV-NA1/11) and regularly appear in clades with OV-NZ2 and OV-

IA82 (Figure 5.1).

Other Protein Findings

Amino acid sequences of MxOV002 (encoding an ankyrin repeat protein), MxOV030

(encoding a telomere-binding protein) and MxOV047 (encoding a putative membrane protein) shared greater identity with goat-associated OV-SA00 (Supplementary Table 5.3).

MxOV037 (encoding a hypothetical protein), MxOV046 (encoding a DNA-binding virion core protein), and MxOV076 and MxOV086 (encoding for immature virion membrane proteins) share the highest amino acid identity with reference strain OV-D1701 which originated from a sheep in Germany (Supplementary Table 5.3).

5.4.5 Whole Genome & Concatenated Protein Tree Comparison

Based on nucleotide percent identity and genomic layout, MxOV has the highest genetic similarity to OV-NZ2 (Figure 5.2; Supplementary Figure 5.1). Phylogenetic analysis using whole- genome amino acid sequences places the closest relative of MxOV as reference strain OV-IA82 identified in a sheep from Iowa, USA (Figure 5.3). Whole-genome phylogenetic trees show greater ultrafast bootstrap support at each node when compared to concatenated protein trees (Figure 5.4).

115

116

Figure 5.1 Amino acid protein trees of key parapoxvirus genes and virulence factors. Ultrafast bootstrap support values are given at the nodes. Trees were rooted on OV-D1701 when PCPV homologue sequences were unavailable. Scale bars indicate amino acid substitutions per site (See previous page).

117

Figure 5.2 Heatmap showing percent identity of MxOV blast hits to related genomes ordered by genomic position. Blast hits with a percent identity > 50% and coverage of > 10% were retained.

118

Figure 5.3 Whole-genome nucleotide comparison of MxOV and parapoxvirus reference sequences. Ultrafast bootstrap support values are given at the nodes.

119

Figure 5.4 Comparison of concatenated gene sequences from MxOV and reference parapoxviruses. Ultrafast bootstrap support values are given at the nodes.

120

5.4.6 Recombination Analysis

Recombination analysis was performed on full nucleotide genome alignments of MxOV and reference sequences. Seven significant recombination events were predicted in MxOV (Table

5.4). These events are graphically represented along the MxOV genome in Figure 5.5.

Two of the three most significant events affect the virulence factors: B2L envelope glycoprotein (MxOV107) recombinant with an unknown minor parent, and the VEGF gene

(MxOV128) recombinant with OV-SA00. The recombination event of the B2L region includes

437 bp of intergenic space between MxOV106 and MxOV107, and 303 bp of the 335 bp annotated

B2L gene (MxOV107). Recombination of the VEGF region of MxOV spans 45 nucleotide bases at the end of MxOV127 (putative membrane protein), an intergenic region of 54 bp, and 431 bp of the 446 bp annotated VEGF gene (MxOV128).

A third major recombination event involves a large 2,958 bp fragment spanning 13 bp at the end of MxOV034 to the first 323 bp of MxOV039, inclusive. OV-D1701 is predicted to be the most likely parent to have contributed this recombinant sequence in MxOV. The gold-standard bioinformatic approach for demonstrating the presence of recombination is a set of statistically incongruent phylogenetic trees (Boni et al., 2010), therefore separate trees were constructed of recombinant and non-recombinant genomic regions (Figure 5.6).

121

Figure 5.5 MxOV genome graphic depicting significant predicted recombination events. Purple indicates the genome of MxOV; grey shading along the genome identifies other regions or recombination excluded by our selection criteria. Names of the most likely donor sequence (minor parent) with approximate recombination region are given in coloured font.

122

Table 5.4 Predicted recombination sites of MxOV with reference orf virus strains. The major parent, or backbone, is the parental strain contributing a larger fraction of sequence. The minor parent is the strain likely involved in the recombination event, contributing a smaller fraction of sequence. Statistical significance at p>0.05.

MxOV Position Detection method (p-value)

Begin End Size Major Minor Protein (s) RDP GENE- Bootscan Maxchi Chimaera SiScan 3seq (bp) Parent(s) Parent (s) CONV

~37735 40693 2958 NA1/11, OV- D1701 MxOV034- 1.57E-08 2.81E-06 2.86E-03 2.46E-04 7.17E-04 1.99E-07 2.78E-04 HN3/12 MxOV039

68210 78990 10780 OV-IA82 Unknown MxOV65- 4.32E-03 ns ns 2.19E-02 3.88E-03 1.86E-02 ns MxOV076

106414 106497 83 NP Unknown intergenic 5.36E-04 2.74E-04 1.79E-04 2.34E-03 1.44E-04 ns ns space - MxOV106

~107526 108266 740 NZ2, Unknown intergenic 2.38E-60 3.50E-52 9.40E-57 3.40E-12 9.50E-09 ns 2.00E-13 NA1/11, OV- space - HN3/12 MxOV107

112052 112259 207 SJ1 PCPV intergenic 1.45E-07 ns 4.14E-06 ns ns 2.81E-03 2.52E-03 space - MxOV112

130346 130908 562 OV-IA82, OV-SA00, MxOV127- 1.00E-131 8.03E-136 1.01E-120 8.25E-21 9.59E-17 5.46E-30 2.69E-13 NZ2 SJ1 128

112360* 112887 527 OV-HN3_12 Unknown MxOV112 3.74E-05 ns 3.03E-02 ns ns 2.98E-03 2.07E-04 * = The actual position is indeterminate (it was most likely obscured by a subsequent recombination event) ~ = It is possible that this event could have been caused by an evolutionary process other than recombination ns = no significant p-value was recorded for this recombination event using this method

123

Non-recombinant region Recombinant region

37735-40693 bp

107526-108266 bp

130346-130908 bp

= major parent

= minor parent

= recombinant

= sequence with evidence of same recombination event

124

Figure 5.6 Phylogenetic trees of the three most significant recombinant regions of MxOV. Branch labels are ML bootstrap support values. Scale bars indicate nucleotide substitutions per site (See previous page).

125

5.5 Discussion

In previous work, orf virus infection was confirmed in a muskox from Victoria Island, NU,

Canada, however, the strain was previously uncharacterized with no closely related reference orf viruses (Tomaselli et al., 2016). Therefore, we sequenced and analyzed the clinical material of orf virus-infected muskoxen from this region to better understand the origin and characteristics of this strain based on the complete genome.

The genome structure of MxOV is similar to known orf virus reference strains and contains

131 predicted genes. The GC content of MxOV is comparable to other orf viruses (large contig), with a drop in GC content at the right terminal end (small contig) as is common in Parapoxviruses

(Mercer et al., 2006).

Since no high-quality reads spanned the gap between the large contig and the right terminal small contig, the MxOV genome is intentionally presented herein as two contigs rather than one full genome. Gaps in genome assembly can arise when dealing with repetitive regions of the genome (Treangen and Salzberg, 2011). These gaps can be closed by developing a PCR targeted to read across repetitive sequences, however, this only works well for small gaps (Ekblom and

Wolf, 2014).

It is probable that the number of viral reads in our clinical samples may be underrepresented using the protein-based taxonomic classifier due to the homology of some viral sequences to host proteins and intergenic regions. Protein-based taxonomic classifiers are more sensitive for classifying viral reads, however, they are generally less accurate in classifying non-coding regions

(Menzel et al., 2016). As such, it is quite likely that a large portion of unclassified reads belong to the host genome and therefore to the kingdom of Eukaryota, whereas some viral reads may also be grouped among the host reads.

126

The same variant in MxOV, 42335T>C, was shared by both muskoxen from Cambridge

Bay on the Nunavut side of Victoria Island. The variant present in both Cambridge Bay muskoxen occurs within the gene sequence of late transcription factor VLTF-1. VLTF-1 is expressed in the intermediate phase of the viral replicative cycle and coordinates with transcription factors to regulate gene expression (Wright et al., 1991). Since the variant does not affect the amino acid sequence of VLTF-1, there is likely no impact on protein function.

Approximately 520 km away from Cambridge Bay, a different variant was found in the muskox from Ulukhaktok on the Northwest Territories side of Victoria Island. The only sequence variant the Ulukhaktok muskox occurs in the intergenic region between an envelope glycoprotein and a hypothetical protein with unknown function. The function of this intergenic region is also not yet known, however, it has been shown that intergenic regions often contain regulatory elements such as promoters or repressors, regulating the expression of the downstream gene (Yang et al., 2004). Mutations in these regulatory elements could play a role in enhanced or repressed virulence of orf virus.

The amino acid sequence of VEGF from MxOV does not share a close phylogenetic relationship with reference orf viruses. VEGF is needed for the characteristic proliferative skin lesions associated with orf virus (Savory et al., 2000). Disruption of the viral VEGF gene and the loss of VEGF activity significantly reduces the severity of orf virus infection in its natural host

(Savory et al., 2000). Fleming et al. (2015) reported that unique VEGF sequences of orf viruses are likely the result of gene capture from specific hosts over the course of evolution. The muskox host is taxonomically grouped among sheep and goats within the subfamily Caprinae, with no extant relatives in the genus Ovibos. The closest related amino acid sequence of MxOV VEGF as per BLAST analysis is that of OV-SA00 from a goat in the USA. Nucleotide recombination

127 analysis performed herein predicted a recombination event of the MxOV VEGF region with OV-

SA00 as the minor parent. Most genes of MxOV analyzed herein share similarity with sheep- associated strains of orf virus.

A large region of approximately 3,000 bp spanning MxOV034-MxOV039 is predicted to have recombined with OV-D1701 as the likely sequence donor. OV-D1701 is known to be licensed for the development of recombinant vaccines for the control of orf virus in infected sheep flocks

(Cottone et al., 1998; Rziha et al., 2019). Although it is unlikely that vaccines used to immunize domestic sheep would encounter muskoxen on the tundra, this raises concerns regarding the likelihood of recombination between a naturally occurring orf strains, or between a vaccine strain and a wild orf virus strain.

Recombination analysis also predicted the recombination of the B2L region of MxOV and

OV-IA82 with an unknown minor parent. It is important to consider sites of recombination when interpreting phylogenetic data since these recombination events led to mismatches between trees constructed of proteins encoded by genome regions of recombinant and non-recombinant origin.

More research is needed to elucidate the frequency of recombination in orf viruses of wildlife

This study shines a light on the variability of MxOV in a small sample of muskoxen from the Canadian Arctic. With continued surveillance and further genomic studies of MxOV in muskoxen and cohabiting animals, we may improve our understanding of viral host range and endemic or introduced strains of orf virus in the Arctic.

5.6 Acknowledgements

Thank you to the hunters and the Hunters and Trappers Organizations/Association of

Cambridge Bay, Kugluktuk, and Ulukhaktok, as well as Matilde Tomaselli, Fabien Mavrot, and

128 the Kutz Research Group at the University of Calgary for providing tissue samples and valuable input in this study. Thank you to James Wasmuth for his insights and guidance during data analysis. Thank you to Richard Pon and Paul Gordon of the Centre for Health Genomics and

Informatics, and the University of Calgary DNA Services for facilitating all sequencing in this study.

5.7 Declaration of Conflicts of Interest

None to declare.

5.8 Funding

This work was supported by the following grants: ArcticNet Inc. Networks of Centres of

Excellence of Canada: Managing Muskox Health for Food Security and Ecosystem and Socio-

Economic Resilience [Grant number 10010793], and the University of Calgary Faculty of

Veterinary Medicine [Grant number 10006449].

5.9 Supplementary Data

See next page

129

Supplementary Table 5.1 Genomes included in phylogenetic and BLASTp analysis.

