Molecular Characterization and Pathogenicity Studies of Canadian Infectious Laryngotracheitis Virus Isolates

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2021-02-02 Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates

Perez Contreras, Ana Paulina

Perez Contreras, A. P. (2021). Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus isolates (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113067 master thesis

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Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis

virus isolates

Ana Paulina Perez Contreras

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIRMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES

CALGARY, ALBERTA

FEBRUARY, 2021

©Ana Paulina Perez Contreras 2021 ABSTRACT

The extensive use of live-attenuated vaccines to control the upper respiratory tract viral infection in chicken known as infectious laryngotracheitis (ILT), has been associated with a surge in vaccine related ILT outbreaks. It is documented that these ILT outbreaks are due to the regaining of virulence of the vaccine viruses due to multiple bird to bird passages following vaccination.

These vaccine-originated infectious laryngotracheitis virus (ILTV) isolates are known as vaccine revertants. An additional concern is that the multiple live attenuated vaccine ILTV and wild-type

ILTV can recombine, resulting in ILTV strains with higher pathogenicity. To date, little is known about the molecular nature of the Canadian ILTV.

The objectives of the present thesis work are to, 1) molecularly characterize the ILTV associated with ILT outbreaks in poultry flocks in Canada using a whole genome sequence approach and 2) study the pathogenicity of representative ILTV isolates in vivo. In achieving objective 1, It was found that most of the ILTV isolates of Canadian origin used in this study were genetically related to chicken embryo origin (CEO) live attenuated vaccine ILTV strains. Evidence of recombination involving commonly used live attenuated ILT vaccines was also detected in an

ILTV isolate belonging to the British Columbia province. A second recombination event was found this time involving an ILTV isolate belonging to Alberta. This Alberta ILTV strain acted as a parental strain along with another live attenuated ILT vaccine strain to give rise to an ILTV strain previously isolated in United states (US) territory. In objective 2, the pathogenicity of two wild- type and one CEO vaccine revertant ILTV isolates was compared, by infecting specific pathogen free chickens along with age matched mock infected controls. We also used naïve contact chickens in order to determine the transmission potential of these ILTV isolates. It was found that the tested

CEO vaccine revertant ILTV isolate can induce not only severe disease but also to transmit more efficiently than the wild-type ILTV isolates used for this study.

Keywords: Poultry, infectious laryngotracheitis virus, whole genome sequencing, recombination, vaccine revertant, pathogenicity, transmission

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PREFACE

The studies described in this thesis were performed at the Department of Ecosystem and

Public health, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada. The in vivo investigations and laboratory analyses were carried out by me, Ana Paulina Perez Contreras, from

January 2019 to January 2021 under the supervision of Dr. M. Faizal Abdul-Careem. Provost

Chantale propagated and isolated ILTV from three samples originated from Quebec. Mohamed

Sarjoon Abdul-Cader supported me with virus propagation. Catalina Barboza Solis supported me with virus propagation, virus titration and animal experiments. Shahnas Mohamed Najimudeen supported me with processing of swab samples and animal experiments. Mohamed S. H. Hassan supported me with animal experiments. The thesis contains the materials already published or to be submitted for publication elsewhere, which are listed below.

Published Manuscripts

Contreras, P.A.; Van der Meer, F.; Checkley, S.; Joseph, T.; King, R.; Ravi, M.; Peters, D.;

Fonseca, K.; Gagnon, C. A.; Provost, C.; Ojkic, D.; Abdul-Careem, M. F., Analysis of Whole-

Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry Flocks in Canada:

Evidence of Recombination. Viruses, 2020. 12(1302).

Manuscript to be published

Contreras, P. A.; Barboza-Solis, C.; Najimudeen, M. S.; Checkley, S. Van der Meer, F.; Abdul-

Careem, M. F.; Joseph, T.; King, R.; Ravi, M.; Peters, D.; Fonseca, K.; Gagnon, C. A.; Ojkic, D.

Pathogenic and transmission potential of chicken embryo origin (CEO) vaccine revertant infectious laryngotracheitis virus. To be submitted to Viruses.

AKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisor, Dr. M. Faizal Abdul-

Careem, for giving me the opportunity to further develop my skills by allowing me to conduct my thesis research as a member in his lab, under his guidance. I am also thankful to him for providing me with tools necessary to accomplish this milestone, for his enormous patience and continuous mentorship, by which I have learned the importance of critical thinking. Most importantly, for bestowing me with his trust and setting a prime example of what hard work can accomplish. I would like to thank the University of Calgary and the Faculty of Veterinary Medicine for welcoming me into their excellent graduate program and community, which has been my home, away from home for the past two years. I am indebted to my supervisory committee members Dr.

Frank van der Meer and Dr. Kevin Fonseca, for their input on this project which helped shape it to the outcome that it is today.

I would like to thank the funding agencies, Egg farmers of Alberta and Alberta Agriculture and Forestry for providing the funding necessary to carry out this work. I would also like to acknowledge the Agri Food Laboratories, from Alberta Agriculture and Forestry (Edmonton, AB), the Animal Health Center (Abbotsford, BC), and the Laboratoire de Diagnostic Moléculaire, the

University of Montreal (QC), for providing me with the ILTV positive clinical samples or sequences.

I would like to thank all the people whose assistance was a milestone in the completion of this project, Mohammed Sarjoon Abdul-Cader for helping me and sharing his knowledge on cell culture, virus propagation and nucleic acid extraction. Upasama de Silva Senapathi for introducing me to quantitative polymerase chain reaction assay and other techniques which I used extensively

v throughout my thesis research. I thank Victor Palomino-Tapia for sharing his knowledge on chicken primary cell culture and the use of embryonated eggs for viral propagation. I am thankful to Mohamed S.H. Hassan for his assistance on animal experiments. I also thank Catalina Barboza-

Solis for helping with the processing of the original ILTV samples, with cell culture, viral propagation, and titration, as well as with animal experiments. I also thank Shahnas Mohamed

Najimudeen for helping me with the nucleic acid extraction process and animal experiments. I am thankful to Mohammad Mostafa Nazari Zanjani for sharing his knowledge and his assistance in the aspects of my research involving bioinformatics. I whole heartedly appreciate the invaluable assistance I received from my lab members. To them and to Sabrina Buharideen, I wish to thank for their support and most of all, for their unvaluable friendship throughout this period.

I wish to acknowledge Lilian Oribhabo and Bukola Ali, from the Prion/Virology Animal

Facility (PVF) for their assistance on animal care and animal facility management. I would like to express my gratitude to Paul Gajda for his assistance on microscopy and digital imaging for cell culture. I would also like to thank Dr. Grace Kwong for her guidance on statistical analyses of this thesis research data. I wish to show my gratitude to Chantale Provost and Carl Gagnon for handling all the sequencing of the ILTV isolates used in this thesis.

Finally, I wish to show my gratitude to Adrian Sacher and my family, whose love, and unconditional support has encouraged me to fulfill this milestone.

DEDICATION

I dedicate this work to my beloved parents Frida, and Pablo.

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TABLE OF CONTENTS

ABSTRACT ...... ii

PREFACE ...... iv

AKNOWLEDGEMENTS...... v

DEDICATION ...... vii

TABLE OF CONTENTS ...... viii

LIST OF TABLES ...... xiii

LIST OF FIGURES ...... xiv

LIST OF ABBREVIATIONS ...... xvi

CHAPTER ONE: INTRODUCTION ...... 1

1.1. General introduction ...... 1

1.2. ILTV Classification and structure ...... 2

1.2.1. Genome structure ...... 3

1.2.2. Viral replication ...... 4

1.2.3. Hosts ...... 5

1.2.4. Transmission ...... 5

1.2.5. ILTV Survival in the environment ...... 5

1.2.6. Tissue tropism and latency establishment...... 6

1.2.7. Clinical signs and lesions ...... 7

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1.3. Control of ILTV infection ...... 8

1.4. Host immune response ...... 9

1.5. ILTV Evolution ...... 10

1.5.1. Mutation ...... 10

1.5.2. Recombination ...... 11

1.5.2.1. ILTV Superinfection ...... 12

1.6. Bioinformatics approach ...... 13

STATEMENT OF RATIONALE ...... 15

HYPOTHESIS ...... 16

OBJECTIVES AND EXPERIMENTAL APPROACH ...... 17

OBJECTIVE 1 ...... 17

OBJECTIVE 2 ...... 17

CHAPTER TWO: MATERIALS AND METHODS ...... 19

2.1. Eggs and animals ...... 19

2.2. ILTV clinical samples ...... 19

2.3. Experimental design ...... 20

2.3.1. Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to identification of recombination events ...... 20

2.3.1.1. Sample submission for WGS and Genome reconstruction ...... 20

2.3.1.2. Genotyping based on Complete ILTV Genome ...... 22

2.3.1.3. Recombination analysis ...... 22

2.3.2. Determination of pathogenicity and transmission potential of wild-type and CEO vaccine revertant ILTV ...... 23

2.3.2.1. Virus ...... 23

2.3.2.2. Experimental infection and contact exposure ...... 23

2.4. Techniques...... 25

2.4.1. ILTV Propagation on CAM and liver cells...... 25

2.4.2. ILTV Titration ...... 27

2.4.3. Viral DNA purification and ILTV genome quantification ...... 28

2.4.4. Data and statistical analysis ...... 29

CHAPTER THREE: RESULTS ...... 30

3.1 Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to

identification of recombination events ...... 30

3.1.1. Flock background information ...... 30

3.1.2. ILTV Whole-genome sequences...... 30

3.1.3. Phylogenetic analysis ...... 34

3.1.4. Recombination analysis ...... 43

3.2. Determination of pathogenicity and transmission potential of wild-type and CEO vaccine

revertant ILTV ...... 47

3.2.1. Clinical manifestations...... 47

3.2.1.1. Experimentally infected chickens ...... 47

3.2.1.2. Contact exposed chickens ...... 48

3.2.2. Weight gains ...... 49

3.2.2.1. Experimentally infected chickens ...... 49

3.2.2.2. Contact exposed chickens ...... 50

3.2.3. Survival rate ...... 51

3.2.3.1. Experimentally infected chickens ...... 51

3.2.3.2. Contact exposed chickens ...... 51

3.2.4. Viral genome loads ...... 52

3.2.4.1. Oropharyngeal swabs ...... 52

3.2.4.1.1. Experimentally infected chickens ...... 52

3.2.4.1.2. Contact exposed chickens ...... 53

3.2.4.2. Cloacal swabs...... 54

3.2.4.2.1 Experimentally infected chickens ...... 54

3.2.4.2.2. Contact exposed chickens ...... 54

3.2.4.3. Feathers ...... 55

3.2.4.3.1. Experimentally infected chickens ...... 55

3.2.4.3.2. Contact exposed chickens ...... 55

3.2.4.4. Trachea and lungs ...... 56

3.2.4.4.1. Experimentally challenged chickens...... 56

3.2.4.4.2. Contact exposed chickens ...... 57

CHAPTER FOUR: DISCUSSION ...... 59

4.1. Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to

identification of recombination events...... 59

4.2. Determination of pathogenicity and transmission potential of wild-type and CEO vaccine

revertant ILTV...... 62

CHAPTER FIVE: GENERAL DISCUSSION ...... 65

5.1. Implications ...... 65

5.1. Limitations and future directions ...... 66

REFERENCES ...... 69

APPENDICES ...... 84

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LIST OF TABLES

Table 1. Relevant background information of the poultry farms from which the 14 Canadian infectious laryngotracheitis virus (ILTV) samples that yielded full genome sequences originated..

...... 31

Table 2. Canadian ILTV full genome sequences (n = 14)...... 33

Table 3. Single nucleotide polymorphisms (SNPs) of the nine Canadian ILTV sequences from the

Alberta and Quebec provinces using the vaccine strain Serva as a reference sequence ...... 40

Table 4. Recombination signals involving Canadian ILTV isolates ...... 45

Table 5. Percentages of nucleotide identity of vaccines CEO-Poulvac ILT® and TCO-LT-IVAX® with British Columbia ILTV isolate (BC-10-1122) indicated by bootscan...... 46

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LIST OF FIGURES

Figure 1. ILTV structure...... 2

Figure 2. ILTV genome...... 3

Figure 3. Clinical signs of ILTV...... 8

Figure 4. Experimental design for pathogenicity and transmission potential evaluation of the three

Canadian ILTV isolates...... 24

Figure 5. Phylogenetic tree of the full genome sequences of 50 ILTV strains from different geographical regions ...... 35

Figure 6. Phylogenetic analysis of 50 ILTV sequences using the unique long region (a), unique short region (b) and the internal repeat regions (c) ...... 37

Figure 7. Bootscan analysis plot of CAN/BC-10-1122 with the suggested parental vaccine strain

TCO LT-IVAX® (red) and Poulvac ILT® strain (blue)...... 46

Figure 8. Bootscan analysis plot of CAN/AB-S20 with the suggested parental vaccine strain

CAN/AB-S20 (blue) and SA2 (red)...... 47

Figure 9. Clinical manifestations of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates ...... 49

Figure 10. Weight gains of chickens infected with two wild-type and a CEO vaccine revertant

ILTV isolates...... 50

Figure 11. Survival percentage of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates...... 52

Figure 12. The viral genome loads quantified in oropharyngeal swabs of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates...... 53

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Figure 13. The viral genome loads quantified in cloacal swabs of chickens infected with two wild- type and a CEO vaccine revertant ILTV isolates ...... 55

Figure 14. The viral genome loads quantified in feathers of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates...... 56

LIST OF ABREVIATIONS

AB Alberta

ABI Applied Biosystems Inc

ANOVA Analysis of variance

ARC Animal Resource Center

BC British Columbia

BHV Bovine herpesvirus

C Celsius

CAM Chorioallantoic membrane

CELIC Chicken embryo liver cells

CEO Chicken embryo origin

CFIA Canadian Food Inspection Agency cm Centimeter

CO2 Carbon dioxide

DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid

DPBS Dulbecco’s phosphate-buffered saline dpi Days post-infection ds Double stranded

FPV Fowl pox virus g Glycoprotein

GHV Gallid herpesvirus h Hours

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HSACC Health Science Animal Care Committee

HSV Herpes simplex virus

HVEM Herpesvirus entry mediator

HVT Herpesvirus of turkeys

ICP Infected cell polypeptide

IFN Interferon

IL Interleukin

ILTV Infectious laryngotracheitis virus

IR Internal repeat

Kbps Kilobase pairs

LAT Latency-associated transcript

LDM Laboratoire de Diagnostic Moléculaire

LMH Leghorn male hepatocellular carcinoma cell line

MAFFT Multiple alignment using fast furrier transform

µL Microliter min Minutes ml Milliliter nm nano meter n Number

NCBI National Center for Biotechnology Information

ON Ontario

ORF Open reading frame

PBL Peripheral blood leukocytes

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PCR Polymerase chain reaction

PK Proteinase K pmol Picomolar

PRV Pseudorabies virus

PsHV Psittacid herpesvirus

PVF Prion Virology Facility

RDP4 Recombination detection program 4

RNA Ribonucleic acid

QC Quebec

SimPlot Similarity Plotting

SNPs Single nucleotide polymorphisms

SPF Specific pathogen free ss Single stranded

TCID Tissue culture infectious dose

TCO Tissue culture origin

TLRs Toll-like receptors

UL Unique long

US Unique short

USA United States of America vCKBP Viral chemokine binding protein

VZV Varicella zoster virus

WGS Whole-genome sequencing

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CHAPTER ONE: INTRODUCTION

1.1. General introduction Infectious laryngotracheitis virus (ILTV), is the cause of the infectious laryngotracheitis

(ILT), an upper respiratory tract disease in chicken. The disease is characterized by respiratory distress, decrease in egg production and body weight with variable mortality. Although ILT was first described in 1925 in Canadian poultry flocks, it is still a source of significant economic losses for the poultry industry worldwide [1]. Control of ILT relies largely on biosecurity measures and vaccination [2]. Chicken embryo origin (CEO) and tissue culture origin (TCO) live-attenuated vaccines are a commonly used for the control of ILT in endemic areas. Although live attenuated vaccines are known for inducing rapid establishment of host immunity for prolonged periods of time, these vaccines possess a number of limitations. These limitations include the potential of vaccine virus to revert to original virulence and increase in virulence due to recombination with circulating vaccine viruses and the wild-type ILTV strains [2, 3]. The recombination events are reported more often among CEO vaccines [4-6].

ILTV infection is endemic in backyard flocks in Canada as such backyard poultry flocks are a constant source of virus. Vaccination of commercial flocks against ILT is practiced in some provinces and most of commercial chickens are naïve. An outbreak of ILT in naïve chickens may bring devastating consequences. During 2017-2019, ILT outbreaks were recorded in commercial poultry operations in Ontario (ON) and Quebec (QC). Although, there were no ILT outbreaks observed in commercial poultry flocks in Alberta, ILT outbreaks are commonly observed in backyard flocks. We do not know the molecular nature and virulence of the ILTV strains associated with these recent ILT outbreaks. The work described in the thesis focused on 1) molecular characterizing the ILTV strains associated with recent ILT outbreaks in three provinces of Canada,

Alberta (AB), British Columbia (BC) and QC and 2) investigating the pathogenicity and transmission potential of three molecularly different ILTV isolates originated from AB.

