Equine Herpesvirus 1: Elucidation of the Core Fusogenic Glycoproteins, Entry Receptors, and Clinical Isolate Analysis

EQUINE HERPESVIRUS 1: ELUCIDATION OF THE CORE FUSOGENIC GLYCOPROTEINS, ENTRY RECEPTORS, AND CLINICAL ISOLATE ANALYSIS

Jekaterina Arnette

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2014

Advisory Committee

Sonja J. Pyott Paulo F. Almeida

Alison R. Taylor Arthur R. Frampton Chair

Accepted by

Dean, Graduate School

This thesis has been prepared in the style and format

consistent with

Cancer Gene Therapy

TABLE OF CONTENTS

ABSTRACT ...... v

ACKNOWLEDGMENTS ...... vii

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

LIST OF ABBREVIATIONS ...... x

INTRODUCTION ...... 1

1: EQUINE HERPESVIRUS 1 (EHV-1) OVERVIEW ...... 1

EHV-1 History ...... 1

EHV-1 Classification and Structure ...... 2

EHV-1 Glycoproteins ...... 4

2: EHV-1 LIFECYCLE ...... 6

EHV-1 Attachment and Entry ...... 7

EHV-1 Replication ...... 11

EHV-1 Assembly, Release, and Cell to Cell Spread ...... 12

3: EHV-1 PATHOGENESIS, VACCINES, and ANTIVIRAL THERAPIES...... 13

EHV-1 Pathogenesis ...... 13

EHV-1 Vaccines ...... 15

EHV-1 Antiviral Therapies ...... 19

CHAPTER 1: CLINICAL ISOLATE ANALYSIS ...... 22

MATERIALS AND METHODS ...... 22

Cells and Viruses ...... 22

Generation of Virus Stocks ...... 23

iii

Viral Replication Analysis of EHV-1 Isolates in EE Cells ...... 24

Plaque Phenotype Analysis of EHV-1 Isolates in EE Cells ...... 24

RESULTS ...... 25

DISCUSSION ...... 26

CHAPTER 2: CORE FUSOGENIC GLYCOPROTEIN SET ...... 30

MATERIAL AND METHODS ...... 30

Cells ...... 30

Generation of EHV-1 Glycoprotein Expression Plasmids ...... 30

PCR Analysis of Glycoprotein Expression in B78H1 Cells ...... 34

Elucidation of the Core Set of EHV-1 Fusogenic Glycoproteins ...... 35

RESULTS ...... 36

DISCUSSION ...... 38

CHAPTER 3: ALTERNATIVE EHV-1 ENTRY RECEPTORS ...... 42

MATERIALS AND METHODS ...... 42

Cells ...... 42

Generation of Equine Nectin-1 and HVEM Expression Plasmids ...... 42

Generation of Stable Nectin-1 and HVEM Receptor Lines in B78H1 Cells ...... 43

RESULTS ...... 43

DISCUSSION ...... 44

REFERENCES ...... 48

ABSTRACT

This project was aimed at gaining a better understanding of equine herpesvirus 1

(EHV-1) infection mechanics via a three-fold study:

The first study was initiated to investigate potential differences between neurologic and non-neurologic EHV-1 strains in terms of viral replication and cell to cell spread, in equine endothelial (EE) cells. Twenty-four hour virus yields were measured for 9 neurologic and 7 non-neurologic clinical isolates. In addition to virus yields, mean plaque size formed after infection with each strain was determined. Results of this study showed that there are no trends in significant differences in terms of both replication and plaque size, suggesting that the aggressive pathology often associated with infection with neurologic EHV-1 strains may not be caused simply by enhanced replication or cell to cell spread of a particular strain.

The second study was focused on identifying the minimum subset of EHV-1 glycoproteins sufficient for the fusion of the virus and host cell membranes. In the cell culture study, a subset of glycoproteins gB, gD, gH, and gL was expressed in one cell type and then these cells were combined with cells that express a known EHV-1 gD receptor,

MHC-I. After mixing, cells were analyzed for their ability to fuse to one another and form multinucleated cells. Results from this study showed that, for EHV-1 strain Ohio 2003, a set of gB, gD, gH, and gL was not sufficient to mediate fusion, suggesting that more glycoproteins or other cell/viral factors may be required for fusion.