Species Strain Animal Country of Year Accession Reference Origin Origin

Orf MxOV Muskox Canada 2019 Awaiting This study virus accession number

SA00 Goat USA 2000 AY386264 Delhon et al., 2004

HN3/12 Sheep China 2012 KY053526 Chen et al., 2017

NA1/11 Sheep China 2011 KF234407 Li et al., 2015

YX Goat China 2012 KP010353 Chi et al., 2015

GO Goat China 2012 KP010354 Chi et al., 2015

NP Goat China 2011 KP010355 Chi et al., 2015

NZ2 Sheep New Zealand 1981 DQ184476 Mercer et al., 2006

SJ1 Goat China 2012 KP010356 Chi et al., 2015

IA82 Sheep USA 1982 AY386263 Delhon et al., 2004

D1701 Sheep Germany 1981 HM133903 Mayr et al., 1981

PCPa VR634 - - Ref NC_013804 Hautaniemi et al., 2010

BPSVb - Calf USA Ref NC_005337 Mercer et al., 2006

aPCPV – Pseudocowpox virus, bBSPV – Bovine papular stomatitis virus

130

Supplementary Table 5.2 Predicted genes of MxOV

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

001 dUTPase 780 1259 480 -

002 ankyrin repeat protein 1322 2872 1551 -

003 hypothetical protein 3037 4293 1257 -

004 putative EEV maturation protein 4423 6354 1932 -

005 putative EEV envelope phospholipase 6424 7560 1137 -

006 hypothetical protein 7589 7777 189 -

007 hypothetical protein 7936 8118 183 -

008 hypothetical protein 8166 8405 240 +

009 modified RING finger protein 8441 8722 282 -

010 hypothetical protein 8725 10344 1620 -

011 hypothetical protein 10414 11172 759 -

012 DNA-binding phosphoprotein 11453 11770 318 +

013 poly-A polymerase catalytic subunit PAPL 11793 13211 1419 -

014 hypothetical protein 13219 15396 2178 -

015 dsRNA-binding PKR inhibitor 15528 16079 552 -

016 RNA polymerase subunit RPO30 16108 16689 582 -

017 hypothetical protein 16776 18479 1704 +

018 putative membrane protein 18500 19318 819 +

019 hypothetical protein 19378 20244 867 +

020 DNA polymerase 20245 23283 3039 -

021 putative IMV redox protein 23301 23591 291 +

022 virion core protein 23588 24001 414 -

023 hypothetical protein 23988 26144 2157 -

024 hypothetical protein 26187 28607 2421 -

131

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

025 hypothetical protein 28625 28780 156 -

026 DNA-binding virion protein 28805 29770 966 -

027 hypothetical protein 29780 29992 213 -

028 DNA-binding phosphoprotein 29999 30856 858 -

029 putative IMV membrane protein 30888 31124 237 -

030 hypothetical protein 31125 32294 1170 -

031 virion core protease 32291 33583 1293 -

032 RNA helicase NPH-II 33589 35640 2052 +

033 putative metalloprotease 35615 37381 1767 -

034 hypothetical protein 37437 37748 312 -

035 late transcription elongation factor 37763 38458 696 +

036 putative glutaredoxin 2 38383 38706 324 -

037 hypothetical protein 38799 40157 1359 +

038 RNA polymerase subunit RPO7 40159 40350 192 +

039 hypothetical protein 40370 40927 558 +

040 virion core protein 40924 42120 1197 -

041 late transcription factor VLTF-1 42153 42953 801 +

042 putative myristylated protein 42944 43966 1023 +

043 putative myristylated IMV envelope protein 43967 44701 735 +

044 hypothetical protein 44747 45019 273 +

045 hypothetical protein 45026 46282 1257 -

046 DNA-binding virion core protein VP8 46312 47091 780 +

047 putative membrane protein 47114 47500 387 +

048 putative IMV membrane protein 47454 47909 456 +

049 poly-A polymerase small subunit VP39 PAPS 47994 48989 996 +

132

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

050 RNA polymerase subunit RPO22 49003 49464 462 +

051 late membrane protein 49415 49918 504 -

052 RNA polymerase subunit RPO147 50002 53871 3870 +

053 putative protein-tyrosine phosphatase 53920 54465 546 -

054 hypothetical protein 54485 55060 576 +

055 putative IMV protein VP55 55065 56057 993 -

056 RNA polymerase-associated protein RAP94 56058 58472 2415 -

057 late transcription factor VLTF-4 58581 59258 678 +

058 DNA topoisomerase type I 59283 60239 957 +

059 hypothetical protein 60232 60648 417 +

060 mRNA capping enzyme large subunit 60683 63208 2526 +

061 virion protein 63170 63625 456 -

062 virion protein 63663 64289 627 +

063 uracil DNA glycosidase 64289 64984 696 +

064 NTPase 64998 67361 2364 +

065 early transcription factor VETFs 67368 69275 1908 +

066 RNA polymerase subunit RPO18 69312 69884 573 +

067 NPH-PPH downregulator 69914 70588 675 +

068 transcription termination factor NPH-I 70578 72494 1917 -

069 hypothetical protein 72558 73124 567 -

070 mRNA capping enzyme small subunit 73171 74040 870 -

071 putative rifampicin resistance protein 74081 75718 1638 -

072 late transcription factor VLTF-2 75739 76191 453 -

073 late transcription factor VLTF-3 76230 76904 675 -

074 thioredoxin-like protein 76901 77149 249 -

133

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

075 virion core protein P4b precursor 77160 78785 1626 -

076 hypothetical protein 78823 79128 306 +

077 virion core protein 79203 79448 246 -

078 hypothetical protein 79549 79848 300 -

079 RNA polymerase subunit RPO19 80264 80782 519 +

080 hypothetical protein 80789 81925 1137 -

081 early transcription factor VETFL 81961 84081 2121 -

082 intermediate transcription factor VITF-3 84149 85060 912 +

083 late virion membrane protein 85029 85310 282 -

084 virion core protein P4a precursor 85325 88042 2718 -

085 hypothetical protein 88057 89067 1011 +

086 hypothetical protein 89102 89797 696 +

087 virion membrane protein 89861 90139 279 -

088 putative IMV phosphorylated membrane 90163 90438 276 - protein

089 putative IMV membrane protein 90455 90616 162 -

090 hypothetical protein 90617 90886 270 -

091 predicted myristylated protein 90873 91949 1077 -

092 putative phosphorylated IMV membrane 91976 92566 591 - protein

093 DNA helicase 92599 94047 1449 +

094 hypothetical protein 94019 94294 276 -

095 hypothetical protein 94305 94631 327 -

096 DNA polymerase processivity factor 94657 95919 1263 +

097 Holliday junction resolvase 95916 96356 441 +

098 intermediate transcription factor VITF-3 96379 97521 1143 +

134

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

099 RNA polymerase subunit RPO132 97546 101031 3486 +

100 A type inclusion protein 101171 101584 414 -

101 A type inclusion protein 102765 104333 1569 -

102 hypothetical protein 104686 105108 423 -

103 RNA polymerase subunit RPO35 105124 106068 945 -

104 virion morphogenesis 106068 106250 183 -

105 hypothetical protein 106251 106400 150 -

106 DNA packaging protein/ATPase 106419 107219 801 -

107 EEV glycoprotein 107963 108298 336 +

108 hypothetical protein 108448 108870 423 +

109 putative chemokine-binding protein 108975 109844 870 +

110 hypothetical protein 109917 110552 636 +

111 hypothetical protein 110596 111636 1041 +

112 hypothetical protein 112254 112994 741 +

113 GM-CSF/IL-2 inhibition factor-like protein 113181 113966 786 +

114 hypothetical protein 114220 114528 309 +

115 hypothetical protein 115006 115572 567 +

116 hypothetical protein 116431 116637 207 +

117 hypothetical protein 116869 117699 831 +

118 hypothetical protein 117753 118724 972 +

119 ankyrin repeat protein 118815 120392 1578 +

120 hypothetical protein 120427 122025 1599 +

121 hypothetical protein 122118 122639 522 +

122 ankyrin repeat protein 122745 124238 1494 +

123 IL-10-like protein 124317 124877 561 +

135

MxOV Predicted Product Genomic Position ORFa Start End Width Strand

124 ankyrin repeat protein 125043 126548 1506 +

125 ankyrin repeat protein 126624 128171 1548 +

126 putative serine/threonine protein kinase 128258 129751 1494 +

127 putative membrane protein 129714 130391 678 +

128 vascular endothelial growth factor-like protein 130477 130923 447 +

129 hypothetical protein 252 509 258 +

130 hypothetical protein 2824 3273 450 -

131 hypothetical protein 3436 3543 108 - a ORF – Open reading frame

136

Supplementary Table 5.3 Blast hits to reference sequences of MxOV ORFs identified using Prokka 1.13.3. The highest scoring blast hits are shown in bold.

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

001 ORF007 - PP136 dUTPase dUTPase dUTPase dUTPase dUTPase ORF007 ORF007 dUTPase dUTPase dUTPase (85.2 %) (94.4 %) (95.6 %) (93.1 %) (100 %) (96.2 %) dUTPase dUTPase (93.1 %) (94.4 %) (68.7 %) (98.1 %) (91.9 %)

002 ankyrin- - PP134 Ankyrin/F- ankyrin/F- Ankyrin/F- Ankyrin/F- ankyrin/F- ORF008 ORF008 Ankyrin/ Ankyrin/F- like protein (97.9 %) box protein box box protein box protein box protein ankyrin ankyrin F-box box protein (59.3 %) (90.7 %) protein (90.5 %) (98.5 %) (98.6 %) repeat repeat protein (90.3 %) (98.6 %) protein protein (90.3 %) (98.3 %) (90.5 %)

003 hypothetica hypotheti PP133 hypothetical hypothetica hypothetical hypothetical hypothetica ORF009 ORF009 hypotheti hypothetica l protein cal (99.1 %) protein (96.2 l protein (99 protein (96.2 protein (99.3 l protein (99 hypothetic hypothetica cal l protein (56.3 %) protein %) %) %) %) %) al protein l protein protein (95.7 %) (88.6 %) (99.5 %) (96.2 %) (95.9 %)

004 EEV EEV PP130 (98 EEV EEV EEV EEV- EEV ORF010 ORF010 EEV EEV maturation maturatio %) maturation maturation maturation maturation maturation putative putative maturatio maturation protein n protein protein (97.8 protein (98 protein (97.8 protein (99.1 protein (98 EEV EEV n protein protein (73.9 %) (91.2 %) %) %) %) %) %) maturation maturation (97.8 %) (97.8 %) protein protein (99.1 %) (98.1 %)

005 palmytilate EEV B2WL EEV EEV EEV EEV EEV ORF011 ORF011 EEV EEV d EEV envelope (97.6 %) envelope envelope envelope phospholipase envelope putative putative envelope envelope membrane phospholi phospholipase phospholip phospholipase (98.9 %) phospholip EEV EEV phospholi phospholip glycoprotei pase (98.4 %) ase (97.9 (97.9 %) ase (97.9 envelope envelope pase ase (97.9 n (84.1 %) (95.2 %) %) %) phospholip phospholip (98.4 %) %) ase (99.5 ase (97.9 %) %)

006 hypothetica hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica ORF012 ORF012 hypotheti hypothetica l protein cal protein (87.1 l protein protein (88.7 protein (93.5 l protein hypothetic hypothetica cal l protein (56.9 %) protein %) (91.9 %) %) %) (91.9 %) al protein l protein protein (82.3 %) (73.1 %) (93.5 %) (85.5 %) (87.1 %)

007 hypothetica hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica - - hypotheti hypothetica l protein (80 cal protein (96.7 l protein protein (98.3 protein (100 l protein cal l protein %) %) (98.3 %) %) %) (98.3 %) (96.7 %)

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 protein protein (93.3 %) (98.3 %)

008 hypothetica hypotheti PP143 hypothetical hypothetica hypothetical hypothetical hypothetica ORF013 ORF013 hypotheti hypothetica l protein cal (94.9 %) protein (89.9 l protein protein (93.7 protein (100 l protein hypothetica hypothetica cal l protein (50.8 %) protein %) (98.7 %) %) %) (98.7 %) l protein l protein protein (92.4 %) (82.3 %) (97.5 %) (96.2 %) (91.1 %)