1.2. ILTV classification and structure

The ILTV, also known as Gallid herpesvirus-1 (GaHV-1), is a member of the family

Herpesviridae and subfamily alphaherpesvirinae. GaHV-1 is classified along with Psittacid herpesvirus-1 (PsHV1), the causative agent of Pacheco’s parrot disease, within the genus Iltovirus

[7-9]. Although GaHV-1 is the new name for ILTV, still literature describe this virus as ILTV.

ILTV is an enveloped virus with a particle size between 200-350 nano meter (nm). It contains an icosahedral nucleocapsid, covered by a tegument of proteins, enveloped by a lipid bilayer, with glycoprotein projections on its surface [10, 11]. Within the nucleocapsid, the virus has a linear double-stranded (ds) deoxyribonucleic acid (DNA) genome (Figure 1) of an approximate length of 155 kilobase pairs (kbps) [12].

Figure 1. ILTV structure. Image created with BioRender.com

1.2.1. Genome structure

The genome is comprised of a unique long (UL) region and a unique short (US) region, which is flanked by two inverted repeat regions, an internal repeat (IR) region and a terminal repeat region (Figure 1). The ILTV genome contains 77 open reading frames (ORFs) coding for proteins, from which 63 are homologous to those of the herpes simplex virus (HSV)-1 [10], including 12 glycoproteins (g) (K, N, H, B, C, M, L, G, J, D, I, E) conserved amongst the alphaherpesviruses

[13]. It has 6 Iltovirus genus-specific genes (ORF A-E, UL (-1)), and one, UL 0 is only found in the ILTV genome. It contains 3 origins of replication, from which, one is located in the UL region, and the other two, in the IR regions [10, 12, 14].

Figure 2. ILTV genome. Adapted from:[10]. Image created with BioRender.com

Of the genes, 62 genes are located in the UL region, and 9 within the US region, where the gG, gD, gI and gE are located [15]. The US region also contains a homolog to the UL47 gene, that like in other herpesviruses encodes for a major tegument protein, but is usually located in the UL region of other herpesviruses [10, 16]. The IR regions hold two copies of the infected cell polypeptide (ICP4) gene, the US10 and the sORF 4/3 [9]. Deletion of UL0, UL23, UL47, UL50,

US5 and US4, has been associated with attenuation of the virus in vitro, and in vivo [10].

1.2.2. Viral replication

Although, little information is known about the specific mechanisms of ILTV cell entry and replication, it is inferred to be similar to that of other alphaherpesviruses. In in vitro studies,

ILTV replication was found to start 8-12 hours (h) post-infection. Expression of a set of genes in a cascade pattern was also found to occur like in other herpesviruses [17].

For most herpesviruses, viral entry is initiated by attachment of the virions to heparan sulfate on the cell surface [11, 18]. Five conserved glycoproteins amongst the herpesviruses, gC, gD, gB, gH, gL are crucial for viral entry, as they play key roles in the process. The entry process is divided in 3 phases. Phase I is characterized by the pairing of both, cell, and viral membranes.

In phase II, there is a mix of membranes and the formation of a hemifusion intermediate. Finally, in phase III, there is formation of a fusion pore to finalize the process [19]. During fusion of the membranes, a requirement for successful viral entry will be the interaction of gD with cell receptors such as herpesvirus entry mediator (HVEM), nectin-1 and -2, or 3-O sulfated heparan sulfate. Where HVEM has been found to be expressed by leukocytes, epithelial cells, and fibroblasts. As for nectins, they are expressed by cells such as fibroblasts, epithelial cells, and neurons. The receptor required will vary depending on the virus and serotype [11, 18]. For ILTV, attachment to the cell surface has been found to be independent of heparan sulfate proteoglycans.

This has been attributed to a 100 amino acid deletion in the first third of gC, and the lack of a heparin-binding amino acid motif in gB [13, 20, 21]. Following phase III of viral entry, the tegument proteins and nucleocapsid are released into the cell cytoplasm [18]. Aided by the cellular microtubules, the nucleocapsid will be transported through the cytosol towards the nuclear pores.

Once the viral DNA is released into the nucleus, transcription, replication, formation of capsids and packaging of DNA, will take place [22].

The process of nuclear egress involves the conserved proteins product of UL31 and UL34 genes and starts with budding and envelopment of the nucleocapsid with the inner leaflet of the nuclear membrane [23]. By fusing with the nuclear membrane, and after losing the primary envelope, the intracytoplasmic capsids will gain a secondary envelopment from the membranes of the trans-Golgi network [24].

1.2.3. Hosts

Infection of ILTV is mostly limited to galliform birds [12]. However occasional infection on pheasants and peafowls has also been reported [25, 26].

1.2.4. Transmission

Transmission of ILTV is facilitated by direct and indirect contact of chickens with ILT and carriers without clinical manifestations [27]. ILTV is mostly shed through aerosolized respiratory secretions and through cloacal route in faeces [28, 29]. Spread of the disease may occur with secretions or droplets of infected birds having contact with the respiratory tract, oral cavity, or eyes of naïve birds [28, 30]. However, it can also be transmitted through contact with vectors such as dust, domestic and wild species of animals, beetles, and fomites [29, 31, 32].

1.2.5 ILTV Survival in the environment

The ILTV can be recovered from swabs left at room temperature, at 0°C and at 5°C for a period of 3-14 days [33]. In chicken carcasses, the virus can remain viable for a period of 3 weeks at room temperature. In chorioallantoic membrane (CAM) and tracheal exudates not exposed to direct light, the virus can persist for up to 3 months [33]. Detectable amounts of ILTV can be found

5 in contaminated litter for a period of 3-20 days [33]. ILTV is sensitive to heat, and it can be inactivated rapidly at 55°C, when exposed for 15 minutes or at 38°C for a period of 48 h. Heating of ILTV contaminated litter at temperatures of 38 °C for a period of 24 h, or 5 days for in house composting, have been found successful in reducing ILTV as indicated by ILTV negative polymerases chain reaction (PCR) results [34]. Sodium bisulfate and hydrogen peroxide have been found to effectively inactivate ILTV from litter [35] and the use of other commercial disinfectants such as benzalkonium chlorides and 2,4-dichloro-meta-xylenol using the manufacturers recommendations has also been found effective for ILTV inactivation [36].

1.2.6. Tissue tropism and latency establishment

The epithelial cells of the larynx, trachea and conjunctiva constitute the main site of viral replication. However, the presence of ILTV has also been detected in other organs such as lungs, harderian gland, esophagus, thymus, pancreas, kidney, caecal tonsils, and cecum [29, 37]. Very small quantities of ILTV have also been detected on feather shafts of infected chicken [38-40].

The presence of ILTV in different organs could suggest migration of the virus via infected immune cells [41, 42], or a broad distribution of permissive cells with essential receptors for ILTV entry

[37].

A key characteristic of herpesviruses is the establishment of lifelong infections through latency in their infected hosts. For ILTV, latency is established in the trachea and in the trigeminal ganglia [28, 43, 44], part of the somatosensory system located in the head of the chicken, in charge of providing innervation to the beak and tongue [45]. The establishment of latency has been detected as soon as 2 days post-infection (dpi) in experimentally infected chicken [41], but in natural occurring infections, can be regularly established after 5 dpi, coinciding with the

6 development of the adaptive immune response [46]. Reactivation from latency to an active replication state can occur intermittently when the bird is subject to periods of stress, coinciding with events such as onset of laying, relocation, introduction of new birds to the flock, and densely populated housing environments [47, 48].

1.2.7. Clinical signs and lesions

The ILT can be manifested as a mild disease or severe disease [38]. In mild presentations of the disease, the clinical signs will include watery eyes, nasal discharge, and mild inflammation of infraorbital sinuses. Severe cases will be characterized by depression, conjunctivitis, respiratory rales, and dyspnea with desquamation of the tracheal epithelium with expectoration of bloody mucous (Figure 2a-c). There can also be presence of fibrinous exudate buildup in the tracheal lumen [26].

Lesions in the tracheal mucosa can be present as soon as 1 dpi and will include hyperplasia and hypertrophy of the goblet cells, destruction of the ciliated epithelium and infiltration of lymphocytes [49]. With the peak of viral replication, severe changes to the mucosal epithelium, manifested with necrosis of the epithelial cells with the formation and desquamation of syncytia will be observed at histopathological examination. Severe congestion and edema with thickening of the tracheal mucosa will also be present (Figure 2d) with an increased infiltration of macrophages, lymphocytes, heterophils and histiocytes, along with hemorrhage and abundant fibrinous exudate in the tracheal lumen [49].

Figure 3. Clinical signs of ILTV. a) Dyspnea manifested by extended neck breathing. b) Open beak respiration. c)

Conjunctivitis. d) Inflammation of larynx and trachea hemorrhage.

1.3. Control of ILTV infection

Control of ILT relies largely on biosecurity and vaccination [2]. Due to the very contagious

nature of ILTV infection, biosecurity has been the choice of prevention of ILT in commercial

flocks in Alberta.

Commonly used and available vaccines for ILTV control are recombinant, with turkey

herpesvirus (HVT) or fowl pox virus (FPV) as vectors for ILTV genes, as well as live attenuated

vaccines. Based on the method used for its attenuation, ILT live attenuated vaccines can be

categorized into either CEO (attenuated through sequential passage on chicken embryonated eggs),

or TCO (attenuated through sequential passage on tissue culture) [3]. Live attenuated vaccines are

usually preferred over recombinant vaccines, given the fact that they provide a much wider

protection span and due to the induction of stronger cellular immunity [50]. The vaccine-mediated

protection can last from a few months up to a year in the case of CEO vaccines whereas for TCO

vaccines, the induced immunity is not as long lasting as that given by the CEO vaccines but can

still help reduce viral shedding when administered in the face of an outbreak [51]. Aside from the

long-lasting protection of live attenuated vaccines, the route of administration also different for

8 live attenuated and recombinant vaccines. For example, live attenuated vaccines are available for administration via eye drop or in drinking water whereas, recombinant vaccines require subcutaneous administration at 1 day of age of the birds or in ovo application at the hatchery, requiring either trained personnel for vaccine administration or special machinery. As such, the use of recombinant vaccines is not accessible for those producers who own small backyard poultry productions. However, live attenuated vaccine virus still possess the potential to establish a state of latency in the vaccinated birds and gain virulence when reactivated and passage through the flock [5], and have some residual virulence (cause vaccinal ILT) [4].

1.4. Host immune response

During ILTV infection, both innate and adaptive immune responses are activated. Innate immune response at the primary site of viral infection plays a crucial role as a defense mechanism against ILTV infections. Cells belonging to the innate immune system as well as epithelial cells of the mucosa recognize ILTV DNA through Toll-like receptors (TLRs) such as TLR1, TLR2,

TLR6 and TLR21. An antiviral response is subsequently triggered through a signaling cascade of cytokines and chemokines [52]. At 3 dpi, and coinciding with the peak of viral replication, cytokines such as chCXCli2, interleukin (IL)-1β, interferon (IFN-α) and IL-10 have been detected in in vivo studies, at primary sites of infection. With chCXCli2 and IL-1β involved in the recruitment of inflammatory cell to the affected organs. Expression of the formerly mentioned cytokines, along with IL-13 and IFN-γ were found to peak at 5 dpi, coinciding with a phase of acute damage on tracheal epithelium. Where IL-10 plays a crucial immunomodulatory role by downregulating inflammatory response, aiding for the repair to the damaged tissue, result of the inflammatory response [53, 54].

The experimental evidence suggests that the cell-mediated immune response is important over antibody-mediated immune response against ILTV infection. This has been shown using surgically and chemically bursectomized chickens, and adoptive cell transfer of peripheral blood leukocytes (PBL) and spleen cells of immune and hyperimmune chickens, followed by experimental ILTV infection. In these studies, it has been shown that the absence of neutralizing antibodies, did not restrict the ability of the chicken to successfully surpass an ILTV infection [55,

56].

Amongst the ILTV immunogenic proteins, gB, gC, gD, gG, gH, gI, gJ, gK, gL and gM, gG stands out as a viral chemokine binding protein (vCKBP) of several alpha herpesviruses, and as a mechanism for immune evasion [10]. However, ILTV gG blocks this signaling cascade by binding to some of these chemokines and inhibiting their activity, hindering cell recruitment at the site of infection [57]. Experiments carried out on gG deleted ILTV mutants, have found that absence of gG has a significant effect on the attenuation of the virus [58].

1.5. ILTV evolution

Different mechanisms are employed by viruses to maintain viral fitness and this is also applicable to ILTV. Relevant to evolution of ILTV, mutation and recombination are common mechanisms involved.

1.5.1. Mutation

Genetic variation comes as a requirement for evolution, and one way to achieve this is through mutation [59]. Being a very well exploited strategy of viruses, and seen more frequently in those with ribonucleic acid (RNA) genomes, reflected on the elevated nucleotide substitution

10 rates, that are 106 times higher than that in DNA viruses [60]. In agreement with the latter statement, the neutral theory states that constraint against nucleotide changes will reduce the evolutionary rate of nucleotide substitutions, which is seen mostly in DNA viruses [60]. The weight of the instability factor tied to the single stranded genomes cannot be denied [61, 62]. Like is the case for some small single stranded (ss)DNA viruses with high nucleotide substitution rate comparable to that of RNA viruses. This theory points towards the ssDNA template affecting the efficiency of the polymerases used. Proofreading mechanisms might not be as effective in these single stranded genomes, and due to the lack of a double helix, they could become more vulnerable to deamination, ultimately leading to mutations [63].

Although ILTV has a dsDNA genome with efficient proof reading capability similar to other herpesviruses [64], occurrence of mutations has been recorded [65].

1.5.2. Recombination

This mechanism is used by viruses as an important tool that allows for the elimination of deleterious mutations off their genome. It plays an important role in the evolutionary process since the outcome of recombination is genome diversification [66]. Recombination is frequently used by DNA viruses and requires host or viral recombinases. Due to their low nucleotide substitution rate, recombination, particularly homologous recombination, comes as a useful mechanism for the evolution of DNA viruses [67], and relies on double stranded break repair systems, which is not exclusive to dsDNA viruses [66]. This process occurs by pairing of related DNA sequences of different viral strains during their replication. Their viral progeny will include genetic segments of each of the parental viral strains [68]. The genome fragment exchange that occurs during the recombination event can give rise to new variants of viruses with very different characteristics

11 than the strains that originated these new viruses such as, increased pathogenicity, different target cell affinity, drug resistance, and immune identity [69].

Recombination between DNA viruses and live attenuated vaccine viruses has been well documented. There has been increasing evidence on the occurrence of these events, frequently between wild-type alpha herpesviruses and vaccine viruses, with reports of an increase in pathogenicity as a product of this recombination with live attenuated vaccines. Such events have been reported in the case of HSV-1 [70, 71], HSV-2 [68, 72], bovine alpha herpesvirus (BHV)-1

[67], varicella zoster virus (VZV) [73], GHV- 2 or Marek’s disease virus [74] and more recently in GaHV-1 or ILTV [69, 75]. Recombination in ILTV with increased pathogenicity has been recorded in Australia [76], China [77], Korea [78] and USA [1].

1.5.2.1 ILTV superinfection

Recombination is feasible when two homologous viruses coinfect a single cell [74, 79, 80].

This event is also known as superinfection. However, for some alpha herpesviruses, infection of a single cell with two viruses, with long er periods of time between infections, is not restrictive for recombination to occur. Such is the case of ILTV, where the in vitro and in vivo asynchronous infection with multiple viruses with up to 4 days difference between infections, still resulted in recombinant progeny. Which is suggestive of low efficiency superinfection inhibition (exclusion) mechanisms for ILTV infection [75, 81]. The specific mechanisms involved in superinfection inhibition of herpesviruses are not yet fully understood. The expression of gD protein in the infected cells membrane was correlated with resistance to infection with other HSV in a study done in vitro [82]. Further studies have found that during HSV-1 latency, the latency-associated transcript (LAT) gene, prevents the latently infected neurons of superinfection with other HSV-1,

12 however, it does not prevent from infection with other RNA or DNA viruses [83]. On recent studies, using HSV-1 and pseudorabies virus (PRV), superinfection inhibition was detected at 2 h post-infection, and was characterized by early protein expression of cellular and viral transcripts, discarding the gD expression dependant mechanism, that was theorized occurred between 4-6 h post-infection. It was also suggested that ICP0, ICP4, ICP22 and ICP27 could be involved in this mechanism [84].

1.6. Bioinformatics approach

Bioinformatics is an interdisciplinary field that encompasses mathematics, computer, physics, and biological sciences to efficiently process and analyze biological data. It is not limited to genomics, instead, it incorporates proteomics and transcriptomics to its field of knowledge [85].

The use of bioinformatics in the virology field holds many advantages, as it can allow for a deeper understanding of the viral genome and its interactions with its host, along with molecular epidemiology, genotyping, and viral genome evolution, through next generation sequencing [86-

88]. Which, amongst other things, can be focused towards the identification of mutations linked with drug resistance, quasispecies or virulence factors [87].

The use of bioinformatics provides valuable tools that can help answer questions related to viral evolutionary dynamics, through detection of recombination events on viral sequences [86].

A prime example is SimPlot software, a bioinformatic tool developed to carry out similarity plotting of sequences believed to be involved in recombination events. Where the detection of recombination signals can be found by identification of distinguishable sequence similarities between a query and the parental sequences. Similarity and its shifts are calculated using a sliding window of adjustable length (base pairs) that scans the alignment and reflects the similarity of the

13 query sequence to the reference sequences in the plot [89]. The software also allows for the detection of approximate recombination crossover points in the query sequence.