The final study examined whether equine cell homologues of the two major alphaherpesvirus receptors, nectin-1 and herpes virus entry mediator (HVEM), are able to serve as entry receptors for EHV-1 and/or contribute to cell to cell spread. Currently, work

v is underway to express each receptor in cells resistant to EHV-1 infection. Once these cell lines are generated, their ability to be infected with EHV-1 will be evaluated.

Overall, this project identified that gB, gD, gH, and gL are not sufficient for cell fusion, and that neurologic and non-neurologic EHV-1 clinical isolates do not differ significantly in replication rate and cell to cell spread.

ACKNOWLEDGMENTS

When I think of the people who have helped me through being in the Master's program, the list unquestionably begins with my advisor, Dr. Art Frampton, who has given me one of the greatest opportunities in my life for intellectual and personal growth by accepting me into his Virology lab. I am grateful for all the important work that I got to do over the past two years, and, equally, for the support and inspiration you have given me along the way. I would like to thank my committee members—Dr. Sonja Pyott, Dr. Alison

Taylor, and Dr. Paulo Almeida—for their support, feedback, and cheer. It has been an honor to work with you.

Every DIS student who has shared with me the ups and downs of generating the expression plasmids, so that the assays of this project were one day made possible—I owe much of the success to your hard work, and I could not thank you enough.

Maria and Stephanie, my lab peers, deserve big thanks (and medals) not only for always helping me in the lab, but also for being true friends. Thank you for all the little things. Thanks to Mom, who encouraged me to go to Graduate school more than anyone, and for always believing in me. My friends—your friendship has been a source of joy and has truly given me the strength and passion in all I do. To all of you, thank you for your love and support.

This research project was financially supported by the grant from the Morris Animal

Foundation. In addition, the project was partially funded by The Bernice Barbour

Foundation, Grayson-Jockey Club Research Foundation, Inc., and North Carolina Horse

Council. Additionally, financial support was provided by University of North Carolina

Wilmington CSURF and eTEAL programs.

vii

LIST OF TABLES

Table Page

1. Titers of stock neurologic and non-neurologic EHV-1 strains...... 61

2. Twenty-four hour replication yields of neurologic and non-neurologic EHV-1 strains .....62

3. PCR primers and amplified target sequence of EHV-1 ...... 63

4. Restriction digest of gB-, gD-, gH-, and gL- TOPO plasmids ...... 63

5. Overview of generated gB-, gD-, gH-, and gL- TOPO plasmids ...... 64

6. Restriction digest of nectin-1- and HVEM- TOPO plasmids ...... 64

viii

LIST OF FIGURES

Figure Page

1. Structure of the Varicellovirus virion ...... 65

2. Twenty four hour virus growth assay in equine endothelial cells...... 66

3. Plaque phenotype of neurologic and non-neurologic EHV-1 strains ...... 67

4. Restriction map of gB-TOPO digested with StuI ...... 68

5. Restriction map of gD-TOPO digested with NdeI...... 69

6. Restriction map of gH-TOPO digested with XmnI ...... 70

7. Restriction map of gL-TOPO digested with PvuII ...... 71

8. Generated gB-, gD-, gH-, and gL- TOPO expression plasmids ...... 72

9. Expression of gB, gD, gH, and gL from Ohio 2003 in B78H1 24 h post-transfection ...... 73

10. Expression of gB from Ab4, L11, and KyA in B78H1 48 h post-transfection ...... 73

11A. Overview of cell to cell fusion assay of HSV and Ohio 2003 gB, gD, gH, and gL ...... 74

11B. Overview of cell to cell fusion assay of HSV and Ohio 2003 gB, gD, gH, and gL ...... 75

11C. Overview of cell to cell fusion assay of HSV and Ohio 2003 gB, gD, gH, and gL ...... 76

12. Cell to cell fusion assay of Ohio 2003 gB, gD, gH, and gL (B78H1-C2) ...... 77

13. Diagnostic EHV-1 infection of B78H1-C2 cell line ...... 78

14. Restriction map of nectin-1-TOPO digested with PvuII ...... 79

15. Generated nectin-1-TOPO expression plasmids ...... 80

16. Restriction map of HVEM-TOPO digested with StuI ...... 81

LIST OF ABBREVIATIONS

β-gal β-galactosidase

2xYT rich yeast-tryptone media

AMV avian myeloblastosis virus

B78H1 mouse melanoma

BoHV-1 bovine herpesvirus 1 bp base pair cDNA complimentary deoxyribonucleic acid