009 ORF014 RING- PP127 RING-H2 RING-H2 RING-H2 RING-H2 RING-H2 ORF014 ORF014 RING- RING-H2 modified H2 motif (97.8 %) motif protein motif motif protein motif protein motif modified modified H2 motif motif RING protein (95.6 %) protein (94.6 %) (100 %) protein RING RING protein protein finger (83.9 %) (96.8 %) (96.8 %) finger finger (94.5 %) (95.6 %) protein protein protein (67.7 %) (98.9 %) (92.5 %)

010 ORF015 hypotheti PP125 (97 hypothetical hypothetica hypothetical hypothetical hypothetica ORF015 ORF015 hypotheti hypothetica hypothetica cal %) protein (95.4 l protein protein (95.9 protein (98.7 l protein hypothetic hypothetica cal l protein l protein (61 protein %) (98.5 %) %) %) (98.7 %) al protein l protein protein (95.2 %) %) (81.8 %) (99.4 %) (96.7 %) (96.1 %)

011 hypothetica hypotheti PP124 hypothetical hypothetic hypothetical hypothetical hypothetica ORF016 ORF016 hypotheti hypothetica l protein cal (93.3 %) protein (91.7 al protein protein (92.9 protein (91.7 l protein hypothetica hypothetica cal l protein (55.2 %) protein %) (97.2 %) %) %) (96.4 %) l protein l protein protein (92.1 %) (70.9 %) (92.1 %) (91.7 %) (93.3 %)

012 ORF017 DNA- DNA- DNA- DNA- DNA binding DNA- ORF017 ORF017 DNA- DNA- DNA- binding binding binding binding phosophopro binding DNA- DNA- binding binding binding phosphop phosophopro phosophopr phosophopro tein (95.2 %) phosophopr binding binding phosoph phosophop phosphopro rotein tein (95.2 %) otein (91.4 tein (95.2 %) otein (91.4 phosphopr phosphopro oprotein rotein (95.2 tein (68 %) (76.5 %) %) %) otein (95.2 tein (94.3 (95.2 %) %) %) %)

013 poly(A) poly(A)- PP123 (97 Poly(A)- poly- Poly(A)- poly-A poly(A)- ORF018 ORF018 Poly(A)- Poly(A)- polymerase polymera %) polymerase polymerase polymerase polymerase polymerase poly-A poly-A polymera polymerase large se subunit catalytic catalytic catalytic catalytic catalytic polymeras polymerase se catalytic subunit (94.3 %) subunit PAPL subunit subunit PAPL subunit (98.9 subunit e catalytic catalytic catalytic subunit (81.1 %) (97.9 %) PAPL (98.1 (97.7 %) %) PAPL (98.1 subunit subunit subunit PAPL (98.1 %) %) PAPL PAPL (98.1 PAPL %) (99.4 %) %) (97.7 %)

014 hypothetica hypotheti PP121 hypothetical hypothetica hypothetical hypothetical hypothetica ORF019 ORF019 hypotheti hypothetica l protein cal (98.5 %) protein (97.4 l protein protein (97.8 protein (99.2 l protein hypothetic hypothetica cal l protein (73.2 %) protein %) (98.2 %) %) %) (98.2 %) al protein l protein protein (97.1 %) (92.1 %) (99.2 %) (97.5 %) (97.7 %)

138

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

015 ORF020 dsRNA- PP120 DsRNA- dsRNA- DsRNA- DsRNA- dsRNA- ORF020 ORF020 DsRNA- DsRNA- dsRNA- binding (94.5 %) binding, binding, binding, binding, binding dsRNA- dsRNA- binding, binding, binding interferon interferon interferon interferon interferon protein binding binding interferon interferon PKR resistance resistance resistance resistance resistance (94.5 %) PKR PKR resistance resistance inhibitor protein (96.7 %) (94.5 %) (94.5 %) (98.9 %) inhibitor inhibitor (96.2 %) (93.4 %) (54.8 %) (74.3 %) (96.7 %) (95.1 %)

016 RNA RNA- PP119 RNA- RNA- RNA- RNA RNA- ORF021 ORF021 RNA- RNA- polymerase polymera (98.4 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase subunit se subunit subunit subunit subunit subunit subunit polymerase polymerase se subunit subunit (80.3 %) RPO30 RPO30 (96.4 RPO30 RPO30 (96.9 RPO30 (100 RPO30 subunit subunit RPO30 RPO30 (91.7 %) %) (98.4 %) %) %) (98.4 %) RPO30 (99 RPO30 (97.4 %) (96.9 %) %) (97.4 %)

017 Hypothetic hypotheti PP152 Pox virus E6 poxvirus E6 Pox virus E6 hypothetical E6 protein ORF022 ORF022 Pox virus Pox virus al protein cal (98.6 %) proteins (98.9 proteins, proteins (99.1 protein (99.5 (98.9 %) hypothetic hypothetica E6 E6 proteins (84.3 %) protein %) virion %) %) al protein l protein proteins (98.9 %) (95.6 %) morphogen (99.6 %) (98.8 %) (98.6 %) esis (99.3 %)

018 hypothetica membran PP153 Membrane membrane Membrane membrane membrane ORF023 ORF023 Membra Membrane l protein e protein (98.9 %) protein (100 protein protein (100 protein (100 protein putative putative ne protein (88.6 %) (96.7 %) %) (100 %) %) %) (100 %) membrane membrane protein (100 %) protein protein (100 %) (100 %) (100 %)

019 ORF024 hypotheti PP154 NF-kappa NF-kappaB NF-kappa hypothetical NF-kappaB ORF024 ORF024 NF- NF-kappa hypothetica cal (91.7 %) pathway pathway pathway protein (97.6 pathway hypothetic hypothetica kappa pathway l protein protein inhibitor (91.8 inhibitor inhibitor (91.8 %) inhibitor al protein l protein pathway inhibitor (65.1 %) (73.3 %) %) (96.5 %) %) (96.5 %) (99.3 %) (91.4 %) inhibitor (92.1 %) (90.1 %)

020 DNA topo DNA- PP114 DNA- DNA- DNA- DNA DNA- ORF025 ORF025 DNA- DNA- polymera (97.8 %) polymerase polymerase polymerase polymerase polymerase DNA DNA polymera polymerase ase (86.9 se (94.1 (99.2 %) (99.5 %) (99.2 %) (99.7 %) (99.6 %) polymeras polymerase se (99.3 (99.3 %) %) %) e (99.8 %) (99 %) %)

021 sulfhydryl ERV/AL PP158 (99 ERV/ALR- ERV/ALR- ERV/ALR- ERV/ALR- ERV/ALR- ORF026 ORF026 ERV/AL ERV/ALR- oxidase R-like %) like like like like protein like protein putative putative R-like like (89.6 %) protein protein(IMV protein protein(IMV (100 %) (100 %) IMV redox IMV redox protein(I protein(IM (99 %) redox (100 %) redox protein protein MV V redox protein) (100 protein) (100 (100 %) (100 %) redox protein) %) %) (100 %)

139

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 protein) (100 %)

022 putative virion PP113 Virion core virion core Virion core virion core virion core ORF027 ORF027 Virion Virion core virion core core (97.8 %) protein (100 protein protein (97.8 protein (99.3 protein virion core virion core core protein protein protein %) (100 %) %) %) (100 %) protein protein protein (97.8 %) (83.2 %) (95.6 %) (98.5 %) (98.5 %) (98.5 %)

023 hypothetica hypotheti PP111 DNA-binding DNA- DNA-binding hypothetical DNA- ORF028 ORF028 DNA- DNA- l protein cal (97.2 %) protein (97.1 binding protein (96.8 protein (99 %) binding hypothetic hypothetica binding binding (65.6 %) protein %) protein %) protein al protein l protein protein protein (85.6 %) (96.9 %) (96.9 %) (99.2 %) (95.3 %) (96.8 %) (96.7 %)

024 ORF029 hypotheti PP109 (97 hypothetical hypothetica hypothetical hypothetical hypothetica ORF029 ORF029 hypotheti hypothetica hypothetica cal %) protein (95.7 l protein protein (95.4 protein (99.6 l protein hypothetic hypothetica cal l protein l protein protein %) (97.4 %) %) %) (97.4 %) al protein l protein protein (95.7 %) (65.9 %) (83.6 %) (99.6 %) (95.4 %) (95.5 %)

026 putative DNA- PP108 DNA-binding DNA- DNA-binding virion core DNA- ORF030 ORF030 DNA- DNA- DNA- binding (98.6 %) virion protein binding virion protein protein (99.7 binding DNA- DNA- binding binding binding virion (99.4 %) virion (99.4 %) %) virion binding binding virion virion virion core core protein protein virion virion protein protein protein protein (99.4 %) (99.4 %) protein protein (99.4 %) (98.4 %) (78.9 %) (93.5 %) (99.7 %) (98.8 %)

027 hypothetica hypotheti B24R (90 hypothetical hypothetic hypothetical hypothetical hypothetic ORF031 ORF031 hypothet hypothetic l protein cal %) protein (100 al protein protein (100 protein (100 al protein hypothetic hypothetic ical al protein (72.9 %) protein %) (100 %) %) %) (100 %) al protein al protein protein (100 %) (97.1 %) (100 %) (100 %) (100 %)

028 DNA- DNA- PP107 DNA-binding DNA- DNA-binding DNA binding DNA- ORF032 ORF032 DNA- DNA- binding binding (93.3 %) phosphoprotei binding phosphoprotei phosphoprot binding DNA- DNA- binding binding phosphopro phosphop n (92 %) phosphopro n (92.6 %) ein (99.3 %) phosphopro binding binding phosphop phosphopro tein (66.4 rotein tein (96.9 tein (96.9 phosphopro phosphopro rotein tein (93 %) %) (87.1 %) %) %) tein (97.9 tein (94.7 (93.7 %) %) %)

029 ORF033 IMV - IMV IMV IMV IMV protein IMV ORF033 ORF033 IMV IMV IMV protein membrane membrane membrane (100 %) membrane putative putative membran membrane membrane (93.6 %) protein (96.2 protein protein (96.2 protein IMV IMV e protein protein protein %) (98.7 %) %) (98.7 %) membrane membrane (96.2 %) (96.2 %) (77.8 %) protein protein (100 %) (94.9 %)

140

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

030 hypothetica telomere- PP105 Telomere- telomere- Telomere- telomere telomere- ORF034 ORF034 Telomer Telomere- l protein binding (98.7 %) binding binding binding binding binding hypothetica hypothetic e- binding (80.5 %) protein protein (99.2 protein (99 protein (99.2 protein (99.5 protein l protein al protein binding protein (94.6 %) %) %) %) %) (98.5 %) (99.2 %) (99.5 %) protein (99.2 %) (99.5 %)

031 ORF035 virion PP104 Virion core virion core Virion core virion core virion core ORF035 ORF035 Virion Virion core virion core core (97.9 %) protease (99.5 protease protease (99.3 protease (100 protease virion core virion core core protease protease protease %) (99.8 %) %) %) (99.8 %) protease protease protease (99.3 %) (84.2 %) (96.7 %) (99.8 %) (99.1 %) (99.5 %)

032 RNA RNA PP171 RNA helicase RNA RNA helicase RNA helicase RNA ORF036 ORF036 RNA RNA helicase helicase (99.1 %) NPH-II (99.6 helicase NPH-II (99.4 (99.9 %) helicase RNA RNA helicase helicase NPH-II NPH-II %) NPH-II %) NPH-II helicase helicase NPH-II NPH-II (78.8 %) (93.4 %) (99.4 %) (99.4 %) NPH-II NPH-II (99.6 %) (99.6 %) (99.4 %) (99.4 %)

033 putative Zn- PP102 Zn-protease, Zn- Zn-protease, Zn-protease, Zn-protease ORF037 ORF037 Zn- Zn- metalloprot protease (98.8 %) virion protease, virion virion (99.2 %) putative putative protease, protease, ease (75.8 (93.2 %) morphogenesi virion morphogenesi morphogenes metalloprot metalloprot virion virion %) s (99 %) morphogen s (99 %) is (99.7 %) ease (99.2 ease (99.2 morphog morphogen esis (99.2 %) %) enesis (99 esis (98.8 %) %) %)