Inference about the viral genome evolution and epidemiological dynamics can be achieved through phylogenetic analysis [90]. Specifically recombination, can be inferred by discrepancies in the topology of the phylogenetic trees of different regions of a given genome, suggestive of a different evolutionary origin for such regions, which could be given by recombination [89]. Boot scanning is a useful bioinformatic tool to examine these phylogenetic discrepancies. In which, using a sliding window of different length (base pair) that will scan a sequence alignment, a plot will reflect the proportion of bootstrapping phylogenies (support), in which the query sequence clustered with the references [89]. Crossover points can also be identified by the later method.

STATEMENT OF RATIONALE

ILTV infection is endemic in backyard poultry flocks in Canada, and sporadic ILT outbreaks are commonly observed in commercial flocks [26]. Reactivation of latent ILTV in these birds is possible during episodes of stress, providing a source of virus for naïve chickens [48]. The vaccines such as CEO and TCO, as well as recombinant vaccines, are currently available in

Canada. For more than 10 years, backyard flock owners were urged to vaccinate their chickens with TCO vaccines in some of the provinces, and it is possible that backyard flocks vaccinated with live attenuated vaccines could be infected with wild-type ILTV. Such situations may facilitate co-circulation of two different ILTV strains and super infection, which could potentially enable recombination, as has been seen in other countries [76, 78, 91, 92]. Few studies have addressed the molecular nature of ILTV circulating in Canada using either complete or partial genes of the

ILTV genome (UL 47, US 8, ORF a and ORF b) [93, 94]. These studies have been successful in differentiating between wild-type and vaccine-related ILTV strains and have reported the presence of both CEO vaccine virus and wild-type ILTV in the Canadian chicken flocks [93, 94]. However, to this date, there are neither whole-genome sequences of Canadian-origin ILTV strains nor any available information to suggest that ILTV recombination has ever occurred in Canada. Whether

ILTV isolates of Canada are higher in transmission potential and virulence that might lead to severe clinical manifestations is also unknown.

HYPOTHESIS

I hypothesized that ILTV strains which originated from diagnostic samples in the past 8 years in Canada are genetically closer to ILT live attenuated vaccine strains than to the wildtype

ILTV strains. I further hypothesized that ILTV Canadian CEO vaccine revertant isolate CAN/AB-

S45, obtained from diagnostic samples from AB will be of higher pathogenicity than wild-type

ILTV isolates, CAN/AB-S20, CAN/AB-S63.

OBJECTIVES AND EXPERIMENTAL APPROACH

Objective 1

To genetically characterize the ILTV isolates obtained from ILTV clinical cases in AB, QC and

BC through whole-genome sequencing (WGS) as well as to provide evidence for recombination events if present in these isolates

Key steps involved:

• Propagation of ILTV positive clinical samples in embryonated eggs and primary liver cell

culture for WGS

• Establishment of ILTV whole genome sequence library including new ILTV sequences of

Canadian origin and published ILTV sequences of different geographic backgrounds

including TCO and CEO vaccine sequences

• Multiple sequences alignment using Geneious software

• Phylogenetic analysis with multiple sequences alignment using Geneious software

• Recombination analysis of multiple sequences alignment of three different regions of the

ILTV genome using RDP4 software

• Bootscan analysis in groups of 4 ILTV sequences

Objective 2

To determine the pathogenicity and transmission potential of three ILTV isolates of AB origin, two wild-type viruses, and one CEO vaccine revertant

Key steps involved:

• Four groups of 8, 3-week-old chickens will be infected with, wild-type CAN/AB-S20,

wild-type CAN/AB-S63 and CEO vaccine revertant CAN/AB-S45 at a dose of 103.5

TCID50, via intratracheal and intraocular maintaining a mock infected control group

• Three groups of 3 naïve chickens (3-week-old), introduced into the isolators and allowed

to get contact with the experimentally infected chickens for a period of 3 days

• Monitoring of disease progression for 14 dpi. The naïve contact chickens were observed

for 11 days after exposure to experimentally infected chickens

• Observation of clinical signs, bodyweight, and mortality

• Collection of oropharyngeal and cloacal swabs and feathers at predetermined time points

• At necropsy, gross lesions were observed, and lung and trachea were collected to quantify

viral load quantification

CHAPTER TWO: MATERIALS AND METHODS

2.1. Eggs and animals

The proposed work involved the use of specific pathogen free (SPF) white leghorn eggs and chicken purchased from Canadian Food Inspection Agency (CFIA), Ottawa, Ontario, Canada.

The work was approved by Health Science Animal Care Committee (HSACC) of the University of Calgary (Protocol number: AC19-0013).

2.2. ILTV clinical samples

Quantitative (q)PCR assays targeting the ICP4 (primers, F: 5′-

GGGTCGGTTCAGTCAGTAA-3′; R: 5′-GGTCATCGACCAAAGACTGT-3′ and probe 5′-

GAGGTCGACGGCCAACGC-FL-3′) and gG genes (primers, F: 5′ -

ATTGCCACCGTTTCCCTAG-3′; R: 5′-CCATTTCACCTCGACTGACACT-3′ and probe, 5’-

CAACCGCACCACGATTGAGG-FL-3′) have been used to quantify ILTV genome in nucleic acids of tracheal swabs and tissues (n = 46) originated from backyard flocks of AB. A qPCR assay targeting ILTV thymidine kinase gene (primers, F: 5′- CGA GAA CGA TGA CTC CGA CTT -3′;

R: 5′- GGC CCG TCG ACG TAA AGA -3′ and TaqMan probe, 5′-6-FAM-CGC CGC GTT GTA

C-MGBNFQ-3′) has been used to detect ILTV genome in clinical samples (n = 9) originated from commercial poultry operations in BC. The qPCR reactions have been carried out using LightCycler

FastStart DNA Master Hybridization Probes Kit (Roche Diagnostics, Laval, QC, Canada) or One-

Step reverse transcriptase (RT)–PCR Master Mix (Life Technologies Inc, Austin, TX, USA) according to manufacturer’s recommendation and run using LightCycler thermocycler (Roche

Diagnostics GmbH, Mannheim, State of Baden-Wurttemberg, Germany) or an Applied

Biosystems Inc (ABI) 7500 Fast Real-Time PCR unit (ABI, Foster City, CA, USA). The

19 amplification conditions consisted of initial denaturation of 50 °C for 2 minutes (min) followed by denaturation of 95 °C for 10 min, then 40 cycles of denaturation of 95 °C for 15 s and annealing at 60 °C for 1 min. A qPCR assay targeting gC gene (primers, F: 5′-

CCTTGCGTTTGAATTTTTCTGT-3’; R: 5′-TTCGTGGGTTAGAGGTCTGT-3′ and TaqMan probe, 5′-6-FAM-CAGCTCGGTGACCCCATTCTA-BHQ1-3′) has been used to screen clinical samples (n = 3) originated in QC commercial operations as has been described previously [95].

These ILTV-positive samples (n = 58) were obtained from the Agri Food Laboratories, Alberta

Agriculture and Forestry in Edmonton, AB, the Animal Health Center in Abbotsford, BC and the

Laboratoire de Diagnostic Moléculaire (LDM), University of Montreal, in St-Hyacinthe, QC, respectively. In order to get enough viral DNA for WGS, the ILTV samples were propagated in either CAM of 9- to 11-day-old SPF chicken embryos or monolayers of hepatocellular carcinoma cell lines (LMH) to increase virus titer. Only 14 of the samples were successfully propagated and each sample required between one to three passages before submission for WGS.

2.3. Experimental design

2.3.1. Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to identification of recombination events

2.3.1.1. Sample submission for WGS and genome reconstruction

The samples with high ILTV genomic content were directly submitted for WGS (LDM,

Faculty of Veterinary Medicine, University of Montreal, QC, Canada) and sequenced using the

MiSeq platform (Illumina Corp, San Diego, CA, USA). To remove Illumina adaptors, automatic adaptor trimming was selected on the MiSeq spreadsheet following CLC Genomic Workbench

(QIAGEN, Redwood, CA, USA). Right before de novo assembly, another step of trimming was

20 performed with the following settings: minus strand only (3’ end trimming); allow internal and end matches with a minimum internal score of 10 and a minimum end score at 4 and alignment scores cost (Mismatch cost at 2 and Gap cost at 3).

For quality filter, settings were set at Q30 reads. Further, using CLC Genomic Workbench

Quality Trimming, with the Trim using quality scores with the limit set at 0.05, the Trim ambiguous nucleotides with a maximum number of ambiguities set at 2 and the discard reads below 15 selected.

For genome reconstruction, all reads were mapped to a complete genome ILTV GenBank reference (Appendix, Table A1) with the Map Reads to Reference tool in CLC Genomic

Workbench, with no reference masking selected, match score at 1, mismatch cost at 2, linear gap cost selected, autodetect paired distance selected, and the map randomly selected in the nonspecific match handling box.

The sequence with the most and longest reads mapped against was used as the reference to perform the mapping of all reads with the same setting as described previously. A second mapping was then used to obtain the consensus sequence with the Extract Consensus Sequence tool with the options as following: low coverage definition threshold set at 5, insert N ambiguity symbols selected, in the conflict resolution vote selected, and the use quality score checked.

Consensus sequences with the Extract Consensus Sequence tool with the options were set as follows: Low coverage definition Threshold set at 5, Insert N ambiguity symbols selected, in the

Conflict resolution vote selected, and the Use quality score checked. Thus, the threshold was set at 5 (Appendix, Table A1).

2.3.1.2. Genotyping based on complete ILTV genome

For phylogenetic analysis, the obtained sequences were aligned with ILTV sequences available in the National Center for Biotechnology Information (NCBI) database. Sequences known to be a product of experimental studies on recombination were not deemed necessary to fulfill the ends of this research and hence, were not included in this study. The sequences included in this analysis mostly comprised of field strains and live attenuated CEO and TCO vaccine sequences from different geographic backgrounds (Appendix, Table A2). Multiple Sequence

Alignment was done with Fast Fourier Transform (MAFFT v7.450 [96]) in Geneious® v10.2.6

[97]. Sequences and alignments were visually inspected. Sites where ambiguous nucleotides were present in the AB ILTV sequences were replaced by the consensus sequence only in sites with no nucleotide variability in the consensus. Next, a phylogenetic tree of the complete sequence alignment was generated using a Bayesian inference method using MrBayes 3.2.6 [98] with default settings. It should also be mentioned that phylogenetic analysis using maximum likelihood resulted in very similar inferences as the Bayesian inference method. Additional phylogenetic trees were constructed using the UL, US, and IR regions of the ILTV genome of the 50 sequences to gather key information on the presence of recombination in any of the sequences. Posterior probability values (given as decimals) indicating the support for any given branch are displayed as branches labels in each of the phylogenetic trees.

2.3.1.3. Recombination analysis

Recombination analysis and detection of crossover points in the 50 aligned genome sequences were conducted in the Recombination Detection Program (RDP4 v.4.80) [99] using default settings. Recombination events suggested by RDP4 software were confirmed by using

Similarity Plotting (Simplot, 3.5.1, SCRoftware, Baltimore, MD, USA) [100], with a window size of 6000 and a step size of 200.

2.3.2. Determination of pathogenicity and transmission potential of wild-type and CEO vaccine revertant ILTV

2.3.2.1. Virus

The ILTV used experimentally were isolated from backyard chicken flocks raised in

Alberta, Canada and affected with ILT. ILT samples (Oropharyngeal swabs and trachea) positive for ILTV genome by PCR assays and corresponding to wild-type and CEO revertant viruses [101], were obtained from the Agri Food Laboratories (Alberta Agriculture and Forestry in Edmonton,

AB). The relevant history of the affected flocks has been described elsewhere [101]. In this study, we used two wild-type ILTV isolates (AB-S20 and AB-S63) and one CEO vaccine revertant ILTV isolate (AB-S45). In order to obtain adequate viral stocks to conduct experimental studies, the

ILTV sample AB-S63 was propagated in CAM of 9 to 11 day-old SPF chicken embryos and isolates AB-S20 and AB-S45 were propagated in monolayers of chicken embryo liver cells

(CELIC).

2.3.2.2. Experimental infection and contact exposure

Three groups of three weeks old chickens (n=8 per group) were infected via combination of intratracheal and intraocular routes with one of the three ILTV isolates, wild-type CAN/AB-

3.5 S20, wild-type CAN/AB-S63 and CEO vaccine revertant CAN/AB-S45, at a dose of 10 TCID50, along with an uninfected control chicken group (n=8). The infected chickens were kept in high containment poultry isolators (Plas Labs Inc., Lansing, MI, USA) at the Prion Virology Facility

(PVF), Foothills Campus, University of Calgary. Three dpi, 3 naïve contact chickens were introduced to each of the isolators exposing them to the experimentally infected chicken and kept there for 3 days. After 3 days of exposure, the contact chickens were placed in separate poultry isolators. Food and water were provided ad libitum. The uninfected control chickens were maintained at the Animal Resource Center (ARC) at Foothills Campus, University of Calgary.

Figure 4. Experimental design for pathogenicity and transmission potential evaluation of the three

Canadian ILTV isolates. Image created with BioRender.com.

Following infection, the chickens were observed for clinical manifestations (Appendix, Table

A3). The observed clinical signs included ruffled feathers, droopy wings, conjunctivitis, depression, and respiratory signs (ranged from mild increase in respiration to severe dyspnea).

Clinical signs were assigned a value between 1-4, depending on their severity. Being 1 the value

24 assigned to mild clinical signs, and 4, the highest value for severe clinical sign presentation.

Clinical signs were monitored twice a day daily, and three times a day on the peak of clinical sign manifestation. The experimentally infected chickens were monitored for a period of 14 dpi and the contact exposed chickens were monitored for 11 days following exposure to the experimentally infected chickens. At 3, 7, 10, and 14 dpi of the experimentally infected chickens, and 3, 7 and 11 dpi of the contact exposed chickens, cloacal and oropharyngeal swabs were taken using sterile polyester swabs in universal transport medium (Puritan, Guilford, Maine, US). After collection, swabs were stored at -80°C until processing. At the same time points, bodyweights were recorded, and three flight feathers were pulled to collect tip of the feathers in RNA Save (BI, Cromwell, CT,

USA) for viral load quantification. At 14 dpi of the experimentally infected chickens, and 10 dpi of the contact chickens, the animals were euthanized. Post-mortem examinations were done to record gross lesions. Trachea and lung samples were collected in 10% formal saline for histopathology examination. In addition, oropharyngeal, cloacal swabs and feather tips were collected in RNASave for ILTV genome load quantification.

2.4 Techniques

2.4.1. ILTV Propagation on CAM and liver cells

The ILTV isolates were propagated on the CAM of SPF eggs on embryo day 10. Briefly, on viable embryonated eggs, the air cell was located by candling. A small mark was placed in the eggshell 0.5-centimeter (cm) right below the bottom edge of the air cell. Carefully, while holding the previously disinfected egg in a horizontal position, and with the previously mentioned mark looking upwards, a puncture was made in the eggshell above the mark using a 25-gauge needle without introducing the needle too deep to avoid damaging the embryo. Another puncture was

25 made in the shell at the top of the air cell while still maintaining the egg in a horizontal position.

Using a rubber bulb covering the orifice made in the air cell, air was taken out of the air cell to create an artificial air cell on the first puncture via negative pressure [102]. The inoculum was then deposited in the artificial air cell and 5 days post-inoculation the CAM is collected from the eggs.

CAMs were placed in 2 milliliter (mL) tubes containing sterile zirconium beads (Benchmark

Scientific, NJ, USA), 1 mL of Dulbecco’s phosphate-buffered saline (DPBS) with 1% of penicillin–streptomycin (Gibco Life Technologies, Burlington, ON, Canada). Shortly after, the

CAMs were homogenized using a microtube homogenizer Bedbug d1030 by Benchmark

Scientific. The supernatant was collected, aliquoted and kept at −80 °C for further use.

For propagation of the ILTV isolates on LMH cell monolayers, T-75 tissue culture flasks

(Greiner Bio One, Kremsmünster, Austria) precoated with 2% gelatin and containing a confluent monolayer of LMH cells were used. Each flask contained 15 mL of Dulbecco’s Modified Eagle

Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco Life

Technologies, Burlington, ON, Canada). The monolayer of LMH cells was inoculated with the

ILTV Canadian isolates and incubated at 37 °C in 5% carbon dioxide (CO2) for 72 h. After the incubation period, flasks were submitted to three freeze- (−80 °C) and thaw- (37 °C) cycles of 30 min each, after which cells and supernatant were collected and taken for ultracentrifugation on a high-speed centrifuge Avanti J-26 (Beckman Coulter Life Sciences, IN, USA) at 50,000× g for three hours. After ultracentrifugation, the supernatant was discarded, and the cell pellets were resuspended in 500 microliters (µL) of DMEM and aliquoted in small vials and stored at −80 °C until further use.