CHO Chinese hamster ovary

CNS central nervous system

CO2 carbon dioxide

DMEM Dulbecco’s modified Eagle’s medium

DNA deoxyribonucleic acid

EBV Epstein-Barr virus

EHM equine herpes myeloencephalopathy

EHV equine herpesvirus g or gp glycoprotein hpi hours post infection

HSV herpes simplex virus

HVEM herpesvirus entry mediator

IE immediate early

MHC-I major histocompatibility complex class I

MOI multiplicity of infection

ONPG o-nitrophenyl-β-D-galactopyranoside

PBMC peripheral blood mononuclear cell

PBS phosphate buffered saline

PCR polymerase chain reaction pfu plaque forming unit

RK13 rabbit kidney 13

RNA ribonucleic acid

RT-PCR reverse transcription polymerase chain reaction

SOC super optimal broth with catabolite repression

SuHV-1 suid herpesvirus 1

UV ultraviolet

VZV varicella-zoster virus

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

INTRODUCTION

Equine herpes virus type 1 (EHV-1) is a major pathogen in the equine that causes clinical disease in horses and substantial economic losses 1. EHV-1 causes a wide range of symptoms including respiratory disease, perinatal foal disease, and periodically may lead to abortion and neurologic disease termed equine herpes myeloencephalopathy (EHM) 1–4.

Section 1 of this literature review will provide background on EHV-1 taxonomy and structure. Section 2 will describe the EHV-1 lifecycle. Finally, section 3 will detail EHV-1 disease pathogenesis, as well as current vaccines and antiviral therapies against EHV-1- induced diseases.

1: EQUINE HERPESVIRUS 1 (EHV-1) OVERVIEW

EHV-1 History

Before EHV-1 became known by its current name, it was known as equine abortion virus, with the name subsequently changed to equine rhinopneumonitis virus 5. EHV-1 was first isolated in the late 1960s–early 1970s, and soon afterwards, strains causing abortion and respiratory disease were distinguished 6–8. Beginning in the early 1980s, EHV-1 and

EHV-4 were no longer considered subtypes of the same virus 9,10. EHV-4 is generally associated with respiratory disease, while EHV-1, in addition to causing respiratory disease, may lead to the development of neurologic disease and abortion in pregnant mares

11. In 1992, the International Committee for the Taxonomy of Viruses designated the family

Herpesviridae, along with its three subfamilies, and EHV-1 was officially recognized 12.

EHV-1 Classification and Structure

EHV-1 belongs to the order Herpesvirales, which includes three families:

Herpesviridae, Alloherpesviridae, and Malacoherpesviridae 13. Apart from structure, all herpesviruses share a number of biological characteristics: (1) they possess enzymes for nucleic acid metabolism, DNA synthesis, and protein processing, (2) their DNA replication and capsid synthesis occurs in the nucleus of the infected cell, (3) they establish latency in the host, and (4) their completed replication cycle usually leads to the destruction of the host cell 14.

Family Herpesviridae, to which EHV-1 belongs, is composed of three subfamilies based on the variation in biological properties: Alphaherpesvirinae, Betaherpesvirinae, and

Gammaherpesvirinae 15. Alphaherpesviruses exhibit a wide host range, relatively short reproductive cycle, rapid spread of the infection, the ability to destroy host cell and to establish latency in the sensory ganglia of the host. Betaherpesviruses are different in that they exhibit a more restricted host range, have a relatively long reproductive cycle, and a slow spread of the infection. Like alphaherspeviruses, betaherpesviruses also establish latency - in lymphoreticular cells and, potentially, secretory cells of the host. Lastly, gammaherpesviruses have the most narrow host range, usually restricted to the family or the order of the natural host. Gammaherpesviruses replicate in lymphoblastoid cells and establish latency in the lymphoid tissue, in either B or T lymphocytes 12.