034 hypothetica hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica ORF039 ORF039 hypotheti hypothetica l protein cal protein (93.3 l protein protein (92.2 protein (98.1 l protein hypothetic hypothetica cal l protein (71.6 %) protein %) (94.2 %) %) %) (94.2 %) al protein l protein protein (93.3 %) (91.4 %) (98.1 %) (91.1 %) (91.1 %)

035 transcriptio late PP176 Late late Late late late ORF038 ORF038 Late Late nal transcript (99.3 %) transcription transcriptio transcription transcription transcriptio late late transcript transcripti elongation ion elongation n elongation factor n transcriptio transcriptio ion on factor (74.2 elongatio factor (100 elongation factor (100 elongation elongation n n elongatio elongation %) n factor %) factor (98.7 %) factor (100 factor (98.7 elongation elongation n factor factor (100 (94 %) %) %) %) factor (99.6 factor (99.1 (99.1 %) %) %) %)

036 glutaredoxi glutaredo PP100 Glutaredoxin- glutaredoxi Glutaredoxin- glutaredoxin glutaredoxi ORF040 ORF040 Glutared Glutaredoxi n-like xin-like (99.1 %) like protein n-like like protein -like protein n-like putative putative oxin-like n-like protein protein (98.1 %) protein (98.1 %) (100 %) protein glutaredox glutaredoxi protein protein (81.9 %) (93.3 %) (97.2 %) (97.2 %) in 2 (100 n 2 (98.1 %) (95.3 %) (99.1 %) %)

141

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

037 Hypothetic hypotheti PP178 (100 hypothetical hypothetica hypothetical hypothetical hypothetica ORF041 ORF041 hypotheti hypothetica al protein cal %) protein (97.3 l protein protein (96.5 protein (99.8 l protein hypothetica hypothetica cal l protein (66.7 %) protein %) (97.6 %) %) %) (97.6 %) l protein l protein protein (97.1 %) (88.6 %) (99.3 %) (96.7 %) (97.3 %)

038 RNA RNA- - RNA- RNA- RNA- RNA RNA- ORF042 ORF042 RNA- RNA- polymerase polymera polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase (84.1 %) se subunit subunit RPO7 subunit subunit RPO7 subunit subunit polymeras polymeras se subunit subunit RPO7 (96.8 %) RPO7 (98.4 (96.8 %) RPO7 (100 RPO7 (98.4 e subunit e subunit RPO7 RPO7 (96.8 (93.7 %) %) %) %) RPO7 (100 RPO7 (100 (98.4 %) %) %) %)

039 ORF043 hypotheti PP181 hypothetical hypothetica hypothetical hypothetical hypothetica ORF043 ORF043 hypotheti hypothetica hypothetica cal (97.8 %) protein (97.3 l protein protein (97.8 protein (100 l protein hypothetic hypothetica cal l protein l protein protein %) (99.5 %) %) %) (99.5 %) al protein l protein protein (97.8 %) (70.1 %) (90.3 %) (100 %) (97.8 %) (97.8 %)

040 putative virion PP95 (96 Virion core virion core Virion core virion core virion core ORF044 ORF044 Virion Virion core virion core core %) protein (99 %) protein protein (98.7 protein (99 %) protein virion core virion core core protein (98 protein protein (98.7 %) %) (98.7 %) protein protein protein %) (66.2 %) (86.1 %) (99.7 %) (98.5 %) (98.7 %)

041 late late PP185 (100 Late late Late late late ORF045 ORF045 Late Late transcriptio transcript %) transcription transcriptio transcription transcription transcripti late late transcrip transcripti n factor ion factor factor n factor factor factor (100 on factor transcripti transcripti tion on factor VLTF-1 (96.6 %) VLTF-1 (100 VLTF-1 VLTF-1 (100 %) VLTF-1 on factor on factor factor VLTF-1 (94 %) %) (99.6 %) %) (100 %) VLTF-1 VLTF-1 VLTF-1 (100 %) (100 %) (100 %) (100 %)

042 poxvirus myristyla PP187 Myristylated myristylate Myristylated myristylprot myristylate ORF046 ORF046 Myristyla Myristylate myristoylpr ted (97.3 %) protein (97.9 d protein protein (98.2 ein (99.7 %) d protein putative putative ted d protein otein (78.1 protein %) (96.4 %) %) (96.4 %) myristylate myristylate protein (98.2 %) %) (86.8 %) d protein d protein (94.6 %) (99.7 %) (94 %)

043 ORF047 IMV PP188 Myristylated myristylate Myristylated IMV protein myristylate ORF047 ORF047 Myristyla Myristylate myristylate protein (98.8 %) IMV d IMV IMV (99.2 %) d IMV putative putative ted IMV d IMV d IMV (92.2 %) envelope envelope envelope envelope myristylate myristylate envelope envelope envelope protein (98 %) protein protein (98 %) protein d IMV d IMV protein protein (98 protein (99.6 %) (99.2 %) envelope envelope (98 %) %) (87.3 %) protein protein (100 %) (98.8 %)

142

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

044 hypothetica hypotheti PP190 (100 hypothetical hypothetic hypothetical hypothetical hypothetica ORF048 ORF048 hypothet hypothetic l protein (64 cal %) protein (100 al protein protein (100 protein (100 l protein hypothetic hypothetica ical al protein %) protein %) (100 %) %) %) (98.9 %) al protein l protein protein (100 %) (92.2 %) (100 %) (98.9 %) (100 %)

045 ORF049 hypotheti PP91 (95.5 hypothetical hypothetica hypothetical hypothetical hypothetica ORF049 ORF049 hypotheti hypothetica hypothetica cal %) protein (96.9 l protein protein (96.7 protein (98.8 l protein hypothetic hypothetica cal l protein l protein protein %) (97.9 %) %) %) (98.3 %) al protein l protein protein (96.2 %) (63.7 %) (81 %) (99 %) (97.4 %) (96.9 %)

046 DNA- virion PP192 DNA-binding DNA- DNA-binding virion core DNA- ORF050 ORF050 DNA- DNA- binding core (99.6 %) virion core binding virion core protein (98.5 binding DNA- DNA- binding binding virion core protein protein VP8 virion core protein VP8 %) virion core binding binding virion virion core protein (93 %) (97.7 %) protein VP8 (97.7 %) protein VP8 virion core virion core core protein VP8 (83.9 %) (99.2 %) (99.2 %) protein VP8 protein VP8 protein (97.3 %) (98.8 %) (97.7 %) VP8 (96.9 %)

047 ORF051 membran - Membrane membrane Membrane membrane membrane ORF051 ORF051 Membran Membrane putative e protein protein (97.7 protein protein (96.1 protein (97.7 protein putative putative e protein protein membrane (90.6 %) %) (97.7 %) %) %) (97.7 %) membrane membrane (97.7 %) (97.7 %) protein (75 protein protein %) (99.2 %) (100 %)

048 ORF052 virion PP194 IMV IMV IMV virion protein IMV ORF052 ORF052 IMV IMV membrane protein (99.3 %) membrane membrane membrane (99.3 %) membrane putative putative membran membrane protein (86.8 %) protein (98.7 protein protein (98.7 protein IMV IMV e protein protein (76.2 %) %) (100 %) %) (100 %) membrane membrane (97.4 %) (98.7 %) protein protein (99.3 %) (98.7 %)

049 poly(A) poly(A)- PP196 Poly(A)- poly- Poly(A)- poly(A) poly(A)- ORF053 ORF053 Poly(A)- Poly(A)- polymerase polymera (99.7 %) polymerase polymerase polymerase polymerase polymerase poly-A poly-A polymera polymerase small se subunit small subunit small small subunit subunit (100 small polymerase polymerase se small small subunit (97.3 %) VP39 PAPS subunit VP39 PAPS %) subunit small small subunit subunit (82.5 %) (99.4 %) VP39 (99.4 %) VP39 (99.4 subunit subunit VP39 VP39 PAPS (99.4 %) VP39 VP39 PAPS PAPS (99.1 %) PAPS (99.7 PAPS (99.1 (99.4 %) %) %) %)

050 ORF054 RNA- PP197 RNA- RNA- RNA- RNA RNA- ORF054 ORF054 RNA- RNA- RNA polymera (98.7 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase polymerase se subunit subunit subunit subunit subunit subunit polymerase polymerase se subunit subunit subunit subunit subunit

143

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 RPO22 RPO22 RPO22 (97.4 RPO22 RPO22 (98 RPO22 (100 RPO22 RPO22 RPO22 RPO22 RPO22 (98 (86.2 %) (95.4 %) %) (99.3 %) %) %) (99.3 %) (99.3 %) (98.7 %) (98 %) %)

051 ORF055 late PP89 (100 Late late Late late late ORF055 ORF055 Late Late late membran %) membrane membrane membrane membrane membrane late late membra membrane membrane e protein protein (99.4 protein protein (99.4 protein (99.4 protein membrane membrane ne protein protein (95.2 %) %) (99.4 %) %) %) (100 %) protein protein protein (99.4 %) (83.7 %) (100 %) (99.4 %) (100 %)

052 ORF056 RNA- PP198 RNA- RNA- RNA- RNA RNA- ORF056 ORF056 RNA- RNA- RNA polymera (99.6 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase polymerase se subunit subunit subunit subunit subunit subunit polymeras polymerase se subunit subunit subunit RPO147 RPO147 (99.4 RPO147 RPO147 (99.4 RPO147 RPO147 e subunit subunit RPO147 RPO147 RPO147 (96.4 %) %) (99.5 %) %) (99.8 %) (99.5 %) RPO147 RPO147 (99.3 %) (99.5 %) (90.3 %) (99.8 %) (99.4 %)

053 ORF057 tyrosine - Tyrosine tyrosine Tyrosine tyrosine tyrosine ORF057 ORF057 Tyrosine Tyrosine protein phosphat phosphatase, phosphatas phosphatase, phosphatase, phosphatas putative putative phosphat phosphatas phosphatas ase (87.8 virus e, virus virus virus e (99.4 %) protein- protein- ase, virus e, virus e (71.6 %) %) assembly assembly assembly assembly tyrosine tyrosine assembly assembly (97.8 %) (99.4 %) (97.8 %) (100 %) phosphatas phosphatas (96.7 %) (97.2 %) e (100 %) e (97.2 %)

054 putative IMV viral PP201 IMV, viral IMV, viral IMV, viral IMV, viral IMV, viral ORF058 ORF058 IMV, IMV, viral viral entry (99.5 %) entry (100 entry (100 entry (100 entry (100 entry (100 hypothetic hypothetic viral entry (99.5 membrane protein %) %) %) %) %) al protein al protein entry %) protein (95.3 %) (100 %) (100 %) (99.5 %) (86.3 %)

055 ORF059 immunod PP83 (94.1 Immunodomi immunodo Immunodomi immunodomi immunodo ORF059 ORF059 Immunod Immunodo IMV ominant %) nant envelope minant nant envelope nant minant putative putative ominant minant protein envelope protein (96.1 envelope protein (94.4 envelope envelope IMV IMV envelope envelope VP55 (62.4 protein %) protein %) protein (98.2 protein protein protein protein protein (95 %) (81.3 %) (97.3 %) %) (97.3 %) VP55 (97 VP55 (95 (94.4 %) %) %) %)

056 ORF060 RNA- PP81 (98.6 RNA- RNA- RNA- RNA- RNA- ORF060 ORF060 RNA- RNA- RNA polymera %) polymerase polymeras polymerase polymerase polymeras RNA RNA polymera polymerase polymerase se associated e associated associated e polymeras polymerase se associated -associated associate protein associated protein RAP94 (99.8 associated e- -associated associate protein protein d RAP94 RAP94 (99 protein RAP94 (98.9 %) protein associated protein d protein RAP94 (99 RAP94 (96.4 %) %) RAP94 %) RAP94 protein RAP94 RAP94 %) (86.8 %) (99.8 %) (99.8 %) (99.1 %) (99.1 %)