For propagation of the ILTV isolates on CELIC, the livers of chicken embryos from SPF on embryo day 14 were extracted. For the preparation of CELIC, embryo livers were minced thoroughly using

26 sterile scissors on a sterile beaker. Minced livers were rinsed three times with enough DPBS (Gibco

Life Technologies, Burlington, ON, Canada) to cover the livers completely. Afterwards, remaining

DPBS and minced livers were placed in a trypsinization flask (Ace Glass Inc, Vineland, NJ, US) with Trypsin-EDTA solution at a ratio 1:1 and incubated at 37°C on a magnetic stirrer at low speed for 15 minutes. Then, the trypsin-EDTA solution was filtered using a sterile cheesecloth (American

Fiber & Finishing Inc., Albemarle, NC, USA) filter over a sterile beaker. DMEM containing 2% calf serum and 2% penicillin/streptomycin (Gibco Life Technologies, Burlington, ON, Canada) was then added to the filtered cell solution at a 1:1 ratio. The cell suspension was then placed into a 50 ml tube

(Froggabio Inc., Toronto, ON, Canada) and centrifuged at 200xg for 10 minutes. The supernatant was discarded, the cell pellet was resuspended in growth media after the corresponding cell count

(2.1x106) and finally seeded into T-75 tissue culture flasks (Greiner Bio One, Kremsmünster,

Austria). The CELIC monolayers were inoculated with the ILTV isolates and incubated at 37°C in

5% CO2 for five days or until the cytopathic effects were evident on an 80% of the CELIC monolayer.

For virus harvesting, the flasks were set to three freeze (-80°C) and thaw (37°C) cycles of 30 minutes each. By the end of the cycles, cells and supernatant were collected using a cell scraper (Corning,

Corning, NY, USA) and aliquoted in small vials and stored at -80°C until further use.

2.4.2. ILTV titration

The CELICs were cultured in 96 well plates (U bottom) (0.01 x 106 cells per well) using

DMEM containing 2% calf serum and 2% penicillin/streptomycin (Gibco Life Technologies,

Burlington, ON, Canada). When the cells were 80% confluent, the propagated ILTV isolates were titrated using 10-fold serial dilution. The plates were incubated at 37°C with 5% CO2. After five

27 days, cytopathic effects were examined and the titer was determined following the Reed and

Muench method [103].

2.4.3. Viral DNA purification and ILTV genome quantification

DNA purification from swabs and tissue samples (feather tips, trachea, and lungs) was carried out using QIAmp DNeasy Kit (QIAGEN GmbH, Hilden, Mettmann, Germany) according to the manufacturer’s instructions. Purity of DNA was assessed, and quantification of DNA was done using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

For ILTV genome quantification, a qPCR assay was carried out using a CFX96-c1000

Thermocycler (Bio-Rad laboratories, Mississauga, ON, Canada) targeting proteinase K (PK) gene

[101, 104]. Total volume per reaction was 20 L, which included genomic DNA as a template, 10

L of SYBR Green Master Mix (Invitrogen, Burlington, ON, Canada), 0.5 L of forward and reverse specific primers with a final concentration of 10 picomolar (pmol)/L targeting the ILTV proteinase K (PK) gene (F: 5'-TAC GAT GAA GCG TTC GAC TG -3' and R: 5'-AGG CGT GAC

AGT TCC AAA GT -3') and DNAse/RNAse free water (Thermo Scientific, Wilmington, DE,

USA). Thermocycler conditions were 95°C for 20 seconds for initial denaturation, then 40 cycles of denaturation to 95°C for 3 seconds, annealing at 60°C for 30 seconds and elongation at 95°C for 10 seconds. Additionally, β-actin genome (F:5'-CAACACAGTGCTGTCTGGTGGTA-3' and

R:5'-ATCGTACTCCTGCTTGCTGATCC-3') was quantified in each of the sample to normalize for the variation in template amount, using the previously mentioned PCR conditions.

2.4.4. Data and statistical analyses

The ILTV genome load quantification was determined using standard curves of the PK gene plasmid and of β-actin, used as housekeeping gene. Starting quantities were determined using the

[Ct−C] following formula: 퐿표푔 푠푡푎푟푡푖푛푔 푞푢푎푛푡푖푡푦 = Starting quantity=10log starting quantity. Ct = m intercept; m= Y-C of the standard curve.

All statistical analysis in this study were carried out using GraphPad Prism 9.0.0 (GraphPad

Software, San Diego, CA, USA). A two-way analysis of variance (ANOVA) and Tukey’s multiple comparison test was used to analyze bodyweight data. Survival curves were analyzed using Log rank (Mantel Cox) Test and Gehan-Breslow-Wilcoxon test. Kruskal Wallis and Dunn’s multiple comparison test were used for clinical score and viral genome load analyses. The comparisons among groups were considered significant at P ≤ 0.05.

CHAPTER THREE: RESULTS

3.1 Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to identification of recombination events

3.1.1 Flock background information

qPCR-confirmed ILT-positive clinical samples originated from AB belonged to small backyard flocks, while the ILT samples originated from QC, and BC belonged to larger commercial flocks. Only two flocks had a history of ILT vaccination: one of the flocks in QC and the other one from AB. All of the relevant flock history and clinicopathological findings are summarized in Table 1 and Appendix Table A4.

3.1.2 ILTV whole-genome sequences

Ten samples from AB, 3 samples from QC and 1 sample from BC yielded enough viral DNA and were directly submitted for WGS (Table 2). The size of the genomes varied between 150,118–

153,648 kbps, with the smallest genome belonging to the BC isolate (CAN/BC-10-1122) due to a

3563-nucleotide deletion on the 5’ end of its genome. Interestingly this deletion was also present in TCO-like and TCO vaccine sequences. The 14 Canadian ILTV sequences were aligned with 36

ILTV whole-genome sequences representing various geographical areas available in the public domain, which included TCO and CEO ILT vaccine sequences (Appendix Table A2). The 14

Canadian ILTV sequences were deposited in the GenBank, and their accession numbers are given in Table 2.

Table 1. Relevant background information of the poultry farms from which the 14 Canadian infectious laryngotracheitis virus (ILTV) samples that yielded full genome sequences originated. Dash lines fill slots where information could not be obtained.

Sample ID Province Age (Weeks) Breed Type Flock ILT Morbidity Mortality Year Size Vaccination # of birds # of birds #1990662 Quebec 4 days - Broiler 19,700 No - - 2017 #2154822 Quebec 8 Ross Broiler 6400 Recombinant 3000 400 2018 #2175807 Quebec - - Broiler 17,000 No - 527 2019 #10-1122 British 11 - Layer 45,000 No - - - Columbia #15 Alberta 6 Heritage Backyard 250 Yes ** 10 10 2014 #20 Alberta 40 Mille fleur Backyard 150 No 4 4 2015 #42 Alberta 60 Heritage Backyard 56 No 22 4 2016 #45 Alberta 24 Barnevelder Backyard 50 No 4 4 2016

#50 Alberta 10 PRS cross * Backyard 475 No 400 40 2016

#61 Alberta 96 Heritage Backyard 50 No 15 15 2017

#63 Alberta 6 Heritage Backyard 150 No 5 5 2017 mixed

#77 Alberta 80 Heritage Backyard 150 No 7 4 2017

#84 Alberta 22 Heritage Backyard 50 No 5 0 2017

#85 Alberta 40 Heritage Backyard 120 No 80 80 2017

* PRS cross = Plymouth-Rhode Island-Sussex cross; ** Further details on the type of vaccine applied could not be gathered.

Table 2. Canadian ILTV full genome sequences (n = 14).

Isolate Genome Province Total Mapped Virus Accession Length Reads Reads Isolation #

CAN/AB- 153,648 Alberta 5,650,374 9220 LMH cells MT797239 15A

CAN/AB- 152,695 Alberta 3,630,632 18,054 LMH cells MT797240 S20

CAN/AB- 153,469 Alberta 3,128,494 75,084 LMH cells MT797241 S42

CAN/AB- 153,630 Alberta 4,144,190 55,821 LMH cells MT797242 S45

CAN/AB- 153,641 Alberta 2,394,680 41,448 LMH cells MT797243 S50

CAN/AB- 153,643 Alberta 3,603,066 15,064 LMH cells MT797244 S61

CAN/AB- 152,703 Alberta 2,660,390 9072 LMH cells MT797245 S63

CAN/AB- 153,633 Alberta 3,245,498 17,863 LMH cells MT797246 S77

CAN/AB- 153,643 Alberta 3,090,720 9735 LMH cells MT797247 S84

CAN/AB- 153,631 Alberta 2,504,122 12,822 LMH cells MT797248 T85

Isolate Genome Province Total Mapped Virus Accession Length Reads Reads Isolation #

CAN/BC- 150,118 British 2,404,772 502,040 LMH cells MT797249 10-1122 Columbia

CAN/QC- 153,598 Quebec 4,669,964 67,376 CAM MT797250 1990662

CAN/QC- 151,326 Quebec 4,233,878 9055 CAM MT797251 2154822

CAN/QC- 153,468 Quebec 8,493,852 50,518 CAM MT797252 2175807

3.1.3 Phylogenetic analysis

Using the multiple sequence alignment of the 50 full genome sequences, a phylogenetic tree was generated. As shown in Figure 5, the first cluster mostly comprised wild-type ILTV isolates and virulent strains considered to be different from vaccines (genotype VI–IX), 3 of the 14

Canadian ILTV sequences clustered among this group (CAN/AB-S20, CAN/QC-2175807,

CAN/AB-S63). The second cluster grouped ILTV isolates with CEO revertant viruses (genotype

V), and 10 of the 14 Canadian ILTV sequences clustered with this group. The remaining Canadian

ILTV sequence, represented by CAN/BC-10-1122, grouped within the cluster with the ILTV strains related to the TCO vaccines (genotype I, II, III).

Wild type

VI-IX

CEO Revertants

TCO vaccines

I, II, III

CEO vaccine

Figure 5. Phylogenetic tree of the complete genome sequences of 50 ILTV strains from

different geographical regions. Tree was generated using the Bayesian inference method

(MrBayes, Geneious software). Posterior probability values are indicated as branch labels in

the tree. Sequences highlighted in orange represent the Alberta ILTV isolates; in blue, the

Quebec ILTV isolates and in purple is the ILTV isolate originated in British Columbia.

Brackets and labels to the right of the tree separate and indicate the suggested genotype

according to the clustering of the sequences in the phylogenetic tree.

Additional information is shown in Appendix Table A5. The last cluster included all the ILTV

CEO commercial vaccine strains (Genotype IV), as well as European origin vaccine strain Serva,

Chinese strains WG, K317, LJS09, Korean strain 40,798, US strains 63,140 and 3.26.90 and

Australian strains CL9 and ACC78. None of the analyzed Canadian ILTV sequences clustered in this group.

Of the 10 Canadian ILTV isolates clustered as CEO revertants following whole-genome sequence analysis, it was interesting to find a high percent nucleotide identity between nine of these sequences (CAN/QC-1990662, CAN/AB-S61, CAN/AB-S50, CAN/AB-S42, CAN/AB-

T85, CAN/AB-S45, CAN/AB-S77, CAN/AB-15A, CAN/AB-S84) and three vaccine strains:

European Serva, Nobilis Laringovac® (an attenuated ILTV Serva strain) and Poulvac ILT® (uses the ILTV Salisbury strain). A comparative analysis was made between these nine ILTV Canadian sequences and the European Serva. On average, these nine sequences shared 99.9% nucleotide identity with 34 SNPs inside the coding sequence in 36 different ORFs (Table 3), including a codon insertion in ORF C, resulting in a frameshift in the amino acid sequence. Fourteen of these

SNPs are synonymous, and 20 non-synonymous. The rest of the Canadian sequences, CAN/QC-

2175807, CAN/AB-S63, CAN/BC-10-1122, and CAN/AB-S20, did not appear to be as closely related to the Serva strain or any of the latter mentioned vaccine strains. Additional phylogenetic analysis was conducted using three separate portions of the ILTV genome, the UL, US, and IR regions (Figure 6a–c, respectively). Overall, the analyzed Canadian ILTV isolates clustered similarly compared to Figure 5 results, with the exception of isolate CAN/QC-2154822, which clustered with CEO vaccines and a US virulent strain on the IR phylogenetic tree.

Wild type Wild type

TCO vaccines

CEO CEO vaccines revertants

CEO vaccines

TCO vaccines CEO revertants CEO vaccines

(a) UL (b) US

Wild type

CEO vaccines

CEO revertants

CEO vaccines

TCO vaccines

CEO vaccines

Figure 6. Phylogenetic analysis of 50 ILTV sequences using the unique long region (a), unique short region (b) and the internal repeat regions (c) using the Bayesian inference method (MrBayes,

Geneious software). Sequences highlighted in orange represent the Alberta ILTV isolates; in blue,

37 the Quebec ILTV isolates and in purple is the ILTV isolate originated in British Columbia.

Brackets and labels to the right of the trees separate and indicate the suggested genotype according to the clustering of the sequences in the phylogenetic trees. Posterior probability values are indicated as branch labels in the tree.

The position within trees of sequence CAN/QC-2175807 remained constant along with

VFAR043, J2 and Canadian CAN/AB-S63. A more detailed analysis of these sequences showed a close relationship among them. Between the first two mentioned strains, only 13 SNPs inside coding sequences differed between them, 7 of them synonymous and 6 non-synonymous. Between

CAN/QC-2175807 and J2, only 10 SNPs inside coding sequences differed between them, 5 synonymous and 5 non-synonymous. The same comparison was made among sequences

CAN/AB-S63, VFAR043 and J2. Between the first two sequences, 37 SNPs inside of coding sequences could be detected, 19 synonymous and 18 non-synonymous and between CAN/AB-63 and US strain J2, only 34 SNPs, 17 synonymous and 17 non-synonymous in 13 different coding sequences (UL2, US4, UL5, US5, UL6, UL9, UL23, UL25, UL27, UL32, UL38, UL39, UL41).

CAN/QC-2154822 ILTV sequence, grouped with CEO vaccine strains (LT BLEN, LARYNGO-

VAC and CEO TRVX) and a US strain 63140/C/08/BR in the IR region phylogenetic tree (Figure

2c), this Canadian isolate seemed to have more discrepancies with the rest of the sequences in the alignment. Hundred and forty-four (144) SNPs were identified inside coding sequences when compared to Serva, 21 SNPs synonymous and 123 non-synonymous, including 2 insertions, one in UL27, the other one in ORFC, and a total of 57 deletions, 36 of them concluding on frameshifts in the amino acid sequences in 21 coding regions. However, it was with Serva and Serva-like sequences that it was more closely related to. The Canadian ILTV isolate CAN/BC-10-1122,

38 which clustered among TCO vaccines in the phylogenetic tree following the whole-genome sequence analysis, was found to cluster along with CEO vaccines in the phylogenetic trees of the

US and IR regions (Figure 6b,c), showing a nucleotide percent identity of 100% with vaccine

Poulvac ILT and Laryngo-vac vaccine sequences in the US multiple sequence alignment, and a

99.8% with Serva and Serva-like vaccine sequences in the IR region.

Table 3. Single nucleotide polymorphisms (SNPs) of the nine Canadian ILTV sequences from the Alberta (CAN/AB-S61, CAN/AB-

S50, CAN/AB-S42, CAN/AB-T85, CAN/AB-S45, CAN/AB-S77, CAN/AB-15A, CAN/AB-S84) and Quebec (CAN/QC-1990662) provinces using the vaccine strain Serva as a reference sequence. Asterisks (*) represent all nine Canadian sequences. RDR = ribonucleoside-diphosphate reductase, DUTN = deoxyuridine 5′-triphosphate nucleotidohydrolase.

Gene Protein CDS Nucleotide Amino Acid Sequence Position Change Change

US7 Envelope glycoprotein 57 C → T CAN/AB-45, 84 and 77 UL39 RDR large subunit 58 G → A D → N CAN/AB-45, 84 and 77 UL35 Large tegument protein 89 C → T T → I CAN/AB-45, 84 and 77 UL1 Uracil-DNA glycosylase 121 C → T D → N CAN/AB-15, 42, 50, 61, 84 and CAN/QC-1990662 UL10 Envelope gM 124 T → C T → A * US10 Virion protein 128 A → G D → G CAN/AB-45, 84 and 77 US10 128 T → C D → G CAN/AB-45, 84 and 77 UL1 161 T → G Q → P * UL27 Envelope gB 347 A → G V → A * ORFB ORF B protein 352 A → C *

ORFA ORF A protein 360 A → C * UL49 Envelope gN 378 T → C CAN/AB -15, 42, 50, 61& 84 ORFE ORF E protein 398 C → G G → A * UL50 DUTN 453 A → G * US8 Envelope gE 629 A → G K → R * UL46 Putative viral tegument 849 A → G * protein UL21 Tegument protein 924 C → T CAN/AB -15, 42, 50, 61& 84 UL5 DNA replication helicase 1027 A → G K → E * US6 Envelope gD 1164 C → T CAN/AB-45, 84 and 77 US3 Protein Kinase 1200 A → G * UL44 Envelope gC 1201 T → C CAN/AB -15, 42, 50, 61& 84 UL9 DNA replication origin- 1428 G → C Q → H CAN/AB -15, 42, 50, 61& 84 binding helicase ORFF ORF F protein 1878 T → C CAN/AB-15, 42, 50, 61, 84 and CAN/QC-1990662 ORFF 1883 C → A S → Y CAN/AB-45, 84 and 77 ORFF 1899 CT → TA GS → GT CAN/AB-15, 42, 50, 61, 84 and CAN/QC-1990662 UL28 Tripartite terminase subunit 1 1913 A → G V → A * UL27 Envelope gB 1931 A → G I → T *

UL52 Helicase-primase primase 2232 A → T F → L CAN/AB -15, 42, 50, 61& 84 subunit UL52 2256 G → A CAN/AB -15, 42, 50, 61& 84 UL52 2325 C → T CAN/AB -15, 42, 50, 61& 84 ICP4 Major viral transcription 2342 T → C H → R * factor ICP4 2342 A → G H → R * UL36 Large tegument protein 2449 G → A R → C CAN/AB-15, 42, 50, 61, 84 and CAN/QC-1990662 UL36 4040 C → T R → H * ICP4 Major viral transcription 4281 C → T * factor ICP4 4281 G → A * UL36 Large tegument protein 7677 T → C * UL36 8349 C → A CAN/AB-45, 84 and 77

3.1.4. Recombination analysis

Of the 14 Canadian ILTV whole-genome sequences examined for potential recombination events, CAN/BC-10-1122 was found to be a recombinant virus with vaccine strains as possible parental strains (Table 4). The first suggested parent is TCO vaccine LT-IVAX®, which shared an identity of 96.3% with the Canadian BC ILTV sequence. The analysis suggested two minor potential parents (i.e., sequences with a minor contribution to the genome of the suggested recombinant), US-CEO origin vaccine Poulvac ILT®, which shared 97.7% identity with the sequence CAN/BC-10-1122. CEO origin vaccine Nobilis Laryngo-vac®, which shared 97.4% identity with the Canadian isolate. Both CEO vaccine strains were almost identical, with 99.9% identity and only 19 nucleotide differences between them in the coding sequence. As shown in

Table 4, the detection of recombination in the CAN/BC-10-1122 isolate using various methods was highly significant.