Herpesviruses belonging to the family Herpesviridae possess a linear, double- stranded DNA contained in a core inside of an icosahedral capsid that is approximately 125 nm in diameter and 15 nm thick 16,17. The capsid is surrounded by an amorphous protein layer, called a tegument, that is in turn enclosed in a lipid envelope containing numerous

2 surface glycoproteins 16 (Figure 1). The capsid of HSV-1 (a prototypical herpesvirus) is composed of 12 pentons and 150 hexons. Each of the hexons is formed by the major capsid protein (encoded by UL19), six molecules of VP5, and six molecules of VP26 (encoded by

UL35). Both the major capsid protein and VP26 form a ring atop of the VP5 subunit. Eleven pentons of the capsid are made of VP5 monomers, whereas the twelfth penton constitutes a cylindrical portal composed of twelve monomers of the UL6 gene product 17. The portal contains an axial channel, through which the DNA enters the capsid during progeny virion assembly, as well as leaves the capsid to enter into the nucleus 17. The tegument contains at least fifteen distinct proteins, some of which are enzymes (ubiquitin-specific protease) and transcription factors 17. The envelope of HSV-1 contains over a dozen proteins and glycoproteins mediating virus binding, entry, egress, cell to cell spread, as well as virulence and pathogenicity 17,18.

EHV-1 belongs to the subfamily Alphaherpesvirinae, which contains five genera and thirty six species 19,20. The virus is classified in the genus Varicellovirus, which also contains the closely related EHV-4 (equine rhinopneumonitis virus), as well as other equid viruses, such as EHV-3 (equine coital exanthema virus), EHV-8 (asinine herpesvirus 3), and EHV-9

(gazelle herpesvirus 1) 19. Three more equine viruses, EHV-2, EHV-5, and EHV-7, have been identified, and all belong to Gammaherpesvirinae 19. The natural host for EHV-1, -2, -3, -4, and -5 is the horse, while for EHV-6, -7, and -8 the natural host is the donkey 21. Within

Alphaherpesvirinae, EHV-1 is closely related to human herpes simplex virus 1 and 2 (HSV-1 and HSV-2), causative agents of orolabial and genital herpes, and varicella-zoster virus

(VZV), a causative agent of chickenpox/shingles. EHV-1 is similar, but less closely related to bovine herpesvirus 1 (BoHV-1) and suid herpesvirus 1 (formerly pseudorabies virus)

(SuHV-1) 19,22. According to the United States Department of Agriculture, EHV-1, EHV-3, and EHV-4 pose the most significant risk to the equine, as these viruses are associated with the most severe clinical disease and economic losses 23.

EHV-1 possesses a typical structure of the Herpesviridae viruses. At its core, EHV-1 contains a linear, double-stranded DNA that is approximately 150,200 base pairs (bp) long

24. The EHV-1 genome is comprised of two regions, long and short. The Unique long (UL,

112,870 bp) and Unique short (US, 11,861 bp) regions are each flanked by inverted repeat regions called the internal repeat (IR) and the terminal repeat (TR) 24. The complete sequence of the EHV-1 genome revealed that the virus contains at least 76 distinct genes; with four of them (gene 64, 65, 66, and 67) duplicated 24. Subsequent studies of the EHV-1 genome revealed that the majority of the 80 translated proteins play a role in viral replication, with over a half of them encoding for structural components of the virus 17,24,25.

EHV-1 Glycoproteins

Envelope glycoproteins of EHV-1 play a crucial role in viral attachment, entry (via fusion of viral envelope with host cell membranes), cell to cell spread, egress, as well as chemokine binding and inflammation 18. EHV-1 encodes for a total of twelve glycoproteins: glycoprotein (g) B (gB), gC, gD, gE, gG, gH, gI, gK, gL, gM, gN, and gp2 18.

Glycoproteins gB, gD, gH, gL, and gN are conserved in all herpesviruses, suggesting their importance 26. Glycoproteins gB, gD, and the gH-gL heterodimer are required for viral growth in cell culture and are therefore considered essential for infection, with gK most likely essential 18.

Glycoproteins gB, gD, and gH-gL compose the Herpesviridae viral entry system and are crucial for the composition of infectious virions 27. Across all herpesviruses, glycoproteins gB and gH-gL constitute a conserved fusion machinery component, with different herpesviruses using additional non-conserved accessory components that aid in the fusion event through binding an entry receptor, gH-gL, and/or through determining cell tropism 28,29. In HSV, cell fusion and entry requires glycoproteins gB, gD, and gH-gL 30–41.

Functionally, gB and gD are crucial for viral penetration and cell to cell spread 42–44.

In HSV-1, viruses with deletions for either gB, gD, or gH could not initiate the infection

31,39,45. In EHV-1, viruses lacking gD have been shown to attach to the target cell, but are unable to penetrate it 46.