144

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 RAP94 (99.8 %)

057 - late PP204 Late late Late late late ORF061 ORF061 Late Late transcript (94.7 %) transcription transcripti transcription transcription transcripti late late transcript transcriptio ion factor factor VLTF4 on factor factor VLTF4 factor on factor transcriptio transcriptio ion factor n factor VLTF4 (89 %) VLTF4 (89.4 %) VLTF4 (97.8 VLTF4 n factor n factor VLTF4 VLTF4 (70 %) (97.8 %) %) (97.8 %) VLTF-4 VLTF-4 (88.5 %) (89.9 %) (96.5 %) (92.1 %)

058 DNA topoisom PP205 DNA DNA DNA topoisomeras DNA ORF062 ORF062 DNA DNA topoisomer erase I (99.4 %) topoisomeras topoisomer topoisomeras e I (100 %) topoisomer DNA DNA topoisom topoisomer ase type I (95.9 %) e type I (99.4 ase type I e type I (99.4 ase type I topoisomer topoisomer erase type ase type I (87.1 %) %) (100 %) %) (100 %) ase type I ase type I I (99.7 %) (98.7 %) (99.7 %) (99.7 %)

059 hypothetica hypotheti PP206 hypothetical hypothetica hypothetical hypothetical hypothetica ORF063 ORF063 hypotheti hypothetica l protein cal (97.8 %) protein (99.3 l protein protein (99.3 protein (100 l protein hypothetica hypothetica cal l protein (64.5 %) protein %) (98.6 %) %) %) (98.6 %) l protein l protein protein (99.3 %) (92 %) (99.3 %) (97.1 %) (99.3 %)

060 ORF064 mRNA PP207 mRNA mRNA mRNA mRNA mRNA ORF064 ORF064 mRNA mRNA mRNA capping (99.9 %) capping capping capping capping capping mRNA mRNA capping capping capping enzyme enzyme large enzyme enzyme large enzyme enzyme capping capping enzyme enzyme enzyme subunit subunit (99.4 large subunit (99.4 subunit (99.8 large enzyme enzyme large large large (96.6 %) %) subunit %) %) subunit large large subunit subunit subunit (99.9 %) (99.9 %) subunit subunit (99 %) (99.6 %) (85.7 %) (99.6 %) (99.4 %)

061 ORF065 virion PP75 (98.7 Virion virion Virion virion virion ORF065 ORF065 Virion Virion virion protein %) protein (99.3 protein protein (99.3 protein (99.3 protein virion virion protein protein protein (88.2 %) %) (99.3 %) %) %) (98.7 %) protein protein (98 %) (99.3 %) (73.3 %) (99.3 %) (99.3 %)

062 ORF066 virion PP210 Virion protein virion Virion protein virion virion ORF066 ORF066 Virion Virion virion protein (93.7 %) (97.1 %) protein (97.6 %) protein (99 protein virion virion protein protein protein (62 (85.2 %) (98.1 %) %) (98.1 %) protein (99 protein (97.6 %) (97.6 %) %) %) (98.1 %)

063 ORF067 uracil- PP211 Uracil DNA uracil DNA Uracil DNA uracil-DNA uracil DNA ORF067 ORF067 Uracil Uracil uracil DNA DNA (98.3 %) glycosidase glycosidase glycosidase glycosylase glycosidase uracil DNA uracil DNA DNA DNA glycosyla (98.3 %) (98.7 %) (98.3 %) (99.6 %) (99.1 %) glycosida

145

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 glycosidase se (96.7 glycosidase glycosidase se (97.4 glycosidase (86.5 %) %) (99.1 %) (98.7 %) %) (98.7 %)

064 ORF068 NTPase PP212 NTPase (99.6 NTPase NTPase (99.6 NTPase (99.9 NTPase ORF068 ORF068 NTPase NTPase NTPase (95.9 %) (99.4 %) %) (99.9 %) %) %) (99.9 %) NTPase NTPase (99.6 %) (99.5 %) (89.1 %) (99.6 %) (99.2 %)

065 ORF069 early PP213 Early early Early early early ORF069 ORF069 Early Early early transcript (99.2 %) transcription transcripti transcription transcription transcripti early early transcript transcripti transcriptio ion factor factor (99.8 on factor factor (99.8 factor (99.5 on factor transcriptio transcripti ion factor on factor n factor (97.5 %) %) (99.8 %) %) %) (99.8 %) n factor on factor (99.7 %) (99.8 %) VETFs VETFs VETFs (92.1 %) (99.7 %) (99.8 %)

066 ORF070 RNA- PP214 RNA- RNA- RNA- RNA RNA- ORF070 ORF070 RNA- RNA- RNA polymera (98.4 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase polymerase se subunit subunit subunit subunit subunit subunit polymeras polymerase se subunit subunit subunit RPO18 RPO18 (96.8 RPO18 RPO18 (96.8 RPO18 (98.9 RPO18 e subunit subunit RPO18 RPO18 RPO18 (89.4 %) %) (95.3 %) %) %) (95.3 %) RPO18 RPO18 (97.9 %) (95.8 %) (82.2 %) (99.5 %) (97.9 %)

067 ORF071 NTP PP215 NPH-PPH NPH-PPH NPH-PPH NTP NPH-PPH ORF071 ORF071 NPH- NPH-PPH NPH-PPH pyrophos (98.4 %) downregulato downregul downregulato pyrophospho downregul NPH-PPH NPH-PPH PPH downregula downregula phohydro r (NTP ator (100 r (NTP hydrolase ator (100 downregul downregula downreg tor (NTP tor (79 %) lase (96.9 pyrophosphoh %) pyrophosphoh (100 %) %) ator (100 tor (99.1 %) ulator pyrophosph %) ydrolase) ydrolase) %) (NTP ohydrolase) (99.6 %) (99.6 %) pyrophos (99.6 %) phohydro lase) (99.6 %)

068 transcriptio NPH-1 PP62 (99.5 Transcriptio transcriptio Transcription NPH-1 (99.8 transcriptio ORF072 ORF072 Transcrip Transcript n (97.3 %) %) n n termination %) n transcripti transcripti tion ion termination termination termination factor NPH-I termination on on terminati terminatio factor factor NPH-I factor (99.7 %) factor NPH- terminatio terminatio on factor n factor NPH-I (99.8 %) NPH-I I (99.4 %) n factor n factor NPH-I NPH-I (87.8 %) (99.5 %) NPH-I NPH-I (99.1 %) (99.8 %) (99.8 %) (99.8 %)

069 ORF073 hypotheti PP61 (94.1 hypothetical hypothetica hypothetical hypothetical hypothetic ORF073 ORF073 hypotheti hypothetica hypothetica cal %) protein (92.6 l protein protein (93.1 protein (95.7 al protein hypothetica hypothetica cal l protein l protein (64 protein %) (96.8 %) %) %) (97.3 %) l protein l protein protein (92.6 %) %) (87.6 %) (96.8 %) (92.6 %) (93.1 %)

146

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

070 ORF074 mRNA PP60 (95.2 mRNA mRNA mRNA mRNA mRNA ORF074 ORF074 mRNA mRNA mRNA capping %) capping capping capping capping capping mRNA mRNA capping capping capping enzyme enzyme small enzyme enzyme small enzyme (98.6 enzyme capping capping enzyme enzyme enzyme (95.5 %) subunit (97.6 small subunit (97.6 %) small enzyme enzyme small small small %) subunit %) subunit small small subunit subunit subunit (98.6 %) (98.6 %) subunit (99 subunit (98.3 %) (97.9 %) (86.2 %) %) (98.3 %)

071 ORF075 rifampici PP59 (98.2 Rifampicin rifampicin Rifampicin rifampicin rifampicin ORF075 ORF075 Rifampic Rifampicin rifampin n %) resistance resistance resistance resistance, resistance putative putative in resistance resistance resistance protein (99.6 protein protein (99.6 membrane protein rifampicin rifampicin resistance protein protein protein %) (99.8 %) %) protein (99.6 (99.8 %) resistance resistance protein (99.6 %) (85.3 %) (95.2 %) %) protein protein (99.5 %) (99.6 %) (99.6 %)

072 late late PP58 (98 Late late Late late late ORF076 ORF076 Late Late transcriptio transcript %) transcription transcriptio transcription transcription transcriptio late late transcript transcriptio n factor ion factor factor VLTF2 n factor factor VLTF2 factor n factor transcripti transcriptio ion factor n factor VLTF-2 VLTF2 (98.7 %) VLTF2 (98.7 %) VLTF2 (100 VLTF2 on factor n factor VLTF2 VLTF2 (85.3 %) (96 %) (99.3 %) %) (99.3 %) VLTF-2 VLTF-2 (98.7 %) (98.7 %) (100 %) (98.7 %)

073 late late PP57 (99.1 Late late Late late late ORF077 ORF077 Late Late transcriptio transcript %) transcription transcripti transcription transcription transcripti late late transcrip transcripti n factor ion factor factor VLTF3 on factor factor factor on factor transcripti transcripti tion on factor VLTF-3 VLTF3 (99.6 %) VLTF3 VLTF3 (100 VLTF3 (100 VLTF3 on factor on factor factor VLTF3 (91.9 %) (99.6 %) (100 %) %) %) (100 %) VLTF-3 VLTF-3 VLTF3 (100 %) (100 %) (100 %) (100 %)

074 ORF078 thioredox - Thioredoxin- thioredoxin Thioredoxin- thioredoxin- thioredoxin ORF078 ORF078 Thioredo Thioredoxi thioredoxin in-like like protein -like like protein like protein -like protein thioredoxi thioredoxin xin-like n-like -like protein (94.9 %) protein (94.9 %) (96.3 %) (92.7 %) n-like -like protein protein protein (72 (86.1 %) (92.7 %) protein protein (94.9 %) (94.9 %) %) (96.3 %) (94.9 %)

075 ORF079 virion PP56 (98.7 Virion core virion core Virion core virion core, virion core ORF079 ORF079 Virion Virion core virion core core %) protein P4b protein P4b protein P4b P4b precursor protein P4b virion core virion core core protein P4b protein P4b protein precursor precursor precursor (99.8 %) precursor protein protein P4b protein precursor precursor P4b (99.1 %) (99.6 %) (99.1 %) (99.4 %) P4b precursor P4b (99.1 %) (76.6 %) precursor precursor (98.9 %) precursor (90.2 %) (100 %) (99.1 %)

147

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

076 putative membran PP223 IMV, viral IMV, viral IMV, viral IMV, viral IMV, viral ORF051 ORF051 IMV, IMV, viral viral e protein (91.3 %) entry (55 %) entry (55 entry (55 %) entry (55 %) entry (55 putative putative viral entry (55 membrane (57.1 %) %) %) membrane membrane entry (55 %) protein (55 protein protein %) %) (57.1 %) (57.1 %)

077 ORF080 virion PP55 (88.9 Virion core virion core Virion core virion core virion core ORF080 ORF080 Virion Virion core virion core core %) protein (87.7 protein protein (87.7 protein (100 protein virion core virion core core protein protein protein %) (96.3 %) %) %) (96.3 %) protein protein protein (87.7 %) (54.3 %) (72 %) (93.8 %) (90.2 %) (86.4 %)

079 ORF081 RNA- PP225 RNA- RNA- RNA- RNA- RNA- ORF081 ORF081 RNA- RNA- RNA polymera (98.8 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase polymerase se subunit subunit subunit subunit subunit subunit polymeras polymerase se subunit subunit subunit RPO19 RPO19 (98.3 RPO19 RPO19 (98.8 RPO19 (99.4 RPO19 e subunit subunit RPO19 RPO19 RPO19 (92.5 %) %) (99.4 %) %) %) (99.4 %) RPO19 RPO19 (98.8 %) (98.8 %) (88.7 %) (100 %) (98.8 %)