A second recombination event was detected in sequence 6.48.88 of US origin. In this suggested recombination event, sequence CAN/AB-S20 was indicated as a minor parent (sequence with a minor contribution to the genome of the suggested recombinant), and vaccine SA2 was suggested as a potential major parent in this event (Table 4). As shown in Table 4, the detection of recombination in 6.48.88 ILTV isolate using various methods was highly significant with p values < 0.05.

Interestingly, RDP4 software pointed to another recombination event with sequence

CAN/QC-2154822 as a major parent for Australian CL9 and minor parent A20. This recombination event had been previously described with Serva as a major parent for the CL9 isolate. After considering the background information on this isolate, belonging to Australia where vaccination with Serva is done with regularity, comparing, and doing a visual verification of the

43 alignment including the Quebec sequence, Serva and the rest of the isolates involved, this event was no longer taken into consideration.

Both recombination events were originally revealed using RDP4 software, confirmed by RDP,

GENCONV, MaxChi, Chimaera, SiScan and 3Seq methods [105-110]. To further confirm the

RDP4 analysis results, a Bootscan analysis using the full genome sequence alignment done with

MAFFT of the parental vaccine strains, suggested by RDP4 software, TCO LT-IVAX® and

Poulvac ILT® was carried out. In this analysis, the US 1874C5ILTV strain was used as a control and CAN/BC10-1122 as the query sequence (Figure 7). Table 5 summarizes the percentage nucleotide identity of the BC ILTV isolate (BC-10-1122) with CEO Poulvac ILT® and TCO LT-

IVAX® in different segments of the ILTV genome (1–15,393; 15,393–37,509; 37,509–113,748;

113,748–end) as indicated by bootscan analysis. It is noteworthy to mention that the same plot was obtained when using Nobilis Laryngo-vac® as a minor parent in the Bootscan analysis.

Table 4. Recombination signals involving Canadian ILTV isolates, recombination analysis carried out on Recombination Detection

Program (RDP4) software.

Potential Potential Potential Detection p-Values Recombination Breaking Recombinant Major Minor Methods Points Parent Parent BC-10-1122 LT-IVAX Poulvac RDP 2.379 × 10−6; 3.356 × 10−9 * 15,393-UL52 ILT GENECONV 1.457 × 10−5; 3.362 × 10−8 * 37,509-UL26 Nobilis MaxChi 3.060 × 10−6; 6.860 × 10−8 * 113,748-ICP4 Laryngo- Chimaera 1.518 × 10−6; 3.337 × 10−8 * vac SiScan 1.476 × 10−5; 1.054 × 10−7 * 3Seq 1.194 × 10−7; 2.190 × 10−3 * 6.48.88 SA2 CAN/AB- RDP 5.375 × 10−20 9,652-ORF F S20 GENCONV 1.864 × 10−18 81,152-UL 19 MaxChi 2.159 × 10−10 104,647-UL 5 Chimaera 1.463 × 10−10 SiScan 2.379 × 10−11 3Seq * p-value attributed to recombination event including LT IVAX and Nobilis Laryngo-vac as a potential minor parent.

Figure 7. Bootscan analysis plot of CAN/BC-10-1122 with the suggested parental vaccine strain

TCO LT-IVAX® (red) and Poulvac ILT® strain (blue). US 1874C5 strain was used as control

(green).

Table 5. Percentages of nucleotide identity of vaccines CEO-Poulvac ILT® and TCO-LT-IVAX® with British Columbia ILTV isolate (BC-10-1122) indicated by Bootscan.

1–15,393 15,393–37,509 37,509–113,748 113,748–End Poulvac ILT 77.09% 99.98% 99.84% 99.52% TCO_IVAX 99.41% 99.91% 99.83% 87.99%

The bootscan analysis also corroborated the recombination event involving CAN/AB-S20

ILTV and Australian SA2 as potential parental strains of US 6.48.88, as suggested by the RDP4 analysis; the results are illustrated in Figure 8. In this analysis, the US-TCO strain was used as a control and US 6.48.88 was used as the query sequence.

Figure 8. Bootscan analysis plot of CAN/AB-S20 with the suggested parental vaccine strain

CAN/AB-S20 (blue) and SA2 (red). US 1874C5 strain was used as control (green).

3.2 Determination of pathogenicity and transmission potential of wild-type and CEO vaccine revertant ILTV

3.2.1. Clinical manifestations 3.2.1.1. Experimentally infected chickens

Peak of clinical sign presentation was observed at 4 dpi in the three groups of infected chickens

(Figure 9a) while the mock infected controls showed no clinical signs. At 4 dpi, the difference in clinical signs between AB-S63 (p=0.02), AB-S45 (p=0.004) and AB-S20 (p=0.04) and mock infected controls were significant. At 5 dpi, the difference in clinical signs between AB-S63

(p=0.009) and AB-S45 (p=0.001) and mock infected controls were significant. The clinical signs of the chicken infected with wildtype strain AB-S20 included mild but persistent inflammation of the periorbital sinuses and increased respiratory rate with occasional open beak breathing. The chickens infected with ILTV AB-S63 developed mild inflammation of periorbital sinuses, increased respiratory rates, difficulty breathing, manifested by open beak respiration with characteristic extended neck posture, and gasping. Clinical signs observed in some of the chickens infected with CEO revertant AB-S45 were conjunctivitis, depression, gasping and open beak respiration with extended neck posture. At 5 dpi, one of the chickens experimentally infected with the CEO revertant ILTV isolate, reached a critical accumulation in the clinical scores and as indicated by the established Animal protocol, the chicken had to be humanely euthanized.

3.2.1. 2. Contact exposed chickens

The naïve chickens exposed to the experimentally infected chickens with CEO revertant virus

AB-S45, manifested severe clinical signs such as watery eyes, conjunctivitis, difficulty breathing, and constant gasping accompanied by depression. The clinical signs peaked at 4 and 5 dpi (Figure

9b). The chickens exposed to CEO vaccine revertant ILTV AB-S45 had significantly higher clinical scores when compared to mock infected controls at 3 dpi (p=0.02), at 4 dpi (p=0.04) and

5 dpi (p=0.03). The chickens exposed to CEO vaccine revertant ILTV AB-S45 had significantly higher clinical scores when compared to AB-S63 at 4 dpi (p=0.04) and 5 dpi (p=0.03). All the

48 chickens belonging to this group had to be humanely euthanized at 5 dpi, due to a critical accumulation of clinical scores. There were no endpoints observed for the groups of chicken challenged with the ILTV wild-type strains during the course of the experiment. The naive chicken exposed to experimentally infected chickens with wild-type viruses, AB-S20 and AB-S63 displayed increase in respiratory rates at 3, 4 and 5 dpi.

Figure 9. Clinical manifestations of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates. a) Clinical scores of the chicken experimentally infected with the ILTV isolates b) Clinical scores of the chickens infected by exposure to the experimentally infected chickens. Mean of the clinical signs scores are plotted along with bars representing standard error.

The clinical signs were scored as indicated in the materials and methods and analyzed statistically using Kruskal Wallis and Dunn’s multiple comparison test.

3.2.2 Weight gains

3.2.2.1 Experimentally infected chickens

Overall, bodyweight gains in the experimentally infected chickens with the two wild-type

ILTV, were similar and remained constant throughout the entire experiment (Figure 10a). At 7 dpi significant difference was found in weight gains between mock infected controls and chickens

49 infected with wild-type AB-S63 (p=0.009), CEO vaccine revertant AB-S45 (p=0.009), and wild- type AB-S20 (P=0.0058). At 10 dpi, AB-S45 ILTV infected group had significantly lower weigh gains when compared to the mock infected controls (p=0.03). At 14 dpi, AB-S63 ILTV infected group had significantly lower weigh gains when compared to the mock infected controls

(p=0.009).

3.2.2.2 Contact exposed chickens

At 3dpi, the chickens exposed to experimentally infected chickens with vaccine revertant

ILTV AB-S45 and wild-type ILTV AB-S63 and AB-S20 had similar weight gains to the controls

(P>0.05) (Figure 10b). Due to severe clinical manifestations, the chickens exposed to AB-S45

ILTV reached the humane end points by 5 dpi precluding further comparison of weight gains with other groups. At this time point, two of the exposed chickens had lost on average 12% of their initial bodyweight, and the third chickens had gained little to no weight. At 7 dpi, the difference in weight gains between AB-S20 and AB-S63 exposed groups were significant (p=0.03).

Figure 10. Weight gains of chickens infected with two wild-type and a CEO vaccine revertant

ILTV isolates. The infected and control chickens were weighed on day 0, 3, 7, 10 or 11 and 14 following ILTV infection and weight gains were calculated. Mean of the weight gains are plotted

50 along with bars representing standard error. A two-way ANOVA and Tukey’s multiple comparison test was used to identify significance. a) Average weight gains of experimentally infected chickens and mock infected controls. b) Weight gains of chickens infected by contact exposure and unexposed controls.

3.2.3. Survival rate

3.2.3.1. Experimentally infected chickens

The survival rate of experimentally infected chickens and their uninfected controls is illustrated in Figure 11a. Only one chicken reached a humane end point at 5dpi, in the group of chicken infected with vaccine revertant ILTV, AB-S45, where due to the severity of clinical manifestation of the disease, humane euthanasia was required.

3.2.3.2. Contact exposed chickens

The survival rate of chickens exposed to experimentally infected chickens and their unexposed controls is illustrated in Figure 11b. The chickens exposed to vaccine revertant ILTV AB-S45 had a survival rate of 0% by 5 dpi. The chickens in any other groups did not reach the end points. The survival rate between vaccine revertant ILTV, AB-S45 and other groups were significantly different (P= 0.01).

Figure 11. Survival percentage of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates. a) Percentage survival of experimentally infected chickens. b) Percentage survival of chickens infected by contact exposure. Following ILTV infection, the chickens were scored for development of clinical signs in order to determine the humane end points. The percentage of surviving chickens were recorded. The survival rates of the groups were compared to identify significant differences using Log rank (Mantel Cox) Test and Gehan-Breslow-

Wilcoxon test.

3.2.4. Viral genome loads

3.2.4.1. Oropharyngeal swabs

3.2.4.1.1. Experimentally infected chickens

The ILTV genome loads in oropharyngeal swabs of experimentally infected chickens is illustrated in Figure 12a. At 3 dpi, the genome load in ILTV AB-S45 infected group was significantly higher than ILTV AB-S20 (p=0.0001). At 7 dpi, the genome load in ILTV AB-S45 infected group was significantly higher than the group experimentally infected with ILTV AB-S20

(p=0.009) and ILTV AB-S63 (p=0.007). However, no viral load was detected in oropharyngeal swabs of ILTV AB-S45 infected groups at 10 and 14 dpi, most likely due to the establishment of latency.

3.2.4.1.2 Contact exposed chickens

The ILTV genome loads in oropharyngeal swabs of chickens exposed to experimentally infected chickens is illustrated in Figure 12b. No ILTV genome in oropharyngeal swabs was quantified in contact chickens exposed to chickens experimentally infected with wild-type ILTV,

AB-S63. In the group of contact chickens exposed to the chicken experimentally infected with wild-type ILTV, AB-S20, the virus was recovered only from the oropharyngeal swabs of one of the chickens at 10 dpi. All the contact chickens exposed to the chickens experimentally infected with CEO vaccine revertant AB-S45 had detectable viral loads at 3 dpi in oropharyngeal swabs and it is significantly higher when compared to ILTV genome load quantified in groups exposed to AB-S63 (p=0.03) and AB-S-20 (p=0.03).

Figure 12. The viral genome loads quantified in oropharyngeal swabs of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates. Mean of the viral genome loads in log10 scale are plotted with bars representing the standard error. ILTV genome loads were quantified targeting proteinase K (PK) gene using SYBR Green method. Kruskal Wallis and Dunn’s multiple comparison test were used for group comparisons. a) ILTV genome loads of the experimentally infected chicken. b) ILTV genome loads of chickens infected by contact exposure.

3.2.4.2. Cloacal swabs

3.2.4.2.1. Experimentally infected chickens

The ILTV genome loads in cloacal swabs of experimentally infected chickens is illustrated in

Figure 11a. At 3 dpi, the genome loads in cloacal swabs of chickens experimentally infected with

ILTV AB-S45 was significantly higher when compared to that in chickens experimentally infected with ILTV AB-S20 (p=0.020) and AB-S63 (P=0.020). At 7 dpi, the genome loads in cloacal swabs of chickens experimentally infected with ILTV AB-S45 was significantly higher when compared to that in chickens experimentally infected with ILTV AB-S20 (p=0.020).

3.2.4.2.2 Contact exposed chickens

The ILTV genome loads in cloacal swabs of chickens exposed to experimentally infected chickens is illustrated in Figure 13b. All contact chickens exposed to the chickens experimentally infected with CEO vaccine revertant AB-S45 had quantifiable genome load in cloacal swabs at 3 and 7 dpi and they are significantly higher when compared to that in ILTV AB-S20 (p=0.050) and

AB-S63 (P=0.050) infected groups. In fact, no virus was recovered from the cloacal swabs of the contact chickens exposed to chickens experimentally infected with wild-type ILTV AB-S20 and

AB-S63.

Figure 13. The viral genome loads quantified in cloacal swabs of chickens infected with two wild- type and a CEO vaccine revertant ILTV isolates. Mean of the viral genome loads in log10 scale are plotted with bars representing the standard error. ILTV genome loads were quantified targeting proteinase K (PK) gene using SYBR Green method. Kruskal Wallis and Dunn’s multiple comparison test were used for group comparisons. a) ILTV genome loads of the experimentally infected chicken. b) ILTV genome loads of chickens infected by contact exposure.

3.2.4.3. Feathers

3.2.4.3.1. Experimentally infected chickens

The ILTV genome loads in feathers of experimentally infected chickens is illustrated in Figure

14a. ILTV genome could not be recovered from the feathers of chickens experimentally infected with wild-type ILTV AB-S20. At 10 dpi, the genome loads in feather tips of chickens experimentally infected with ILTV AB-S63 was significantly higher when compared to that in chickens experimentally infected with ILTV AB-S20 (p=0.01).

3.2.4.3.2. Contact exposed chickens

The ILTV genome loads in feathers of contact exposed chickens is illustrated in Figure 14b.

ILTV genome could not be recovered from the feathers of chickens exposed to chickens

55 experimentally infected wild-type ILTV AB-S20. At 7 dpi, the genome loads in feather tips of chickens exposed to chickens experimentally infected with ILTV AB-S63 was significantly higher when compared to that in chickens exposed to chickens experimentally infected with ILTV AB-

S20 (p=0.01).

Figure 14. The viral genome loads quantified in feathers of chickens infected with two wild-type and a CEO vaccine revertant ILTV isolates. Mean of the viral genome loads in log10 scale are plotted with bars representing the standard error. ILTV genome loads were quantified targeting proteinase K (PK) gene using SYBR Green method. Kruskal Wallis and Dunn’s multiple comparison test were used for group comparisons. a) ILTV genome loads of the experimentally infected chicken. b) ILTV genome loads of chickens infected by contact exposure.

3.2.4.4. Trachea and lungs

3.2.4.4.1. Experimentally infected chickens

The ILTV genome loads in trachea and lungs of experimentally infected chickens are illustrated in Figure 15a and b. At the end of experiment (14 dpi), no ILTV genome was quantifiable from trachea and lungs originated from chickens experimentally infected with wild- type ILTV AB-S20. At the same timepoint, the genome loads in trachea of chickens

56 experimentally infected with ILTV AB-S45 was significantly higher when compared to that in chickens experimentally infected with ILTV AB-S20 (p=0.0002) and AB-S63 (P=0.001). In lungs,

ILTV genome was quantifiable only from the chickens experimentally infected with ILTV AB-

S45 and the group differences were not significantly different (p>0.05).