Across herpesviruses, gH has been shown to be essential for viral entry, infectivity, and cell to cell spread 47. Structural analysis of HSV-2 gH-gL revealed that the two glycoproteins have a strong interaction, require each other for proper folding and therefore form a tight complex that varies among herpesviruses 28. In HSV-1, the gH-gL heterodimer was able to cause a hemifusion event in the absence of gB, suggesting the fusogenic property of gH 48. Glycoprotein gL is required for processing and incorporation of gH onto the surface of the host cell, from which the viral envelope is derived. A study has shown that the envelopes of HSV mutants lacking gL contained no gH or gL on the envelope surfaces 40.

Studies on a number of alphaherpesviruses (namely, HSV-1, SuHV-1, duck enteritis virus, and EHV-1) have demonstrated the importance of glycoprotein gK in viral entry and replication, as well as cell to cell spread and viral egress 49–54. In 2012, Azab and colleagues discovered that gK is essential for EHV-4 viral replication in vitro 55.

Despite being non-essential for virus replication in cell culture, glycoproteins gC, gE, gG, gI, gM, gN, and gp2 play important roles in other viral infectious processes within the host 25. For instance, the gI-gE complex is essential for efficient cell to cell spread of EHV-1

56. Glycoprotein gM plays a role in virus penetration, as well as its cell to cell spread 57. In

HSV-1, gM has been shown to be used for incorporation of gH-gL into virus particles, as viruses lacking gM showed significantly reduced amounts of gH-gL expressed on the envelope surface 26. It has been found that gM aids in internalization of gH-gL to the cytoplasmic membranes during infection, as gM-deficient HSV-1 incorporated gH-gL less successfully, resulting in less efficient entry and cell to cell spread 26. No other glycoprotein is believed to have a similarly strong effect that gM has on gH-gL heterodimer localization and, subsequently, assembly of infectious HSV-1 26,58. In EHV-1, enveloped progeny virions lacking either gM or gp2 have been shown to be severely impaired for egress, but cell to cell spread is not affected 59.

Glycoprotein gG has been found to bind a broad range of chemokines and is therefore speculated to play an important role in the viral immune evasion during the infection 60. It has been shown that gG, when administered in low doses, exacerbated respiratory disease in mice infected with EHV-1 61.

Glycoprotein gN has been associated with processing of gM, while gp2 has been found to play a role in inflammation 59,62.

2: EHV-1 LIFECYCLE

Alphaherpesvirus life cycle can be divided into three major stages: (1) attachment and entry, (2) replication, and (3) egress. The life cycle of EHV-1 has been extensively

6 characterized and is used as a primary source for the overview below, with relevant alphaherpesvirus references cited when applicable.

EHV-1 Attachment and Entry

In permissive cells, closely related alphaherpesvirus HSV-1 requires 18 to 20 hours to complete viral replication 16.

The initial step in EHV-1 replication is attachment to the target cell surface, a process that involves glycoproteins gB, gC, gD, and possibly the gH-gL complex 42–44,63,64.

Initial attachment is achieved via glycoproteins gB and gC binding to glycosaminoglycans on the host cell plasma membrane, which are polysaccharides consisting of repeating units of disaccharides 64,65. As this interaction is unstable, a subsequent interaction of gD with cell surface receptor is required 44,66,67. The process of binding of gD to the cell surface entry receptor triggers conformational changes in the glycoproteins and leads to viral entry

68.

Two major gD entry receptors for a number of alphaherpesviruses are nectin-1

(also known as herpesvirus entry mediator C, HveC) and herpesvirus entry mediator

(HVEM) (also known as herpesvirus entry mediator A, HveA). Nectin-1 belongs to an immunoglobulin superfamily of adherens junction transmembrane glycoproteins that play a role in cell to cell adhesion and neuronal synapse formation 69. Nectin superfamily currently includes nectin-1, -2, -3, and -4, all of which share low homology (35% of amino acids identical) and have been grouped together based on the interaction of the receptor cytoplasmic domains with F-actin-binding protein afadin 69–72. Additionally, the superfamily includes poliovirus and vitronectin receptor CD155 (formerly poliovirus