080 ORF082 hypotheti PP54 (98.7 hypothetical hypothetic hypothetical hypothetical hypothetica ORF082 ORF082 hypotheti hypothetica hypothetica cal %) protein (97.9 al protein protein (97.9 protein (97.3 l protein hypothetica hypothetica cal l protein l protein protein %) (98.9 %) %) %) (98.9 %) l protein l protein protein (97.9 %) (73.3 %) (89.7 %) (97.9 %) (97.9 %) (97.6 %)

081 ORF083 early PP53 (98.6 Early early Early early early ORF083 ORF083 Early Early early transcript %) transcription transcriptio transcription transcription transcriptio early early transcript transcriptio transcriptio ion factor factor (99.4 n factor factor (99.3 factor (99.6 n factor transcripti transcriptio ion factor n factor n factor (96.2 %) %) (99.6 %) %) %) (99.6 %) on factor n factor (99.4 %) (99.4 %) VETFL VETFL VETFL (88.9 %) (99.9 %) (99.3 %)

082 ORF084 intermedi PP229 Intermediate intermedia Intermediate intermediate intermedia ORF084 ORF084 Intermedi Intermediat intermediat ate (98.3 %) transcription te transcription transcription te intermediat intermediat ate e e transcript factor VITF-3 transcripti factor VITF-3 factor (99.3 transcripti e e transcript transcriptio transcriptio ion factor (99.3 %) on factor (99 %) %) on factor transcriptio transcriptio ion factor n factor n factor (94.4 %) VITF-3 VITF-3 n factor n factor VITF-3 VITF-3 VITF-3 (99.7 %) (99.7 %) VITF-3 VITF-3 (99.3 %) (99.3 %) (83.9 %) (99.3 %) (98.3 %)

083 ORF085 late - Late virion late virion Late virion virion late virion ORF085 ORF085 Late Late virion late virion virion membrane membrane membrane membrane membrane late virion late virion virion membrane membrane membran protein (100 protein protein (100 protein (100 protein membrane membrane membra protein protein e protein %) (100 %) %) %) (100 %) protein protein ne (100 %) (89.2 %) (88.3 %) (100 %) (97.8 %) protein (100 %)

148

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

084 ORF086 virion PP48 (96.8 Virion core virion core Virion core virion core virion core ORF086 ORF086 Virion Virion core virion core core %) protein P4a protein P4a protein P4a protein P4a protein P4a virion core virion core core protein P4a protein P4a protein precursor precursor precursor (98 precursor precursor protein P4a protein P4a protein precursor precursor P4a (97.8 %) (98.9 %) %) (99.4 %) (98.9 %) precursor precursor P4a (97.9 %) (75.4 %) precursor (99.3 %) (98.1 %) precursor (90.3 %) (98.1 %)

085 ORF087 virion PP233 Virion virion Virion virion virion ORF087 ORF087 Virion Virion hypothetica formation (97.3 %) formation formation formation formation formation hypothetica hypothetica formation formation l protein protein (99.4 %) (99.4 %) (99.4 %) (99.7 %) (99.4 %) l protein l protein (99.4 %) (99.7 %) (86.7 %) (98.2 %) (99.4 %) (99.4 %)

086 ORF033 IMV PP234 IMV IMV IMV IMV protein IMV ORF033 ORF033 IMV IMV IMV protein (86.2 %) membrane membrane membrane (58.3 %) membrane putative putative membran membrane membrane (58.3 %) protein (58.3 protein protein (58.3 protein IMV IMV e protein protein protein %) (58.3 %) %) (58.3 %) membrane membrane (58.3 %) (58.3 %) (58.3 %) protein protein (58.3 %) (58.3 %)

087 ORF089 virion PP46 (98.9 Virion virion Virion virion virion ORF089 ORF089 Virion Virion virion membran %) membrane membrane membrane membrane membrane virion virion membran membrane membrane e protein protein (98.9 protein protein (98.9 protein (100 protein membrane membrane e protein protein protein (92.4 %) %) (98.9 %) %) %) (98.9 %) protein protein (97.8 %) (97.8 %) (65.4 %) (98.9 %) (98.9 %)

088 ORF090 IMV PP45 (100 IMV IMV IMV IMV IMV ORF090 ORF090 IMV IMV IMV membran %) phosphorylate phosphoryl phosphorylate membrane phosphoryl putative putative phosphor phosphoryl phosphoryl e protein d membrane ated d membrane protein (100 ated IMV IMV ylated ated ated (82.6 %) protein (98.9 membrane protein (98.9 %) membrane phosphoryl phosphoryl membran membrane membrane %) protein %) protein ated ated e protein protein protein (78 (98.9 %) (98.9 %) membrane membrane (98.9 %) (98.9 %) %) protein protein (98.9 %) (97.8 %)

089 ORF091 putative PP44 (98.1 IMV IMV IMV putative IMV ORF091 ORF091 IMV IMV IMV IMV %) membrane membrane membrane virulence membrane putative putative membra membrane membrane virulence protein (100 protein protein (100 factor, IMV protein IMV IMV ne protein protein factor %) (98.1 %) %) (100 %) (98.1 %) membrane membrane protein (100 %) (84.9 %) (86.8 %) protein protein (100 %) (100 %) (100 %)

090 hypothetica hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica ORF092 ORF092 hypotheti hypothetica l protein cal protein (96.6 l protein protein (95.5 protein (98.9 l protein hypothetica hypothetica cal l protein (65.2 %) %) (97.8 %) %) %) (97.8 %) (95.5 %)

149

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 protein l protein l protein protein (92.1 %) (97.8 %) (96.6 %) (95.5 %)

091 ORF093 myristyla PP43 (99.2 Myristylated myristylate Myristylated myristylated myristylate ORF093 ORF093 Myristyla Myristylate myristylate ted %) protein (98.6 d protein protein (98.6 protein (99.7 d protein predicted predicted ted d protein d protein protein %) (98.9 %) %) %) (98.6 %) myristylate myristylate protein (98.9 %) (84.1 %) (92.2 %) d protein d protein (98.9 %) (100 %) (99.4 %)

092 ORF094 phosphor PP42 (97.4 phosphorylate phosphoryl phosphorylate phosphorylate phosphoryl ORF094 ORF094 phosphor phosphoryl phosphoryl ylated %) d IMV ated IMV d IMV d IMV ated IMV putative putative ylated ated IMV ated IMV IMV membrane membrane membrane membrane membrane phosphoryl phosphoryl IMV membrane membrane membran protein (98.5 protein (98 protein (98.5 protein (99.5 protein (98 ated IMV ated IMV membran protein protein e protein %) %) %) %) %) membrane membrane e protein (98.5 %) (81.4 %) (90 %) protein protein (98.5 %) (100 %) (100 %)

093 ORF095 DNA PP239 DNA helicase DNA DNA helicase DNA helicase DNA ORF095 ORF095 DNA DNA DNA helicase (93.6 %) (99 %) helicase (99 %) (99.8 %) helicase DNA DNA helicase helicase helicase (96.9 %) (99.6 %) (99.6 %) helicase helicase (99 %) (98.8 %) (88.2 %) (99.6 %) (99.4 %)

094 ORF096 Zn-finger PP39 (95.6 Zn-finger Zn-finger Zn-finger Zn-finger Zn-finger ORF096 ORF096 Zn-finger Zn-finger hypothetica protein %) protein (96.7 protein protein (96.7 protein (100 protein hypothetic hypothetica protein protein l protein (76.8 %) %) (100 %) %) %) (100 %) al protein l protein (96.7 %) (96.7 %) (69.1 %) (100 %) (96.7 %)

095 hypothetica hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica ORF098 ORF098 hypotheti hypothetica l protein cal protein (95.4 l protein protein (96.3 protein (97.2 l protein hypothetica hypothetica cal l protein (76.1 %) protein %) (97.2 %) %) %) (97.2 %) l protein l protein protein (96.3 %) (88.9 %) (97.2 %) (96.3 %) (96.3 %)

096 ORF097 DNA- PP240 DNA- DNA- DNA- DNA DNA- ORF097 ORF097 DNA- DNA- DNA polymera (98.6 %) polymerase polymerase polymerase polymerase polymerase DNA DNA polymera polymerase polymerase se processivity processivity processivity processivity processivity polymeras polymerase se processivity processivity processiv factor (97.6 factor (99 factor (97.9 factor (99 %) factor (99 e processivity processiv factor (97.1 factor (70.4 ity factor %) %) %) %) processivit factor (98.1 ity factor %) %) (92.4 %) y factor %) (98.1 %) (99.3 %)

097 ORF099 resolvase PP243 (100 Holliday Holliday Holliday resolvase Holliday ORF099 ORF099 Holliday Holliday Holliday (98.6 %) %) junction junction junction (100 %) junction Holliday Holliday junction junction junction resolvase resolvase resolvase resolvase junction junction resolvase resolvase (100 %) (100 %) (100 %) (100 %) (100 %) (100 %)

150

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 resolvase resolvase resolvase (95.2 %) (100 %) (100 %)

098 ORF100 intermedi PP244 Intermediate intermediat Intermediate intermediate intermediat ORF100 ORF100 Intermedi Intermediat intermediat ate (98.9 %) transcription e transcription transcription e intermedia intermediat ate e e transcript factor VITF-3 transcriptio factor VITF-3 factor VITF3 transcriptio te e transcript transcriptio transcriptio ion factor (98.7 %) n factor (98.9 %) (100 %) n factor transcripti transcriptio ion factor n factor n factor VITF3 VITF-3 VITF-3 on factor n factor VITF-3 VITF-3 VITF-3 (97.6 %) (98.7 %) (98.7 %) VITF-3 VITF-3 (98.9 %) (98.7 %) (85.2 %) (100 %) (98.9 %)

099 ORF101 RNA- PP245 RNA- RNA- RNA- RNA RNA- ORF101 ORF101 RNA- RNA- RNA polymera (99.7 %) polymerase polymerase polymerase polymerase polymerase RNA RNA polymera polymerase polymerase se subunit subunit subunit RPO132 (99.8 subunit polymeras polymerase se subunit subunit subunit RPO132 RPO132 (99.5 RPO132 RPO132 (99.7 %) RPO132 e subunit subunit RPO132 RPO132 RPO132 (98.1 %) %) (99.7 %) %) (99.8 %) RPO132 RPO132 (99.7 %) (99.7 %) (93.2 %) (99.9 %) (99.7 %)

100 A-type A-type PP33 (92 A-type A-type A-type A-type A-type ORF102 A ORF102 A A-type A-type inclusion inclusion %) inclusion inclusion inclusion inclusion inclusion type type inclusion inclusion protein protein/fu protein/fusion protein/fus protein/fusion protein/fusion protein/fus inclusion inclusion protein/fu protein/fusi (75.9 %) sion peptide ion peptide peptide peptide ion peptide protein protein sion on peptide peptide hybrid (92 %) hybrid hybrid (91.2 hybrid (96.3 hybrid (96.3 %) (97.1 %) peptide hybrid hybrid (97.1 %) %) %) (97.1 %) hybrid (91.2 %) (86.1 %) (94.2 %)

101 ORF103 A A-type - A-type A-type A-type A-type A-type ORF103 A ORF103 A A-type A-type type inclusion inclusion inclusion inclusion inclusion inclusion type type inclusion inclusion inclusion protein protein (51.5 protein protein (51.3 protein (50.9 protein inclusion inclusion protein protein (96 protein (83.6 %) %) (98.9 %) %) %) (98.9 %) protein protein (68.3 %) %) (58.6 %) (97.7 %) (51.5 %)

102 ORF105 IMV - IMV surface IMV IMV surface IMV surface IMV ORF105 ORF105 IMV IMV hypothetica surface protein (99.3 surface protein (99.3 protein (99.3 surface hypothetic hypothetica surface surface l protein (90 protein %) protein %) %) protein al protein l protein protein protein %) (95 %) (99.3 %) (99.3 %) (100 %) (99.3 %) (99.3 %) (99.3 %)