3.2.4.4.2. Contact exposed chickens

At the end of experiment (11 dpi for contact exposed chickens), no ILTV genome was quantifiable from trachea or lungs originated from the chickens exposed to the experimentally infected chickens with wild-type ILTV AB-S20 and AB-S63. Since all the contact chickens exposed to chickens experimentally infected with CEO vaccine revertant ILTV AB-S45 reached the end points at 5 dpi, the ILTV genome load was quantified at this point in trachea and lungs.

ILTV genome was quantifiable from the trachea of all three contact exposed chickens (median,

2x105) and from the lungs of the three contact exposed chickens (median, 1.4x104).

Figure 15. The viral genome loads quantified in trachea and lungs of chickens experimentally infected with two wild-type and a CEO vaccine revertant ILTV isolates and sampled on 14 days post-infection. Mean of the viral genome loads in log10 scale are plotted with bars representing the standard error. ILTV genome loads were quantified targeting proteinase K (PK) gene using SYBR

Green method. Kruskal Wallis and Dunn’s multiple comparison test. A) ILTV genome loads in trachea, b) ILTV genome loads lungs.

CHAPTER FOUR: DISCUSSION

4.1 Genetic characterization of ILTV isolates obtained from ILTV clinical cases leading to identification of recombination events

The use of live attenuated vaccines is a common practice that is frequently done in Canada, just as in many other countries around the world. However, reversion to virulence of attenuated vaccines, especially of chicken embryo origin, and the circulation of these vaccine revertant strains is no longer a strange and isolated event, as it has been documented on many occasions [41, 65,

111, 112]. The aim of the current study was to genetically characterize the ILTV isolates linked to

ILT-positive cases in Canada using WGS.

Among the highlights of this research was finding that most of the obtained Canadian sequences (n = 10) are genetically related to CEO vaccines. This observation of CEO vaccine- related ILT virus circulating in Canada is consistent with observations in other studies [1, 112,

113], and more recently, coinciding with findings on a study on the molecular characterization of circulating Western Canadian ILTV using Iltovirus specific genes, ORF a and b [93], where 84%

ILTV isolates were characterized as genotype V, CEO revertants. It is noteworthy that some of the

ILTV isolates belonging to AB and BC used in this study were also included in the previously mentioned study [93]. However, molecular characterization using partial ORF a and b genes did not classify any of the examined Canadian ILTV isolates as TCO vaccine-related. Instead, using partial ORF a and b genes, classified one of the isolates in this study, BC-10-1122 (considered to be TCO vaccine-related using WGS) as genotype IV, with the CEO vaccines, along with other two

BC isolates [93].

Thanks to previous studies that documented the vaccination practices in the British Columbia province, in 1971, there is substantial information on three live attenuated CEO vaccines, and one

TCO vaccine, being used extensively by the BC poultry industry, for the vaccination of their flocks against ILT. The same records also mention the BC poultry industry encountered the emergence of several ILT outbreaks, related to the use and inadequate practices of vaccination with live attenuated vaccines, which involved 25–50% of the vaccinated flocks, during 1971–1973 [8]. The introduction of live attenuated vaccines during the 70’s, probably led to the circulation of the vaccine viruses in the province.

Current practices of inadequate vaccination with TCO vaccines (for example, administering the vaccine via drinking water, when it is indicated by the manufacturers for eyedrop administration only) may facilitate TCO vaccine virus reversion to virulence [6] and its circulation among the flocks in BC. The concurrent circulation of vaccine-related ILTV strains and wild-type

ILTV probably facilitated the recombination event found in the BC ILTV isolate.

In the case of AB, all the ILT samples in this study obtained from this province, originated from noncommercial back yard flocks. In the province, these flocks are generally comprised by laying hens and other susceptible avian species of varying ages, with reports of vaccination in less than half of these flocks. These birds are usually kept under a combination of indoor and outdoor housing, potentially facilitating contact with other predatory species. Additionally, new birds are frequently introduced to these flocks without adhering to proper biosecurity measures. With the majority of producers being unexperienced and recently introduced to production, adequate and strict biosecurity measures are rarely followed [114]. Contact of these birds with other wild avian species and insects [32], which could be carriers of the disease, is likely. Along with contact with visitors, and the transportation of show birds to various exhibitions and competitions, these set of

60 conditions create an ideal situation for ILTV transmission and maintenance since strict biosecurity in such flocks is rarely maintained effectively [26, 31].

Similar to other viral families, recombination has been documented among different members of the alpha herpesvirinae subfamily, with many of the recombination events involving the use of attenuated vaccines [68, 115-118]. In the case of ILTV, the attenuated CEO vaccines are usually involved in recombination events with more frequency than the TCO vaccines, which could be influenced by the higher degree of attenuation of the TCO vaccine strains. Recombination between attenuated ILT vaccines (either CEO or TCO) depends on a number of factors including, but not limited to, the quantity and ratio of the vaccines used [75]. However, naturally occurring ILTV recombination events are common, and the results of this work agree with these studies [69, 77,

78]. However, the characteristics of these recombinant field ILTV isolates have not been inquired yet; it is important to know the frequency of these recombination events and whether they lead to increased virulence, as has been documented for other members of the alpha herpesvirinae subfamily such as the varicella-zoster virus (VZV) [68], herpes simplex virus 1 (HVS-1), herpes simplex virus 2 (HVS-2) [68], and other ILTV recombinants [69, 77, 78].

It is clear that Canada is not exempt from ILTV recombination involving vaccine strains, as two of the 14 Canadian ILTV isolates (CAN/BC-10-1122 and CAN/AB-S20) appeared to be associated in recombination events, and in both cases with CEO or TCO live attenuated vaccine viruses, even though vaccination with CEO vaccines is no longer recommended to be used in the

Western part of Canada.

Highlighted by the results from the recombination event involving Australian vaccine strain

SA2 and Canadian isolate CAN/AB-20, the results of this work are also in agreement with previous

61 work suggesting a US origin for the Australian vaccine strain SA2, which could have later diversified and spread into different geographical areas [119]. This could explain its involvement in the later mentioned recombination event with the Canadian isolate. This view is based on sequence similarity of Canadian ILTV sequence CAN/AB-S20 with the Australian vaccine strains

SA2 and A20, US field strains 6.48.88 and S2 816 and Russian strain

RU/CK/TATARSTAN/2009/1043 and pointed out by the phylogenetic analysis results [119].

Overall, using a WGS approach, a successful molecular characterization of 14 ILTVs of

Canadian origin was achieved, and it also aided for the identification of naturally occurring ILTV recombination. The CEO vaccine viruses that circulate actively within the chicken flocks in

Canada, added to the inadequate vaccination practices with live attenuated vaccines in some of the provinces, favors recombination events between vaccine and wild-type ILTV, as has been observed in the current study.

4.2 Determination of pathogenicity and transmission potential of wild-type and CEO vaccine revertant ILTV

Although ILT control heavily relies on vaccination using live attenuated vaccines, vaccine revertant ILTV strains are increasingly gaining attention due to their involvement in ILT outbreaks, and for the characteristics of these strains such as enhanced pathogenicity and increased mortality [41, 120].

With the molecular characterization of 14 ILTV isolates using WGS, product of this work, and previous studies conducted in Canada, there is sufficient evidence of circulating ILT viruses

62 genetically related to live attenuated vaccines [93, 94, 101]. In a proportion of CEO vaccine revertant ILTV isolates higher (71%) than wild-type ILTV Canadian isolates (21%).

The third objective of this study was to compare the pathogenicity and transmission potential of three ILTV isolates that originated in Alberta, Canada. Two of them characterized as wild-type, and one of them was characterized as CEO vaccine revertant ILTV isolates. The results of this study revealed a greater transmission potential for the CEO vaccine revertant ILTV isolate,

AB-S45 when compared to the wild-type ILTV isolates AB-S63 and AB-S20. The transmission potential of ILTV was evaluated by using contact exposure of naïve chickens to experimentally infected chickens, as has been done by previous studies [121, 122].

Supported by the critical accumulation of the clinical signs score, and the significantly lower weight gains in the groups of chicken challenged with the CEO vaccine revertant ILTV isolate AB-S45, this strain of Canadian origin, showed a higher pathogenicity potential than the tested wild-type ILTV isolates. The observation in this regard, agrees with previous studies [6,

111, 120], where severity of clinical sign manifestation and an increase in mortality rates have been a constant characteristic observed in chicken infected with CEO revertant ILTV isolates.

An additional finding of this study is that two out of three tested ILTV isolates target feather follicles providing suggestive evidence that some ILTV strains could be shed to environment via feather dander in addition to oropharyngeal and cloacal routes.

Compared to wild-type, the CEO vaccine revertant ILTV showed a higher transmission potential. Evidenced by ILTV genome, still present in the trachea and lung tissues of the chicken experimentally challenged, suggesting of longer shedding periods of this virus. This observation agrees with other studies on vaccine derived ILTV with similar observations [123]. Also,

63 indicative of a higher transmission potential of the CEO revertant ILTV, was the ILTV genome data in the swabs and tissues of the contact exposed chickens [121]. It was not determined as to why CEO vaccine revertant had a higher transmission potential, but it could be due the shedding of higher ILTV amount and/or higher number of routes as well as a higher cell to cell spread efficiency.

Historically, ILTV is known to confine its replication in the respiratory tract tissues and now it has been shown that certain strains of ILTV can replicate in multiple body organ systems such as the respiratory tract, gastrointestinal tract, skin and immune system organs, excreting the virus into the environment in feces and feather dander in addition to respiratory and oropharyngeal secretions [37, 39, 41]. In agreement with the observation of Davidson and colleagues [39] it was also found that some wild-type and CEO vaccine revertant ILTV could replicate in feather tips but at a lower rate. This observation has implication since viral replication feather tips may add another route of shedding of ILTV to the environment. Although, the presence of ILTV in feather dander was not valuated in this study, the possibility of presence of ILTV genome in feather dust has been shown [29].

Overall, the ILTV isolate AB-S45, CEO vaccine revertant, originated from back yard chickens in Alberta, Canada has a higher pathogenicity and transmission potential compared to the wild-type ILTV isolates, AB-S63 and AB-S20. Further studies are required to confirm these observations. It is noteworthy to mention that is not possible to generalize these conclusions, by assumption and attributing these characteristics to all the CEO revertant isolates of Canadian origin that were used in this work. To be able to make such conclusions, this evaluation should also be applied to a representative number of isolates of Canadian origin, belonging to this genotype.

CHAPTER FIVE: GENERAL DISCUSSION

5.1. Implications

Recombination between homologous alpha herpesviruses as an evolutionary strategy is not uncommon in ILTV. Relevant to ILTV, two scenarios may promote the recombination. First, being a herpesvirus, ILTV establishes lifelong infection in the host and this allows wild-type virus circulation in multi age birds poultry flocks. Second, the use of ILT live attenuated vaccines also promotes the circulation of ILT vaccine virus since, similar to wild-type ILTV, ILT vaccine virus also establishes latency [43] and establishes lifelong infection in the host. Since ILTV infection has poor exclusion mechanisms [75, 81], this will aid coinfection with two different ILTV strains promoting recombination. In AB and elsewhere in Canada, ILTV infection is endemic in backyard flocks with multi age birds. Further, provincial authorities recommend vaccination of unaffected birds on the face of ILT outbreaks. In the first part of my thesis work, I found that most of ILTV associated with ILT outbreaks in Canada are CEO vaccine revertants. I also found evidence of recombination of ILTV involving frequently used ILT vaccines. In the second part of my thesis, I also found CEO vaccine revertant ILTV is more virulent when compared to the wild-type ILTV.

There is an indication that CEO vaccine revertant ILTV is efficient in transmission through direct contact when compared to tested wild-type ILTV strains. These findings imply that live attenuated

ILT vaccines have created new issues rather than controlling ILT. This issue of reliance on ILT live attenuated vaccines can be addressed by multiple ways. First, we can recommend switching the vaccine to recombinant ILT vaccines. Since recombinant ILT vaccines are administered at the hatchery via parenteral or in ovo routes, this vaccine is not suitable for backyard flocks. Also, the efficacy of recombinant vaccines is comparatively lower than the live attenuated vaccines [2, 51,

124]. Second, giving priority for biosecurity is an alternative but biosecurity lapse are common.

Recent ILT outbreaks in commercial flocks in QC and ON [94] suggest that biosecurity lapse could occur even under commercial poultry management standards. Third, research should be focused to develop gene deleted efficacious ILT vaccines that lacks latency establishment or reactivation

[125], and this will minimize the cocirculation of ILTV strains, super infections and hence, recombination.

5.2. Limitations and future directions

Although my thesis work yielded significant data that is applicable to poultry industry in

Canada and elsewhere and scientific community, there are gaps that need further investigations.

Of the 58 clinical samples received, we were successful in studying only 14 ILTV isolates via

WGS. One of the issues we faced was the lower ILTV genome content in clinical materials. We had to propagate some of the samples few times either in CAM or liver cells. We did not propagate more than 3 times just to minimise culture induced changes in the ILTV genome. We believe it is important to molecular characterise the rest of the Canadian ILTV isolates and for which we require to optimise techniques that increase percentage of viral genome within total DNA. To be successful in WGS, a sample should provide Ct value of <20 in ILTV qPCR.

Although it was conducted an in vivo experiment using naïve contact chicken to compare the transmission potential of CEO revertant and wild-type ILTV isolates of Canadian origin, the experimental design did not allow to identify the transmission route as respiratory, conjunctival, or gastrointestinal. In the current study, the naïve chickens shared the same poultry isolator as experimentally infected chickens for 3 days. Therefore, during these 3 days the chickens would have been infected with ILTV via multiple routes. To determine airborne respiratory transmission, we would need to modify our poultry isolators to direct air from the poultry isolators containing the experimentally infected chickens to the clean poultry isolator with naïve chickens, as has been described previously [126]. Since ILTV can be infected via respiratory tract and conjunctiva, this later approach will indicate that the transmission is airborne, and the routes of infection is either respiratory or conjunctival. To determine whether, the infection of naïve contact chickens took place via fecal oral route, we would have introduced the naïve chickens into dirty poultry isolators inhabited by experimentally infected chickens following removal of experimentally infected chickens. It is also important to determine the ILTV in feces, feed, and water in this later experiment. This is another area worth investigating and the generated data will be valuable for indicating the source of ILTV for transmission to naïve flocks.

The technique used for quantification of ILTV in the in vivo study is qPCR and it does not quantify infectious viral particles [127]. As such, we do not know if the ILTV genome loads that was quantified in oropharyngeal swabs, cloacal swabs, feather tips and tissues were originated from infectious viral particles. Therefore, we cannot tell with certainty that the

ILTV genome load data is attributable to infectious ILTV hence involved in transmission.

Primary isolation of ILTV can be done in CAM or CELIC [128], however, these methods require embryos and primary cells and also are less sensitive than the qPCR technique [129].

Although my thesis characterised the ILTV associated with clinical cases in Canada, one question remains to be answered is whether the currently available ILT vaccines are protective against our ILTV isolates. It is interesting to note that one of the broiler chicken flocks in QC was vaccinated with recombinant ILT vaccine and suffered ILT loss at the rate of 46% morbidity and

6% mortality in 2018 (Table 1). This shows that the efficacy of current vaccines is depending on the ILTV strain involved in the infection. As such, efficacy of current ILT vaccines should be determined against challenge with our ILTV isolates with high virulence as determined in our objective 2 work.

REFERENCES

1. Menendez, K.R., et al., Molecular epidemiology of infectious laryngotracheitis: a review.

Avian Pathol, 2014. 43(2): p. 108-17.

2. Garcia, M. and G. Zavala, Commercial Vaccines and Vaccination Strategies Against

Infectious Laryngotracheitis: What We Have Learned and Knowledge Gaps That Remain.

Avian Dis, 2019. 63(2): p. 325-334.

3. Oldoni, I. and M. Garcia, Characterization of infectious laryngotracheitis virus isolates

from the US by polymerase chain reaction and restriction fragment length polymorphism

of multiple genome regions. Avian Pathol, 2007. 36(2): p. 167-76.

4. Groves, P.J., et al., Uptake and spread of infectious laryngotracheitis vaccine virus within

meat chicken flocks following drinking water vaccination. Vaccine, 2019. 37(35): p. 5035-

5043.

5. Maekawa, D., et al., Protection Efficacy of a Recombinant Herpesvirus of Turkey Vaccine

Against Infectious Laryngotracheitis Virus Administered In Ovo to Broilers at Three

Standardized Doses. Avian Dis, 2019. 63(2): p. 351-358.

6. Guy, J.S., H.J. Barnes, and L. Smith, Increased virulence of modified live laryngotracheitis

vaccine virus folliwing bird to bird passage. Avian Diseases, 1991. 35: p. 348-355.

7. Ou, S.C. and J.J. Giambrone, Infectious laryngotracheitis virus in chickens. World J Virol,

2012. 1(5): p. 142-9.

8. Hayes LB, M.K., Newby WC, Wood CW, Gilchrist EW, MacNeill AC, Epizootiology of

infectious laryngotracheitis in British Columbia 1971-1973. Canadian Veterinary Journal,

1976. 17: p. 101-108.