7 receptor), along with Tage4, the putative rodent homologue 73,74. CD155 has been included due to its high homology to nectin-2 (54% amino acids identical). Nectins are localized at cadherin-based junctions and, except for Nectin-4, are expressed in a wide range of adult tissues, including hematopoietic, neuronal, endothelial and epithelial cells 71,75. Nectin-1 is the primary entry receptor for HSV-1 and HSV-2 70,76,77. In addition, nectin-1 mediates entry of BoHV-1, SuHV-1, and macacine herpesvirus 1 75,78,79. Nectin-2 is used for entry of HSV-1 containing amino acid substitutions in gD at positions 25 or 27, SuHV-1, and provides a weak entry activity for HSV-2 80,81. Nectin-3 has been added to the superfamily in 2000, and it is currently unknown whether it can mediate alphaherpesvirus entry 71. Nectin-4 is mainly expressed in the embryonic tissue, and it is re-expressed in breast carcinoma 72,82.

Nectin-4 has been shown to mediate entry of three viruses of Morbillivirus genus: measles virus, canine distemper virus, and peste-des-petits-ruminants virus 20,83–86.

HVEM is a membrane integral protein belonging to tumor necrosis factor receptor superfamily87. HVEM acts as one of the primary entry receptors for HSV-1 88. HVEM also provides a low-efficiency entry for HSV-2 89. It is presently unknown whether nectin-1 and

HVEM are able to act as entry receptors for EHV-1.

In 2010, Equus caballus major histocompatibility complex class I (MHC-I) was identified by our lab to be a novel gD entry receptor for EHV-1 67,90. While MHC-I acts as an entry receptor for EHV-1, others, as yet to be identified, entry receptors are hypothesized to exist as EHV-1 can enter some cell types even when the MHC-I receptor is blocked with antibodies.

Binding to the entry receptor leads to the fusion of the viral envelope with cell membranes, a process that is mediated by viral glycoproteins and possibly cell factors.

Pertel and colleagues established that the minimum set of glycoproteins required for HSV-1 to fuse with cell membranes of permissive cell lines (Vero, HeLa, and PEAK) comprise gB, gD, and gH-L; the group established that cell surface heparan sulfates are not required for the fusion event to occur 91. Although the subset of glycoproteins required for fusion of

EHV-1 with cell membranes is considered to comprise glycoproteins gB, gD, and gH-gL, to date, no study has been published that specifically addresses the subject of the minimum subset of EHV-1 glycoproteins required for the fusion event 92.

Two entry pathways have been characterized for alphaherpesviruses – fusion of the viral envelope at the plasma membrane (non-endocytic pathway) or at the endosomal membrane (endocytosis) 16. The process of selection of the entry pathway is unclear, most likely due to the variation among herpesviruses in the factors that influence the entry process. In EHV-1, viral factors (such as glycoproteins), cell factors (gD receptors), as well as the target cell type all influence the entry route 93–97. For instance, EHV-1 enters rabbit kidney (RK-13) and equine dermal (ED) cells via fusion at the plasma membrane at neutral pH, while CHO-K1, peripheral blood mononuclear cells (PBMCs), and equine brain microvasculature endothelial cells are entered via pH-dependent or -independent endocytosis 98–100. It is important to note that with different cell types, in addition to the differences in the entry mode, herpesviruses may also require a different subset of glycoproteins for entry. Such variations have been observed in the gammaherpesvirus

Epstein-Barr virus (EBV), which, with distinct cell types, uses both endocytic and non- endocytic entry pathways, and has been found to require a particular subset of glycoproteins for each process 101–103.

Regardless of the mode of entry, in HSV, the set of glycoproteins required for endocytic and non-endocytic pathway has been found to be nearly identical 96. For fusion at the plasma membrane and production of infectious virus particles, in Vero cells, HSV requires gB, gD, and gH-gL 31,34,38,40. For endocytosis and successful infection, in both HeLa and CHO cells expressing nectin-1 (CHO-nectin-1), HSV also requires gB, gD, gH-gL, and not gC 96.

Transition from the virus attachment to entry is rapid and efficient 16. In Vero cells, infection with HSV yields naked capsids observed in the host cell cytoplasm within minutes

104. Following entry into the cell, dynein transports de-enveloped EHV-1 capsids via microtubules to the nuclear pore complexes where the capsid docks at the nuclear pore complex and releases the viral DNA into the nucleus through the capsid portal, which leads to gene expression 16,105. Kinetics of the capsid transport are rapid, with capsid reaching its destination within one hour of entry 16.