103 ORF106 RNA- PP29 (96.4 RNA- RNA- RNA- RNA RNA- ORF106 ORF106 RNA- RNA- RNA polymera %) polymerase polymeras polymerase polymerase polymeras RNA RNA polymera polymerase polymerase se subunit subunit e subunit subunit subunit e subunit polymerase polymerase se subunit subunit subunit RPO35 RPO35 (98.4 RPO35 (99 RPO35 (97.1 RPO35 (98.4 RPO35 (99 subunit subunit RPO35 RPO35 RPO35 (95.8 %) %) %) %) %) %) RPO35 RPO35 (98.4 %) (98.7 %) (79.5 %) (98.7 %) (98.1 %)

151

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

104 ORF107 putative - Virion virion Virion virion virion ORF107 ORF107 Virion Virion virion virion morphogenesi morphoge morphogenesi morphogenes morphoge virion virion morphog morphogen morphogen morphog s (95 %) nesis (100 s (98.3 %) is (100 %) nesis (100 morphoge morphogen enesis esis (95 %) esis (76.7 enesis %) %) nesis (100 esis (95 %) (96.7 %) %) protein %) (93.2 %)

105 - hypotheti - hypothetical hypothetica hypothetical hypothetical hypothetica - - hypotheti hypothetica cal protein (91.8 l protein protein (93.9 protein (100 l protein cal l protein protein %) (91.8 %) %) %) (91.8 %) protein (91.8 %) (69 %) (91.8 %)

106 ORF108 DNA PP26 (93.8 DNA DNA DNA ATPase, DNA ORF108 ORF108 DNA DNA DNA packagin %) packaging packaging packaging DNA packaging DNA DNA packagin packaging packaging g protein/ATPa protein/AT protein/ATPa packaging protein/AT packaging packaging g protein/AT protein/AT protein/A se (94.5 %) Pase (95.9 se (94.5 %) (98.5 %) Pase (95.9 protein/AT protein/AT protein/A Pase (94.9 Pase (93.9 TPase %) %) Pase (100 Pase (94.5 TPase %) %) (96.9 %) %) %) (94.5 %)

107 ORF110 EEV - - EEV EEV EEV EEV ORF110 - EEV EEV EEV glycoprot glycoprotei glycoprotein glycoprotein glycoprotei EEV glycoprot glycoprotei glycoprotei ein (78.4 n (80.2 %) (79.3 %) (78.4 %) n (80.2 %) glycoprotei ein (80.2 n (95.5 %) n (56.6 %) %) n (98.2 %) %)

108 hypothetica hypotheti PP253 (95 hypothetical hypothetica hypothetical hypothetical hypothetica ORF111 ORF111 hypotheti hypothetica l protein cal %) protein (93.6 l protein protein (92.9 protein (95.7 l protein hypothetic hypothetica cal l protein (60.4 %) protein %) (95.7 %) %) %) (95.7 %) al protein l protein protein (92.9 %) (86.4 %) (97.9 %) (93.6 %) (95 %)

109 - chemokin PP255 chemokine chemokine chemokine chemokine chemokine ORF112 ORF112 chemokin chemokine e-binding (81.2 %) binding binding binding binding binding putative putative e binding binding protein protein (79.7 protein protein (85.1 protein (95.2 protein chemokine- chemokine- protein protein (53 %) %) (95.2 %) %) %) (95.5 %) binding binding (78.1 %) (81.7 %) protein protein (82.4 %) (84.5 %)

110 - hypotheti PP256 hypothetical hypothetica hypothetical hypothetical hypothetica ORF113 ORF113 hypotheti hypothetica cal (87.3 %) protein (80.6 l protein protein (86.7 protein (92.9 l protein hypothetic hypothetica cal l protein protein %) (87.3 %) %) %) (87.3 %) al protein l protein protein (84.7 %) (62.6 %) (95.3 %) (81.1 %) (82 %)

111 ORF114 hypotheti PP257 hypothetical hypothetica - hypothetical hypothetica ORF114 ORF114 hypotheti hypothetica hypothetica cal (96.4 %) protein (94.8 l protein (98 protein (98 %) l protein (98 hypothetic hypothetica cal l protein %) %) %) (94.5 %)

152

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 l protein protein al protein l protein protein (64.9 %) (91.6 %) (98.3 %) (94.5 %) (94.5 %)

112 - - PP260 hypothetical hypothetic - hypothetical hypothetic ORF116 ORF116 hypotheti hypothetica (75.4 %) protein (55.8 al protein protein (67.5 al protein hypothetica hypothetica cal l protein %) (85.2 %) %) (85.2 %) l protein l protein protein (56.3 %) (79.7 %) (51.2 %) (51.5 %)

113 - GM- PP261 GM-CSF/IL- GM- - GM-CSF/IL- GM- ORF117 ORF117 - GM- CSF/IL-2 (93.4 %) 2 inhibition CSF/IL-2 2 inhibition CSF/IL-2 GM- GM- CSF/IL-2 inhibition factor (94.6 inhibition factor (98.5 inhibition CSF/IL-2 CSF/IL-2 inhibition factor %) factor (97.7 %) factor (97.3 inhibition inhibition factor (93.8 (87.3 %) %) %) factor-like factor-like %) protein protein (98.8 %) (93.8 %)

114 - - PP264 hypothetical hypothetica - hypothetical hypothetica ORF118 ORF118 - hypothetica (96.1 %) protein (97.1 l protein protein (98 l protein hypothetica hypothetica l protein %) (97.1 %) %) (95.1 %) l protein l protein (97.1 %) (97.1 %) (97.1 %)

115 ORF119 putative PP266 hypothetical hypothetica IMV hypothetical hypothetica ORF119 ORF119 IMV hypothetica hypothetica IMV (95.2 %) protein (95.7 l protein membrane protein (97.9 l protein hypothetic hypothetica membran l protein l protein virulence %) (97.1 %) protein (71.4 %) (90.9 %) al protein l protein e protein (89.2 %) (55.3 %) factor %) (98.4 %) (94.1 %) (71.4 %) (71.4 %)

116 - - PP267 hypothetical hypothetica - hypothetical hypothetica ORF120 ORF120 hypotheti hypothetica (84.6 %) protein (82.6 l protein (83 protein (97.1 l protein (83 hypothetica hypothetica cal l protein %) %) %) %) l protein l protein protein (82.6 %) (95.7 %) (82.6 %) (82.6 %)

117 hypothetica hypotheti PP268 (83 NF-kappa NF-kappaB NF-kappa hypothetical NF-kappaB ORF121 ORF121 NF- NF-kappa l protein (53 cal %) pathway pathway pathway protein (95.7 pathway hypothetica hypothetica kappa pathway %) protein inhibitor (84.5 inhibitor inhibitor (83.3 %) inhibitor l protein l protein pathway inhibitor (75.5 %) %) (89.4 %) %) (89.8 %) (94.9 %) (83.3 %) inhibitor (84.1 %) (83 %)

118 hypothetica hypotheti PP269 hypothetical hypothetica hypothetical hypothetical hypothetica ORF122 ORF122 hypotheti hypothetica l protein cal (92.5 %) protein (94.1 l protein protein (93.8 protein (99.7 l protein hypothetic hypothetica cal l protein (55.5 %) protein %) (96.9 %) %) %) (96.9 %) al protein l protein protein (93.5 %) (88.8 %) (99.4 %) (93.8 %) (93.8 %)

153

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12

119 ankyrin- Ankyrin/ PP272 (96 Ankyrin/F- ankyrin/F- Ankyrin/F-bx Ankyrin/F- ankyrin/F- ORF123 ORF123 Ankyrin/ Ankyrin/F- like protein F-box %) box protein box protein protein (95.8 box protein box protein ankyrin ankyrin F-box box protein (62 %) protein (95.6 %) (97.3 %) %) (98.7 %) (97.3 %) repeat repeat protein (95.8 %) (82.6 %) protein (99 protein (95.8 %) %) (95.8 %)

120 hypothetica hypotheti PP274 hypothetical hypothetica hypothetical hypothetical hypothetica ORF124 ORF124 hypotheti hypothetica l protein cal (92.3 %) protein (94.2 l protein protein (96.2 protein (98.9 l protein hypothetica hypothetica cal l protein (54.7 %) protein %) (96.4 %) %) %) (96.6 %) l protein l protein protein (94.2 %) (89 %) (98.3 %) (95.9 %) (95.3 %)

121 hypothetica hypotheti PP275 Apoptosis apoptosis Apoptosis hypothetical apoptosis ORF125 ORF125 Apoptosi Apoptosis l protein cal (96.5 %) inhibitor (94.2 inhibitor inhibitor (94.8 protein (98.8 inhibitor hypothetica hypothetica s inhibitor (66.5 %) protein %) (99.4 %) %) %) (99.4 %) l protein l protein inhibitor (94.8 %) (80.3 %) (98.8 %) (92.5 %) (94.2 %)

122 ankyrin- Ankyrin/ PP276 (98 Ankyrin/F- ankyrin/F- Ankyrin/F- Ankyrin/F- ankyrin/F- ORF126 ORF126 Ankyrin/ Ankyrin/F- like protein F-box %) box protein box protein box protein box protein box protein ankyrin ankyrin F-box box protein (59.5 %) protein (96.6 %) (99.2 %) (96.8 %) (99.4 %) (99.2 %) repeat repeat protein (96.6 %) (87.9 %) protein protein (97.2 %) (99.2 %) (96.6 %)

123 ORF127 - PP277 IL-10-like IL-10-like IL-10-like interleukin IL-10-like ORF127 ORF127 IL-10- IL-10-like IL-10 (76.4 (92.4 %) protein (97.8 protein protein (97.8 10 (100 %) protein IL-10-like IL-10-like like protein %) %) (97.3 %) %) (97.3 %) protein protein protein (97.8 %) (94.1 %) (96.2 %) (97.3 %)

124 ankyrin- Ankyrin/ PP278 Ankyrin/F- ankyrin/F- Ankyrin/F- Ankyrin/F- ankyrin/F- ORF128 ORF128 Ankyrin/ Ankyrin/F- like protein F-box (94.2 %) box protein box protein box protein box protein box protein ankyrin ankyrin F-box box protein (57.4 %) protein (96.4 %) (96.6 %) (96.6 %) (97.2 %) (96.4 %) repeat repeat protein (95.6 %) (84.8 %) protein protein (96.4 %) (97.5 %) (96.6 %)

125 ankyrin- Ankyrin/ PP280 (95 Ankyrin/F- ankyrin/F- Ankyrin/F- Ankyrin/F- ankyrin/F- ORF129 ORF129 Ankyrin/ Ankyrin/F- like protein F-box %) box protein box protein box protein box protein box protein ankyrin ankyrin F-box box protein (60.5 %) protein (92.6 %) (96.9 %) (92.8 %) (97.5 %) (96.9 %) repeat repeat protein (92.4 %) (77.4 %) protein protein (92.2 %) (98.6 %) (91.8 %)

126 Ser/Thr protein PP281 (99 Serine/threoni serine/threo Serine/threoni protein serine/threo ORF130 ORF130 Serine/thr Serine/thre kinase (86.6 kinase %) ne protein nine protein ne protein kinase (99.8 nine protein putative putative eonine onine %) (94.4 %) kinase (99 %) kinase (99.2 kinase (99.2 %) kinase (99.2 serine/threo serine/threo protein protein %) %) %) nine protein nine protein kinase kinase (99.2 (99.2 %) %)

154

MxOV BPSV PCPV OV-D1701 OV-GO OV- OV-NP OV-NZ2 OV- OV-IA82 OV-SA00 OV-SJ1 OV-YX ORF NA1/11 HN3/12 kinase (99.6 kinase (99 %) %)

127 ORF131 membran PP282 (94 Membrane membrane Membrane membrane membrane ORF131 ORF131 Membran Membrane putative e protein %) protein (93.4 protein protein (95.2 protein (96 protein putative putative e protein protein membrane (93.2 %) %) (93.6 %) %) %) (93.6 %) membrane membrane (93.8 %) (90.6 %) protein protein protein (77.9 %) (95.5 %) (93.8 %)

128 - VEGF - VEGF-like - VEGF-like - - - ORF132 VEGF- - (57.1 %) protein (86.5 protein (83.5 VEGF like %) %) (90.3 %) protein (85 %)

129 - - - - virion virion virion - - - - membrane membrane membrane protein (70 protein (70 protein (70 %) %) %)

130 hypothetica hypotheti PP286 hypothetical hypothetica hypothetical hypothetical hypothetica ORF134 ORF134 hypotheti hypothetica l protein cal (84.6 %) protein (81.2 l protein protein (80.5 protein (98.7 l protein hypothetic hypothetica cal l protein (60.2 %) protein %) (94.6 %) %) %) (94.6 %) al protein l protein (74 protein (81.2 %) (73.3 %) (98.7 %) %) (87.2 %)

155

Supplementary Figure 5.1 Heatmap ordered by percent identity of MxOV blast hits to related reference genomes. Blast hits with a percent identity > 50% and coverage of > 10% were retained.