9. Thureen, D.R. and C.L. Keeler, Jr., Psittacid herpesvirus 1 and infectious laryngotracheitis

virus: Comparative genome sequence analysis of two avian alphaherpesviruses. J Virol,

2006. 80(16): p. 7863-72.

10. Fuchs, W., et al., Molecular biology of avian infectious laryngotracheitis virus. Vet Res,

2007. 38(2): p. 261-79.

11. Spear, P.G. and R. Longnecker, Herpesvirus entry: an update. J Virol, 2003. 77(19): p.

10179-85.

12. Ziemann, K., T. C. Mettenleiter, W. Fuchs, Gene Arrangement within the Unique Long

Genome Region of Infectious Laryngotracheitis Virus Is Distinct from That of Other

Alphaherpesviruses. JOURNAL OF VIROLOGY, 1998: p. p. 847–852.

13. Pavlova, S.P., et al., In vitro and in vivo characterization of glycoprotein C-deleted

infectious laryngotracheitis virus. J Gen Virol, 2010. 91(Pt 4): p. 847-57.

14. Griffin, A.M.B., M. E. G;, Analysis of the nucleotide sequence of DNA from the region of

the thymidine kinase gene of infectious laryngotracheitis virus; potential evolutionary

relationships between the herpesvirus subfamifies. Journal of General Virology (1990),

1990. 71: p. 841-850.

15. WILD, M.A.C., S.; COCHRAN, M., A Genomic Map of Infectious Laryngotracheitis

Virus and the Sequence and Organization of Genes Present in the Unique Short and

Flanking Regions. Virus genes, 1996: p. 107-116.

16. Helferich, D., et al., The UL47 gene of avian infectious laryngotracheitis virus is not

essential for in vitro replication but is relevant for virulence in chickens. J Gen Virol, 2007.

88(Pt 3): p. 732-742.

17. Prideaux, C.T.K., K.; Johnson, M. A.; Sheppard, M.; Fahey, K. J., Infectious

laryngotracheitis virus growth, DNA replication, and protein synthesis. Arch Virol, 1992.

123: p. 181-192.

18. Akhtar, J. and D. Shukla, Viral entry mechanisms: cellular and viral mediators of herpes

simplex virus entry. FEBS J, 2009. 276(24): p. 7228-36.

19. Subramanian, R.P.G., R. J., Herpes simplex virus type 1 mediates fusion through a

hemifusion intermediate by sequential activity of glycoproteins D, H, L, and B. PNAS,

2007. 104(8): p. 2903-2908.

20. Kingsley, D.H.K., C. L., Infectious Laryngotracheitis Virus, an Alpha Herpesvirus That

Does Not Interact with Cell Surface Heparan Sulfate. Virology, 1999. 256: p. 213-219.

21. Poulsen, D.J. and C.L. Keeler, Jr., Characterization of the assembly and processing of

infectious laryngotracheitis virus glycoprotein B. J Gen Virol, 1997. 78 ( Pt 11): p. 2945-

51.

22. Sodeik, B.E., M. W.; Helenius, A., Microtubule-mediated Transport of Incoming Herpes

Simplex Virus 1 Capsids to the Nucleus. journal of Cell Biology, 1997. 136: p. 1007-1021.

23. Mettenleiter, T.C., Herpesvirus assembly and egress. J Virol, 2002. 76(4): p. 1537-47.

24. Granzow, H., et al., Egress of alphaherpesviruses: comparative ultrastructural study. J

Virol, 2001. 75(8): p. 3675-84.

25. Crawshaw G, J.B.B., R, Infectious Laryngotracheitis in Peafowl and Pheasants. Avian

Diseases, 1982. 26 (2): p. 397-401.

26. Morris, S., The Early History of Infectious Laryngotracheitis. Avian Diseases, 1996. 40(2):

p. pp. 494-500.

27. Guy, M.G.S.S.J.S., Infectious Laryngotracheitis. Diseases of Poultry, 2013. 13th edition:

p. 161-179.

28. Bagust, T.J. and M.A. Johnson, Avian infectious laryngotracheitis: virus-host interactions

in relation to prospects for eradication. Avian Pathol, 1995. 24(3): p. 373-91.

29. Roy, P., et al., Real-time PCR quantification of infectious laryngotracheitis virus in chicken

tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals

high levels in faeces and dust. J Gen Virol, 2015. 96(11): p. 3338-3347.

30. Hidalgo, H., Infectious Laryngotracheitis: A Review. Brazilian Journal of Poultry Science

2003. v.5 / n.3: p. 157-168.

31. Johnson, Y.J.C., M.M.; Tablante, N. L.; Hegngi, F. N.; Salem, M.; Gedamu, M.; Pope, C.,

Application of commercial and backyard poultry geographic information system databases

for the identification of risk factors for clinical Infectious laryngotracheitis in a cluster of

cases on Delmarva Peninsula. International Journal of Poultry Science, 2004: p. 201-205.

32. Ou, S.C., J.J. Giambrone, and K.S. Macklin, Detection of infectious laryngotracheitis virus

from darkling beetles and their immature stage (lesser mealworms) by quantitative

polymerase chain reaction and virus isolation. Journal of Applied Poultry Research, 2012.

21(1): p. 33-38.

33. Dufour-Zavala, L., Epizootiology of Infectious Laryngotracheitis and Presentation of an

Industry Control Program. Avian Dis, 2008. 52: p. 1-7.

34. Giambrone, J.J., O. Fagbohun, and K.S. Macklin, Management Practices to Reduce

Infectious Laryngotracheitis Virus in Poultry Litter. Journal of Applied Poultry Research,

2008. 17(1): p. 64-68.

35. Ou, S., J.J. Giambrone, and K.S. Macklin, Infectious laryngotracheitis vaccine virus

detection in water lines and effectiveness of sanitizers for inactivating the virus. Journal of

Applied Poultry Research, 2011. 20(2): p. 223-230.

36. ELLERY, B.W.H., D. W., INACTIVATION OF INFECTIOUS

LARYNGOTRACHEITIS VIRUS BY DISINFECTANTS. Australian Veterinary Journal,

1971. 47.

37. Wang, L.G., et al., Dynamic distribution and tissue tropism of infectious laryngotracheitis

virus in experimentally infected chickens. Arch Virol, 2013. 158(3): p. 659-66.

38. Davidson, I., et al., Detection of wild- and vaccine-type avian infectious laryngotracheitis

virus in clinical samples and feather shafts of commercial chickens. Avian Dis, 2009.

53(4): p. 618-23.

39. Davidson, I., et al., Infectious laryngotracheitis virus (ILTV) vaccine intake evaluation by

detection of virus amplification in feather pulps of vaccinated chickens. Vaccine, 2016.

34(13): p. 1630-1633.

40. Davidson, I., Diverse Uses of Feathers with Emphasis on Diagnosis of Avian Viral

Infections and Vaccine Virus Monitoring. Brazilian Journal of Poultry Science, 2009. 11:

p. 139-148.

41. Oldoni, I., et al., Pathogenicity and growth characteristics of selected infectious

laryngotracheitis virus strains from the United States. Avian Pathol, 2009. 38(1): p. 47-53.

42. Calnek, B.W.F., K. J.; Bagust, T. J, In vitro studies with Infectious Laryngotracheitis Virus.

Avian Dis, 1986. 30: p. 327-336.

43. Bagust, T.J., Laryngotracheitis (Gallid-1) herpesvirus infection in the chicken. 4. Latency

establishment by wild and vaccine strains of ILT virus. Avian Pathol, 1986. 15(3): p. 581-

95.

44. Williams, R.B., M.; Bradbury, J.; Gaskel, R; Jones, R. & Jordan, F., Demonstration of sites

of latency of Infectious laryngotracheitis virus using the polymerase chain reaction.

General Virology, 1992. 73: p. 2415-2420.

45. Dubbeldam, J.L., The sensory trigeminal system in birds: input, organization and effects

of peripheral damage. A review. Arch Physiol Biochem, 1998. 106(5): p. 338-45.

46. Garcia, M., et al., Genomic sequence analysis of the United States infectious

laryngotracheitis vaccine strains chicken embryo origin (CEO) and tissue culture origin

(TCO). Virology, 2013. 440(1): p. 64-74.

47. Huges, C.S.G., R. M.; Jones, R. C.; Bradbury, J. M.; Jordan, F. T. W. , Effects of certain

stress factors on re-excretion of infectious laryngotracheitis virus from latently infected

carrier birds. Research in Veterinary Science, 1989. 49: p. 274-276.

48. HUGHES, C.S.G., R. M.; JONES, R. C.; BRADBURY, J. M; JORDAN, F. T. W, Effects

of certain stress factors on the re-excretion of infectious laryngotracheitis virus from

latently infected carrier birds. Research in Veterinary Science, 1989: p. 274-276.

49. Hayashi, S.O., Y.; Kotani, T.; Horiuchi, T., Pathological Changes of Tracheal Mucosa in

Chickens Infected with Infectious Laryngotracheitis Virus. Avian Dis, 1985. 29(4): p. 943-

950.

50. Schijns, V.E.J.C., et al., Practical Aspects of Poultry Vaccination, in Avian Immunology.

2014. p. 345-362.

51. Palomino-Tapia, V.A., et al., Long-term protection against a virulent field isolate of

infectious laryngotracheitis virus induced by inactivated, recombinant, and modified live

virus vaccines in commercial layers. Avian Pathol, 2019. 48(3): p. 209-220.

52. Goraya, M.U., L. Ali, and I. Younis, Innate Immune Responses Against Avian Respiratory

Viruses. Hosts and Viruses, 2017. 4(5).

53. Vagnozzi, A.E., et al., Cytokine gene transcription in the trachea, Harderian gland, and

trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus

(ILTV) strain. Avian Pathol, 2018. 47(5): p. 497-508.

54. Wu, Z., et al., Analysis of the function of IL-10 in chickens using specific neutralising

antibodies and a sensitive capture ELISA. Dev Comp Immunol, 2016. 63: p. 206-12.

55. Honda, T., et al., The Role of Cell-Mediated Immunity in Chickens Inoculated with the

Cell Associated Vaccine of Attenuated Infectious Laryngotracheitis Virus. Journal of

Veterinary Medical Science, 1994. 56 (6): p. 1051-1055.

56. Fahey, K.J., J.J. York, and T.J. Bagust, Laryngotracheitis herpesvirus infection in the

chicken. II. The adoptive transfer of resistance with immune spleen cells. Avian Pathol,

1984. 13(2): p. 265-75.

57. Coppo, M.J., C.A. Hartley, and J.M. Devlin, Immune responses to infectious

laryngotracheitis virus. Dev Comp Immunol, 2013. 41(3): p. 454-62.

58. Devlin, J.M., et al., Glycoprotein G is a virulence factor in infectious laryngotracheitis

virus. J Gen Virol, 2006. 87(Pt 10): p. 2839-47.

59. Bonhoeffer, S. and P. Paul Sniegowski, The importance of being erroneous. Nature, 2002.

420: p. 367-369.

60. Gojobori, T., E. Moriyama, and M. Kimura, Molecular clock of viral evolution, and the

neutral theory. PNAS, 1990. 87 No.24: p. 10015-10018.

61. S., A.-B., P. L., and I. N., Viral Long-Term Evolutionary Strategies Favor Stability over

Proliferation. Viruses, 2019. 11(8).

62. Hanada, K., Y. Suzuki, and T. Gojobori, A large variation in the rates of synonymous

substitution for RNA viruses and its relationship to a diversity of viral infection and

transmission modes. Mol Biol Evol, 2004. 21(6): p. 1074-80.

63. Shackelton, L., et al., High rate of viral evolution associated with the emergence of

carnivore parvovirus. PNAS, 2004. 102(2): p. 379–384.

64. Crutel, J.J.L., I. R., Herpes Simplex- 1 DNA Polymerase. Journal of Biological Chemistry,

1989. 264(32): p. 19266-19270.

65. Piccirillo, A., et al., Full Genome Sequence-Based Comparative Study of Wild-Type and

Vaccine Strains of Infectious Laryngotracheitis Virus from Italy. PLoS One, 2016. 11(2):

p. e0149529.

66. Sanjuán, R. and P. Domingo, Genetic Diversity and Evolution of Viral Populations, in

Reference Module in Life Sciences. 2019, ELSevier: Valencia, Spain.

67. Thiry, E., et al., Recombination in the alphaherpesvirus bovine herpesvirus 1. Vet

Microbiol, 2006. 113(3-4): p. 171-7.

68. Norberg, P., et al., Divergence and recombination of clinical herpes simplex virus type 2

isolates. J Virol, 2007. 81(23): p. 13158-67.

69. Lee, S.W., et al., Attenuated vaccines can recombine to form virulent field viruses. Science,

2012. 337(6091): p. 188.

70. Bowden, R., et al., High recombination rate in herpes simplex virus type 1 natural

populations suggests significant co-infection. Infect Genet Evol, 2004. 4(2): p. 115-23.

71. Javier, R.T.S., F.; Stevens, J. G., Two Avirulent Herpes Simplex Viruses Generate Lethal

Recombinants in Vivo. Science, 1986. 234: p. 746-748.

72. Koelle, D.M., et al., Worldwide circulation of HSV-2 x HSV-1 recombinant strains. Sci

Rep, 2017. 7: p. 44084.

73. Norberg, P., et al., Recombination of Globally Circulating Varicella-Zoster Virus. J Virol,

2015. 89(14): p. 7133-46.

74. Jarosinski, K., Dual infection and superinfection inhibition of epithelial skin cells by two

alphaherpesviruses co-occur in the natural host. PLoS One, 2012. 7(5): p. e37428.

75. Fakhri, O., et al., Genomic recombination between infectious laryngotracheitis vaccine

strains occurs under a broad range of infection conditions in vitro and in ovo. PLoS One,

2020. 15(3): p. e0229082.

76. Agnew-Crumpton, R., et al., Spread of the newly emerging infectious laryngotracheitis

viruses in Australia. Infect Genet Evol, 2016. 43: p. 67-73.

77. Zhao, Y., C. Kong, and Y. Wang, Multiple Comparison Analysis of Two New Genomic

Sequences of ILTV Strains from China with Other Strains from Different Geographic

Regions. PLoS One, 2015. 10(7): p. e0132747.

78. La, T.M., et al., Comparative genome analysis of Korean field strains of infectious

laryngotracheitis virus. PLoS One, 2019. 14(2): p. e0211158.

79. Muylkens, B., et al., Coinfection with two closely related alphaherpesviruses results in a

highly diversified recombination mosaic displaying negative genetic interference. J Virol,

2009. 83(7): p. 3127-37.

80. Meurens, F., et al., Superinfection prevents recombination of the alphaherpesvirus bovine

herpesvirus 1. J Virol, 2004. 78(8): p. 3872-9.

81. Fakhri, O., et al., Superinfection and recombination of infectious laryngotracheitis virus

vaccines in the natural host. Vaccine, 2020. 38(47): p. 7508-7516.

82. Johnson, R.M.S., P. G., Herpes Simplex Virus Glycoprotein D Mediates Interference with

Herpes Simplex Virus Infection. Jounal of Virology, 1989. 63(2): p. 819-827.

83. Mador, N., A. Panet, and I. Steiner, The latency-associated gene of herpes simplex virus

type 1 (HSV-1) interferes with superinfection by HSV-1. J Neurovirol, 2002. 8 Suppl 2:

p. 97-102.

84. Criddle, A., et al., gD-Independent Superinfection Exclusion of Alphaherpesviruses. J

Virol, 2016. 90(8): p. 4049-58.

85. Bayat, A., Bioinformatics. BMJ 324, 2002: p. 1018-1022.

86. Ibrahim, B., et al., A new era of virus bioinformatics. Virus Res, 2018. 251: p. 86-90.

87. Barzon, L., et al., Next-generation sequencing technologies in diagnostic virology. J Clin

Virol, 2013. 58(2): p. 346-50.

88. Capobianchi, M.R., E. Giombini, and G. Rozera, Next-generation sequencing technology

in clinical virology. Clin Microbiol Infect, 2013. 19(1): p. 15-22.

89. Lam, T.T., C.C. Hon, and J.W. Tang, Use of phylogenetics in the molecular epidemiology

and evolutionary studies of viral infections. Crit Rev Clin Lab Sci, 2010. 47(1): p. 5-49.

90. Yang, Z. and B. Rannala, Molecular phylogenetics: principles and practice. Nat Rev Genet,

2012. 13(5): p. 303-14.

91. Loncoman, C., Hartley CA, Coppo MJC, Vaz PK, Diaz-Méndez A, Browning GF, García

M, Spatz S, Devlin JM., Genetic Diversity of Infectious Laryngotracheitis Virus during In

Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots

within the Genome. Appl Environ Microbio 83:e01532-17., 2017.

92. Bayoumi, M., et al., Molecular characterization and genetic diversity of the infectious

laryngotracheitis virus strains circulating in Egypt during the outbreaks of 2018 and 2019.

Arch Virol, 2020. 165(3): p. 661-670.

93. Barboza-Solis, C., et al., Genotyping of Infectious Laryngotracheitis Virus (ILTV) Isolates

from Western Canadian Provinces of Alberta and British Columbia Based on Partial Open

Reading Frame (ORF) a and b. Animals (Basel), 2020. 10(9).