Additionally, the virus establishes a life-long reservoir in its host via latency in the reported sites of trigeminal ganglia, circulating lymphocytes, and lymphoid tissue 106–112.

While it is well established that stress can cause the virus to reactivate, specific details on the contributing factors, as well as the events leading up to EHV-1 establishing latency, are in need of more research 107,113. Latent EHV-1 has been successfully confirmed by polymerase chain reaction 106. Similarly to related alphaherpesviruses, EHV-1 efficiently evades host immunity by spreading from an infected cell to the adjacent, uninfected cell, among other immune system evasion strategies, such as antibody-dependent cell lysis, cytotoxic T lymphocyte-mediated lysis of infected cells, as well as disruption of host cytokine activity 114,115. As outlined in section 1 of this Introduction, glycoproteins gB, gD,

10 gI, gE, gK, and gM have been found to aid, to a varying degree, in cell to cell spread of EHV-

1, with gI and gE playing the key role in cell to cell spread process. The latter has been shown in a study where RK13 cells infected with EHV-1 strain RacL11ΔgIΔgE (with deleted sequences for glycoproteins gI and gE) yielded a small plaque phenotype comparing with a parent strain RacL11, which yielded a large plaque phenotype 116.

EHV-1 Replication

Once the DNA enters the nucleus, transcription of the viral genome takes place, which is temporally and sequentially controlled 16. Viral genes are grouped into three categories, according to the timing of their transcription in the infected cells: (1) the immediate early (IE) or α genes, (2) the early or β genes, and (3) the late or γ genes 117.

First, a solo EHV-1 IE gene is transcribed into a single mRNA, a process crucial for virus replication 117–120. The IE gene is expressed independently of de novo viral protein synthesis, with the aid of a viral tegument protein acting as a transactivator 16. In HSV,

VP16 fulfills the role, while in EHV-1, it is ETIF (also known as VP16-E and α-trans-inducing factor (α-TIF)) 16,121. The IE gene product ensures a proper subsequent expression of the early and late genes. Following the expression of the IE gene, E genes are expressed, which are responsible for the viral DNA replication 16. Finally, the late genes are expressed last and generally encode for viral structural proteins, which allows for the production of progeny virus particles 16.

EHV-1 Assembly, Release, and Cell to Cell Spread

Once the L genes have been expressed, virus capsids are constructed within the nucleus of the host cell 16. Prior to exiting the cell, the virus undergoes an envelopment— de-envelopment—envelopment process 122. The process begins when capsids are filled with viral DNA and bud off through the host cell inner nuclear membrane, thereby acquiring a primary envelope 122. The capsids are then de-enveloped via fusion with the outer nuclear membrane, and undergo a secondary envelopment in the cytoplasm of the host cell. The exact location of herpesvirus secondary envelopment is not clear; evidence suggests that viral membranes originate from Golgi, trans-Golgi network and/or endosomal membranes 123. As with the other alphaherpesviruses, the complication of EHV-

1 assembly and egress stems from the fact that the processes differ not only between the members of the same subfamily, but also between cell systems 124–126.

Once released, the virus may also spread directly from cell to cell, thereby evading neutralizing antibodies of the host immune system 16. A number of cell and viral factors are essential for facilitating such cell to cell spread. In HSV, this process requires gD and a cell entry receptor 127. In HSV-1, the process is aided by gI and gE, which form a complex and are conserved in all alphaherpesviruses 122. In EHV-1, glycoproteins required for virus entry (gB, gD, gH-gL) have also been shown to be essential in cell to cell spread 42,44,63.

Glycoproteins gI and gE have been shown to be important in facilitating cell to cell spread in EHV-1 56,128. Additionally, glycoproteins gM and gK aid in cell to cell spread as well 51,57.

Lastly, infected cells generally do not survive the infection due to viral cytopathic effects and undergo cell lysis, releasing the virions 16.

3: EHV-1 PATHOGENESIS, VACCINES, AND ANTIVIRAL THERAPIES

EHV-1 Pathogenesis

The primary modes of EHV-1 spread are through virus-shedding in-contact horses, air, and fomites 25. Horses exposed to EHV-1 for the first time have been found to secrete the virus through nasal shedding from day 1 for as long as 15 days from the onset of the infection 129. EHV-1 affects horses at a very young age, and may affect a wide range of age groups 130.