CHAPTER 6: DISCUSSION AND CONCLUSION

Viruses circulating in wildlife can have a direct impact on the health and well-being of other wildlife species, domestic animals, and humans; however, our knowledge of these viruses is still limited. Viral infections in wildlife are not always obvious; clinically affected individuals might be killed by predators before clinical signs can be observed by researchers or veterinarians, or disease may be inapparent or confused with other ailments (Levinson et al., 2013; Ryser-

Degiorgis, 2013). One way we can learn more about viruses in wildlife is through molecular surveillance, whereby genetic data of the virus, the host, or both are explored. Molecular techniques can differentiate viruses based on genetic material and can confirm the presence or exposure of a wildlife host to a viral pathogen.

The goals of this thesis were to use molecular techniques to identify dsDNA viruses present in wildlife, seek out evidence of coevolution of dsDNA viruses and their hosts, characterize the association of viral presence and clinical disease, contribute to the database of viral gene and genome sequences, and to evaluate the benefits and limitations of using tissues for molecular surveillance. The preceding chapters have explored the use of various PCRs, gene and genome sequencing, and phylogenetic analyses to characterize HV in multiple wildlife species, and orf virus in muskoxen. This chapter highlights the key contributions of this research and discusses potential avenues for future research on wildlife dsDNA viruses.

Part I: Molecular surveillance of HV

HV are challenging to study as different HV can induce similar tumors and lesions, individual viral species can produce a variety of equivocal syndromes, and infection may be widespread in the absence of overt disease (Das Neves et al., 2010; Smith and Whitley, 2017). In

157 the case of many viruses, there is a lack of surveillance data on the incidence or prevalence of HV in wild populations (Thomas et al., 2015). Viral DNA is minimal compared to an overwhelming amount of genetic material from the infected host and other microbiota, therefore sensitive methodologies are needed for effective molecular surveillance studies (Nelson et al., 2019).

Chapters 2, 3, and 4 have shown that pan-HV nested PCRs using degenerate primers offer an effective tool for the characterization of HV strains in a variety of tissues from many different species. The fragment amplified from the HV DNA polymerase gene (DPOL) is genetically sufficiently specific to distinguishing alpha-, beta-, and gamma-HV at a subfamily level and delineating related sequences. Although infection status was not evaluated, the presence of HV genetic material indicates exposure (and therefore lifelong infection) to HV and allows us to evaluate the distribution of naturally occurring HV. In Chapter 2 we utilized pan-HV degenerate primers in nested PCRs to identify several species of HV present in wildlife animals. We were able to demonstrate efficacy in screening for previously uncharacterized as well as known HV using previously described degenerate primers directly on DNA extracted from archived frozen tissues.

The DPOL of HV is a robust target, however, it is small, conserved, and lacking in variation. Specific and sensitive methods are required for the study of more variable regions of the

HV genome. A major challenge in the characterization of HV is the lack of pan-HV primers targeting other genes, and very few representative HV genomes are available for novel primer design. The development of other pan-HV degenerate primers targeting other regions of the viral genome aside from the DPOL gene would tremendously benefit the practice of molecular surveillance and the generation of a database of comparable HV sequences. Furthermore, there is

158 a continued need for the adaptation and validation of molecular diagnostic tools for the study of wildlife health and disease.

The choice of phylogenetic inference can influence the results and interpretation of evolutionary relationships between entities in a phylogenetic tree. Two commonly used strategies for phylogenetic analysis are Maximum Likelihood (ML), and Bayesian inference. Bayesian analysis uses Markov Chain Monte Carlo (MCMC) algorithms to obtain a large sample of trees that maximizes both the model parameters and the tree (Lemey et al., 2009). Each step in the search builds upon the current tree. The resulting tree is a consensus tree of all of the trees sampled.

The aim of ML analysis is to find the one maximum (highest) likelihood tree (Lemey et al., 2009).

A ML tree optimizes the model parameters in a separate step, then finds the most optimal tree

(conditioned on previously optimized model parameters) (Lemey et al., 2009). This thesis includes both ML and Bayesian inference to generate phylogenetic trees where applicable. With some exception, the majority of phylogenetic trees generated by theses gave similar results with essentially the same topology and differences largely in branch lengths rather than branching order.

Future studies should continue to incorporate various phylogenetic strategies so that results can interpreted with minimal bias.

The methodologies described herein can be used, and improved upon, to perform retrospective studies on other viral pathogens which are implicated in wildlife health where frozen tissues or DNA extracts are available. Identifying which viruses are present in nature is essential for the understanding of the health of a wildlife species. Knowing which viruses exist and circulate naturally in wildlife can help target molecular surveillance efforts.

In Chapter 3, we investigated the prevalence, variance, and coevolution of HV in caribou and marten. Tissue sampling was targeted towards the detection of lymphatic gamma-HV because

159 of their limited host range and ability to remain in asymptomatic latency in an apparently healthy host. Based on the commonly targeted fragment of the viral DPOL gene, Reindeer gamma-HV 1 from Canadian caribou were not different from those in reindeer from Greenland, Alaska, and

Norway. Caribou of a migratory ecotype had in our sample set a higher prevalence than other caribou, but this is skewed by the larger sample size of available archived tissues from migratory populations. Future studies would benefit from screening for HV in archived tissues from geographically or behaviourally isolated caribou and marten to determine if different HV strains are affecting these populations. Virus isolation and full genome sequencing from tissues would provide larger genetic fragments to analyze for evidence of ancestral links among Rangifer tarandus populations in the Holarctic region, or Martes americana populations across Canada, and will broaden the understanding of HV evolution in free-ranging animals.

A major limitation of this study is the varying quality of tissue samples as thoroughly discussed in Chapter 3. Poor sample quality greatly reduced the success of DNA-based diagnostic tools and downstream sequencing efforts. The amplification of host mtDNA suggested that the

DNA sample does not have inherent inhibitors to the PCR. More caribou samples were PCR positive for mtDNA 54/90 (60%) compared to 51/209 (24%) marten. The differences in success between caribou and marten tissues imply differences in primer affinity, however, the primers used herein have been successful in tissues from rodents, carnivores, primates and fish (Kocher et al.,

1989). Marten tissues were generally necrotic tissues that were subject to multiple freeze-thaw events and conveniently sampled after the pelt was removed by the trapper. Caribou tissues, on the other hand, were often samples taken and shipped for scientific research. Like Thomas et al.

(2015), we experienced less sensitivity to viral detection in hunt-harvested tissues compared to diagnostic specimens. This likely relates to variability in time before samples were taken, how

160 tissues were collected and stored, and the duration of transport before arriving at the laboratory.

Branching patterns of host species in taxonomical trees resembled the branching of HV at the species level in viral trees. However, due to the difficulty in producing large numbers of sequences from PCR products (likely a result of low product yield following multiple purification steps), we were unable to adequately interrogate the coevolution of HV and host mtDNA beyond a species level.

It is important to set high standards for the collection of wildlife tissues, comparable to those for the study of livestock or human infectious diseases. Care must be taken to properly preserve wildlife tissues when collected with sequencing and molecular diagnostics in mind.

Future surveillance initiatives should implement the immediate and maintained cold storage of tissue samples to minimize degradation and interference with downstream sequencing and delivering the samples for processing as soon as possible.

Sequences generated from this research may provide the necessary context for the study of other new or emerging pathogens in the future. Additional surveillance is needed to better understand the prevalence and geographic distribution of HV in wild populations. Future studies are needed to clarify the ecology, epidemiology, and pathobiology of Reindeer gamma-HV 1 in caribou and the bouquet of HV in marten.

Part II: Molecular surveillance of orf virus

Orf virus was recently described in a case study of one muskox from 2014 on Victoria

Island, NU, Canada with visible lesions on its mouth, nose, and skin (Tomaselli et al., 2016). Since the primary case study, several muskoxen were discovered dead around the Cambridge Bay and southern region of Victoria Island (Chapter 4). This prompted a molecular investigation into the

161 health of muskoxen in the Canadian Arctic, and the impact of orf virus. In Chapter 4, we screened for orf virus in muskoxen from Victoria Island and mainland NU to establish a baseline of prevalence in our study population and determine the efficacy of these molecular methodologies to detect orf virus in hosts with and without clinical signs. Orf virus was present in male and female muskoxen of all age groups, sourced from different geographical areas (Ulukhaktok in the northwest area of Victoria Island managed by the NT, to southern Victoria Island, NU, and multiple areas along the adjacent mainland NU), and spanning different years of collection.

Muskoxen with clinical lesions were also PCR-positive for orf virus, and in many cases, sequences were successfully produced. While the wide geographic distribution and high prevalence of orf virus indicate that this virus represents a disease threat for muskoxen, there is still limited knowledge on the ecology, epidemiology, and pathobiology of MxOV. Continued surveillance of orf virus in muskoxen will help elucidate if orf virus is endemic to muskoxen at a high rate of sustained infection, or whether this data represents an outbreak currently depicted by clusters of infection. Additionally, future studies should survey for MxOV in wild species that cohabit or come in contact with muskoxen (caribou, mainland moose, elk, bighorn sheep, mountain goat, deer).

We have shown in Chapter 5 that viral genome sequencing of orf virus can be accomplished directly from DNA purified from frozen tissues. These methodologies eliminate the need for the propagation of viral particles in model organisms or cell-lines and avoid an opportunity to adapt to a new host cell environment. The presence of clinical signs in orf-infected muskoxen corresponds to a high amount of virus particles available for genome sequencing using the Illumina

NextSeq platform. Phylogenetic analyses identify the orf virus in these muskoxen to belong to one strain, MxOV, and place the genome of MxOV as a unique strain among other orf viruses in the

162 family Parapoxviridae. Future studies could use these methodologies to sequence the genome of orf virus from archived clinical material to evaluate if MxOV is implicated in any historical outbreaks or case studies.

Recombination analysis in Chapter 5 suggested likely recombination of two major virulence factors (B2L, and VEGF) with NZ2 and OV-IA82, respectively. Additionally, a roughly

3,000 bp segment of MxOV (also OV-IA82 and OV-NZ2) was predicted to be recombinant with

OV-D1701, the orf virus strain currently administered to infected domestic animals as part of outbreak control measures (Rziha et al., 2019). This raises concern regarding the likelihood of recombination between a vaccine and a naturally occurring orf virus strain, or between closely related naturally occurring strains, and emphasizes the need for more molecular surveillance in wild populations.

Molecular surveillance, via sequencing and phylogenetic analysis of genetic material, has proved to be a valuable tool for identifying viral pathogens in wildlife. This research utilized molecular tools to identify HV and orf virus present in wildlife, describe the variation in strains both within and among populations of wildlife and contribute to the database of viral sequences.

Although the field of wildlife health research has grown in interest beyond human cases, more needs to be done to generate molecular data from wildlife surveillance programs. Wildlife health and disease surveillance must be approached with interdisciplinary tools and techniques for the integrated comparison of health and ecological data over time and among geographical regions

(Ryser-Degiorgis, 2013). It is important to set high standards for the collection of wildlife tissue samples so to preserve genetic material for the detection of pathogens. Knowing which pathogens are present in wildlife and likely to be transmitted between wildlife, or to livestock or humans, can help target surveillance more efficiently, and to inform control and management strategies.

163

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