94. Ojkic, D., et al., Characterization of field isolates of infectious laryngotracheitis virus from

Ontario. Avian Pathol, 2006. 35(4): p. 286-92.

95. Callison, S.A., et al., Development and validation of a real-time Taqman PCR assay for the

detection and quantitation of infectious laryngotracheitis virus in poultry. J Virol Methods,

2007. 139(1): p. 31-8.

96. Katoh, K. and D.M. Standley, MAFFT multiple sequence alignment software version 7:

improvements in performance and usability. Mol Biol Evol, 2013. 30(4): p. 772-80.

97. Kearse, M., et al., Geneious Basic: an integrated and extendable desktop software platform

for the organization and analysis of sequence data. Bioinformatics, 2012. 28(12): p. 1647-

98. Huelsenbeck and Ronquist, MRBAYES: Bayesian inference of phylogenetic trees.

Bioinformatics, 2001. 17: p. 754-755.

99. Martin, D.P.L., P.; Lott, M.; Moulton, V.; Posada, D.; Lefeuvre, P., RDP3: a flexible and

fast computer program for analyzing recombination. Bioinformatics, 2010. 26(19): p.

2462-3.

100. Lole KS, B.R., Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard

HW, Ray SC., Full-Length Human Immunodeficiency Virus Type 1 Genomes from

Subtype C-Infected Seroconverters in India, with Evidence of Intersubtype

Recombination. JOURNAL OF VIROLOGY, 1999: p. 152-60.

101. Contreras, P.A.V.d.M., F. .; Checkley, S.; Joseph, T.; King, R.; Ravi, M.; Peters, D.;

Fonseca, K.; Gagnon, C. A.; Provost, C.; Ojkic, D.; Abdul-Careem, M. F., Analysis of

Whole-Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry

Flocks in Canada: Evidence of Recombination. Viruses, 2020. 12(1302).

102. Cunningham, C.H., A Laboratory Guide in Virology. 1973. 7th. ed.

103. Reed, L.J. and H. Muench, A Simple Method for Estimating Fifty Per Cent Endpoints.

1938: Lancaster Press, Incorporated.

104. Thapa, S., E. Nagy, and M.F. Abdul-Careem, In ovo delivery of Toll-like receptor 2 ligand,

lipoteichoic acid induces pro-inflammatory mediators reducing post-hatch infectious

laryngotracheitis virus infection. Vet Immunol Immunopathol, 2015. 164(3-4): p. 170-8.

105. Martin, D.R., E., RDP: detection of recombination amongst aligned sequences.

Bioinformatics, 2000. 16: p. 562-563.

106. Padidam, M., Sawyer, S. & Fauquet, C. M., Possible emergence of new geminiviruses by

frequent recombination. Virology, 1999. 265: p. 218-225.

107. Maynard Smith, J., Analyzing the mosaic structure of genes. J Mol Evol, 1992. 34: p. 126-

129.

108. Posada, D. and C. K. A., Evaluation of methods for detecting recombination from DNA

sequences: Computer simulations. Proc Natl Acad Sci 2001. 98: p. 13757-13762.

109. Gibbs, M.J., J.S. Armstrong, and A.J. Gibbs, Sister-scanning: a Monte Carlo procedure for

assessing signals in recombinant sequences. Bioinformatics, 2000. 16(7): p. 573-82.

110. Boni, M.F., D. Posada, and M.W. Feldman, An exact nonparametric method for inferring

mosaic structure in sequence triplets. Genetics, 2007. 176(2): p. 1035-47.

111. Shehata, A.A., et al., Chicken embryo origin-like strains are responsible for Infectious

laryngotracheitis virus outbreaks in Egyptian cross-bred broiler chickens. Virus Genes,

2013. 46(3): p. 423-30.

112. Chacon, J.L., et al., Persistence and spreading of field and vaccine strains of infectious

laryngotracheitis virus (ILTV) in vaccinated and unvaccinated geographic regions, in

Brazil. Trop Anim Health Prod, 2015. 47(6): p. 1101-8.

113. Blacker, H.P., et al., Epidemiology of recent outbreaks of infectious laryngotracheitis in

poultry in Australia. Aust Vet J, 2011. 89(3): p. 89-94.

114. Mainali, C. and I. Houston, Small Poultry Flocks in Alberta: Demographics and Practices.

Avian Dis, 2017. 61(1): p. 46-54.

115. Previdelli, R.L., et al., The Role of Marek's Disease Virus UL12 and UL29 in DNA

Recombination and the Virus Lifecycle. Viruses, 2019. 11(2).

116. He, L., et al., Genomic analysis of a Chinese MDV strain derived from vaccine strain

CVI988 through recombination. Infect Genet Evol, 2020. 78: p. 104045.

117. Casto, A.M., et al., Large, Stable, Contemporary Interspecies Recombination Events in

Circulating Human Herpes Simplex Viruses. J Infect Dis, 2020. 221(8): p. 1271-1279.

118. Cudini, J., et al., Human cytomegalovirus haplotype reconstruction reveals high diversity

due to superinfection and evidence of within-host recombination. Proc Natl Acad Sci U S

A, 2019. 116(12): p. 5693-5698.

119. Lee, S.W., et al., Phylogenetic and molecular epidemiological studies reveal evidence of

multiple past recombination events between infectious laryngotracheitis viruses. PLoS

One, 2013. 8(2): p. e55121.

120. Guy, J.S., H.J. Barnes, and L.M. Morgan, Virulence of Infectious Laryngotracheitis

Viruses: Comparison of Modified-Live Vaccine Viruses and North Carolina Field Isolates.

Avian Diseases, 1990. 34(1): p. pp. 106-113.

121. Lee, S.W., et al., Growth kinetics and transmission potential of existing and emerging field

strains of infectious laryngotracheitis virus. PLoS One, 2015. 10(3): p. e0120282.

122. Rodriguez-Avila, A., et al., Replication and transmission of live attenuated infectious

laryngotracheitis virus (ILTV) vaccines. Avian Dis, 2007. 51(4): p. 905-11.

123. Vagnozzi, A.R., S. M.; Williams, S. M.; Zavala, G.; Garcıa, M., Infection of Broilers with

Two Virulent Strains of Infectious Laryngotracheitis Virus: Criteria for Evaluation of

Experimental Infections. Avian Diseases 59, 2015: p. 394-399.

124. Vagnozzi, A., et al., Protection induced by commercially available live-attenuated and

recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler

chickens. Avian Pathol, 2012. 41(1): p. 21-31.

125. Thilakarathne, D.S., et al., Attenuated infectious laryngotracheitis virus vaccines differ in

their capacity to establish latency in the trigeminal ganglia of specific pathogen free

chickens following eye drop inoculation. PLoS One, 2019. 14(3): p. e0213866.

126. Spekreijse, D., et al., Quantification of dust-borne transmission of highly pathogenic avian

influenza virus between chickens. Influenza Other Respir Viruses, 2013. 7(2): p. 132-8.

127. Rodriguez, R.A., I.L. Pepper, and C.P. Gerba, Application of PCR-based methods to assess

the infectivity of enteric viruses in environmental samples. Appl Environ Microbiol, 2009.

75(2): p. 297-307.

128. Dufour-Zavala, L., A Laboratory Manual for the Isolation, Identification, and

Characterization of Avian Pathogens. American Association of Avian Pathologists,

Jacksonville, FL, USA. 5th edition, 2018.

129. Hamza, I.A.B., K. , Journal of Virological Methods 266 2019: p. 11-24.

APPENDICES

Table A1. Depth variation along the genome of the 14 ILTV Canadian sequences. GenBank Positions with Reference Consensus Minimum Maximum Median Average Sequence Reference coverage Length length coverage coverage coverage coverage accession # below 5 CAN/QC- VHTSL 153629 153590 2 178 67 69,43 39 1990662 sequence* CAN/QC- MF417811 151327 151297 1 45 9 9,54 30 2154822 CAN/QC- MG775218 152701 152535 1 120 34 35,44 166 2175807 CAN/BC-10- VHTSL sequence 151495 151452 1 1295 652 622,34 43 1122

CAN/AB-S15 KP677885 153653 151802 1 35 10 10.64 1851 CAN/AB-S20 JX466898 153630 153169 1 91 25 25.64 461 CAN/AB-S42 KP677885 153653 153469 1 251 119 118.69 184 CAN/AB-S45 MF417811 153629 153539 1 208 81 81.87 90 CAN/AB-S50 KP677885 153653 153389 1 145 67 66.02 264 CAN/AB-S61 KP677885 153653 153005 1 58 23 23.24 648 CAN/AB-S63 MG775218 152701 151703 1 46 15 15.14 998

CAN/AB-S77 MF417811 153629 153133 1 77 26 26.18 496 CAN/AB-S84 KP677885 153662 151963 1 46 14 14.63 1699 CAN/AB-S85 MF417811 153629 152795 1 59 22 22.13 834 *Veterinary high-throughput sequencing laboratory ILTV sequence.

Table A2. The representative ILTV whole genome sequences (n = 36) that were obtained from public domain and their genome length and Genbank accession numbers for the purpose of aligning with the Canadian ILTV whole genome sequences.

Strain Country Genome Length Accession Number TCO LOW USA 155,465 JN580315 TCO IVAX USA 155,465 JN580312 S2816 USA 154,001 MF417807 J2 USA 153,711 MF417808 USDA REF USA 151,756 JN542534 CEO TRVX USA 153,647 JN580313 6.48.88 USA 154,022 MF417810 81658 USA 150,335 JN542535 63140/C/08/BR USA 153,633 JN542536 1874C5 USA 149,682 JN542533 3.26.90 USA 153,655 MF417809 14.939 USA 153,629 MF417811 SERVA Europe 152,630 HQ630064 O Russia 153,634 KU128407 CK/TATARSTAN/2009/1643 Russia 153,933 MF405079 2013/2701 Russia 153,634 MF405080 VFAR043 Peru 153,634 MG775218 NOBILIS LARYNGOVAC USA 152,701 KP677881 LT BLEN USA 153,623 JQ083493 LARYNGOVAC USA USA 153,624 JQ083494 40798/10 Korea 153,649 MH937566 30678 Korea 153,659 MH937565 0206/14 Korea 153,645 MH937564 POLUVAC ILT Italy 153,650 KP677882 193435/07 Italy 153,662 KP677883 4787/80 Italy 153,653 KP677885

Strain Country Genome Length Accession Number 757/11 Italy 153,662 KP677884 WG China 153,505 JX458823 K317 China 153,639 JX458824 LJS09 China 153,201 JX458822 V199 Australia 153,630 JX646898 SA2 Australia 152,975 JN596962 CL9 Australia 152,635 JN804827 ACC78 Australia 152,632 JN804826 A20 Australia 152,978 JN596963 CSW-1 Australia 151,671 JX646899

Table A3. Clinical monitoring sheet. All clinical signs are assigned a score from 1-4 depending on the severity of the sign manifested. Monitoring of

the clinical signs was done twice a day, three times on the peak of clinical sign presentation. Nonspecific signs Specific signs (dyspnea) Other signs Depression Ruffled with Mild Moderate Blood- Bodyweight loss feathers and Droopy Severe lowered (increased (increased stained =1 point per 5% Date/Time Huddling wings (increased Conjunctivitis head and no respiration but respiration, mucus bodyweight loss together respiration as No=0; movement beaks constant open compared to =2 marked by (Endpoint) No=0; yes=1 remained beak original weight No=0; gasping) =3 =4 yes=1 closed) =1 breathing) =2 of the bird yes=1 Day AM PM

Day AM PM

Table A4. Clinicopathological history of the ILTV infected chickens where the 14 Canadian ILTV samples that yielded full genome sequences

originated. Dash lines fill slots where information could not be obtained.

Sample Province of Clinical Signs Gross lesions Histopathological lesions ID origin Dyspnea and respiratory #1990662 Quebec - - rales Dyspnea and respiratory #2154822 Quebec - - rales Conjunctivitis and #2175807 Quebec Hemorrhagic tracheitis - respiratory rales British #10-1122 - - - Columbia Lymphoplasmacytic laryngotracheitis with syncytial Lethargy, extended neck Hemorrhagic tracheitis #15 Alberta respiration with respiratory cells and intranuclear viral inclusions

rales and gasping Lymphoplasmacytic laryngotracheitis with syncytial Periorbital inflammation, Catarrhal laryngotracheitis #20 Alberta cells and intranuclear viral inclusions dyspnea and sudden death

Fibrinohemorrhagic and necrotizing, and Fibrinohemorrhagic and Gasping and dyspnea with lymphoplasmacytic and heterophilic laryngotracheitis #42 Alberta necrotizing laryngotracheitis respiratory rales with syncytial cells and intranuclear viral inclusions

Fibrino necrotizing, lymphoplasmacytic and Fibrinonecrotizing Periorbital inflammation, heterophilic laryngotracheitis with syncytial cells and laryngotracheitis #45 Alberta ocular secretion and intranuclear viral inclusions conjunctivitis

Fibrinohemorrhagic and necrotizing, and Fibrinohemorrhagic and Dyspnea and extended neck lymphoplasmacytic and heterophilic laryngotracheitis necrotizing laryngotracheitis #50 Alberta respiration with respiratory with syncytial cells and intranuclear viral inclusions rales

Fibrinohemorrhagic and necrotizing, and Fibrinonecrotizing Breathing with respiratory lymphoplasmacytic and heterophilic laryngotracheitis #61 Alberta laryngotracheitis rales with syncytial cells and intranuclear viral inclusions

Fibrinohemorrhagic Lymphoplasmacytic laryngotracheitis with syncytial Nasal and ocular secretions #63 Alberta laryngotracheitis cells and intranuclear viral inclusions and facial edema, depression

Catarrhal and necro- Catarrhal, fibrinohemorrhagic and necrotizing, Periorbital edema and hemorrhagic lymphoplasmacytic and heterophilic laryngotracheitis #77 Alberta extended neck respiration laryngotracheitis with syncytial cells and intranuclear viral inclusions and gasping

Fibrinonecrotizing Lymphoplasmacytic and heterophilic fibrinonecrotic Periorbital edema and #84 Alberta laryngotracheitis laryngotracheitis sneezing

Fibrinohemorrhagic and necrotizing, Fibrinonecrotizing #85 Alberta Lethargy and gasping lymphoplasmacytic and heterophilic laryngotracheitis laryngotracheitis

Table A5. Proposed genotype for the 14 Canadian ILTV sequences adressed in this study, and average % identity with the rest

of the sequences belonging in the corresponding groups.

Canadian ILTV Genotype Average % Identity ILTV sequences belonging to this genotype sequences CAN/QC-2175807 Wild-type 99.7% VFAR043, VI99, CSW-1, J2, 1874C5 and 6.48.88, CAN/AB-S63 (VI- IX) CK/TATARSTAN, Ko-30678/14 CAN/AB-S20 CAN/QC-1990662 CEO 99.8% US/14.939, IT-4787/80, RU-2013/2701, IT757/11, IT- CAN/AB-S61 revertant 193435/07 CAN/AB-S50 (V) CAN/AB-S42 CAN/AB-T85 CAN/AB-S45 CAN/AB-S77 CAN/AB-15A CAN/AB-S84 CAN/QC-2154822

CAN/BC-10-1122 TCO 97.7% TCO-IVAX, TCO-Low, USDA ref, US-81658 vaccine (I,II,III)

As the co-authors that contributed to the paper “Contreras, P.A.; Van der Meer, F.; Checkley, S.;

Joseph, T.; King, R.; Ravi, M.; Peters, D.; Fonseca, K.; Gagnon, C. A.; Provost, C.; Ojkic, D.;

Abdul-Careem, M. F., Analysis of Whole-Genome Sequences of Infectious laryngotracheitis Virus

Isolates from Poultry Flocks in Canada: Evidence of Recombination. Viruses, 2020. 12(1302).”

We permit using this paper as Chapter 2 of Ana Paulina Perez Contreras’s thesis entitled

“Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus (ILTV)” that will be submitted to the Faculty of Graduate Studies at the University of Calgary in January 20201.

Co-Author Signature Date

Frank Van der Meer

Sylvia Checkley

Tomy Joseph

Robin King

Madhu Ravi

Delores Peters

Kevin Fonseca

Carl Gagnon

Chantale Provost

Davor Ojkic

Mohamed F. Abdul-Careem

As the co-authors that contributed to the paper “Contreras, P. A.; Barboza-Solis, C.; Najimudeen,

M. S.; Checkley, S. Van der Meer, F.; Joseph, T.; King, R.; Ravi, M.; Peters, D.; Fonseca, K.;

Gagnon, C. A.; Ojkic, D.; Abdul-Careem, M. F.;. Comparative study of CEO revertant and wildtype Infectious laryngotracheitis virus isolated in Canada.” We permit using this paper as

Chapter 2 of Ana Paulina Perez Contreras’s thesis entitled “Molecular characterization and pathogenicity studies of Canadian infectious laryngotracheitis virus (ILTV)” that will be submitted to the Faculty of Graduate Studies at the University of Calgary in January 20201.

Co-Author Signature Date

Catalina Barboza-Solis

Shahnas M. Najimudeen

Frank Van der Meer

Sylvia Checkley

Tommy Joseph

Robin King

Madhu Ravi

Delores Peters

Kevin Fonseca

Carl Gagnon

Davor Ojkic

Mohamed F. Abdul-Careem