EHV-1 affects at least three different cell types in three different organ systems, causing a systemic response 18. Initially, EHV-1 infects and replicates in the epithelial cells of the upper respiratory tract and the draining lymph nodes, resulting in a fever and nasal shedding. Within the 24 hours from the onset of infection, EHV-1 breaches the respiratory epithelium and infects the second cell type, PBMCs of the immune system, in the deep connective tissue of the respiratory tract 131,132. The infection of the immune system cells and the leukocyte-associated viremia occurs 4 to 6 days from the onset of the infection and lasts on average until 9 to 14 days post infection; viremia is also responsible for disseminating EHV-1 to the internal organs, the tertiary site of virus replication 18,129,133.

With access to PBMCs, EHV-1 is able to spread to the uterus of a pregnant mare or to the central nervous system where the virus replicates in the third susceptible cell type, endothelial cells 18. Resulting vasculitis and ischemic thrombosis can lead to the late-term abortion of the fetus in pregnant mares or to the development of neurological disease, EHM

134,135. The extent of the clinical signs of EHM is highly variable, EHM generally occurs sporadically, and reaches the peak intensity within two to three days of the onset of the infection 25. Manifestation of EHM ranges from temporary ataxia, limb weakness,

13 proprioceptive deficiency, stumbling and falling to complete paralysis requiring euthanasia for humane reasons 25.

While research has shown that all EHV-1 strains have the potential to induce respiratory disease and abortion in pregnant mares, only particular (neurologic) strains will lead to EHM outbreaks 136,137. The group of strains that is said to have high neurologic potential (P<0.0001) is distinguished by a point mutation in an open reading frame 30

(ORF30), which encodes for the viral DNA polymerase 137,138. The genetic marker consists of a non-synonymous A-to-G substitution at nucleotide 2254 (A→G2254) and results in a change from asparagine to aspartic acid residue at amino acid 752 (N→D752) 137,139,140.

According to the findings of the study, EHV-1 strains containing adenine at nucleotide 2254

(A2254) are considered to be non-neurologic (also referred to as abortigenic) and are associated with the development of respiratory disease and/or abortion, while strains containing guanine at the same position (G2254) are mainly, but not always, associated with the development of neurologic disease (strains also referred to as paralytic). Findings of a comprehensive real-time PCR study have shown that weanling foals inoculated intranasally with neurologic EHV-1 strains have in all cases expressed an earlier PBMC- associated viremia that was also of a greater magnitude and longer duration, compared to the viremia with non-neurologic strains 136. Delivery of a greater and longer-lasting EHV-1 viral load to the endothelium of the equine host increases the risk for the subsequent development of EHM by 5 times, when compared with the risk in the foals infected with non-neurologic EHV-1 strains 136. Potentially neurologic and non-neurologic EHV-1 strains can be accurately distinguished using allelic discrimination via real-time PCR assay 141,142.

While the point mutation in the viral DNA polymerase is often associated with neurovirulence of EHV-1, additional factors, such as induction of the host inflammatory response and variation in viral structural components, determine neurovirulence as well.

Infection of equine endothelial cells with particular EHV-1 strains may lead to the upregulation of specific host inflammatory genes resulting in a higher magnitude of vasculitis. Increased vasculitis may, in turn, lead to the greater damage of the surrounding neuronal tissue and, subsequently, a higher risk of the equine developing EHM.

Glycoproteins with specific amino acid composition contributing to virulence may lead to the particular EHV-1 strain gaining selective advantage in terms of attachment to the host cell, entry, cell to cell spread, and other functions in which the particular glycoprotein plays a role. Such advantage could manifest itself in a variety of ways to ultimately induce severe disease in a horse. Specifically, the virus may be able to attach to certain cell types more efficiently, or it may recognize a broader range of entry receptors and thus infect a broader range of cell types, when compared to the virus containing no advantageous amino acid sequences in the glycoprotein structure. Structural changes in one or more glycoproteins may lead to enhanced replication and/or cell to cell spread, which could all lead to an earlier onset of viremia and deliver a greater viral load to the endothelium, a cell type that, when damaged, is closely associated with the development of EHM.

EHV-1 Vaccines

Among the vaccines against EHV-1, the two major groups include (1) modified live virus (MLV) and killed virus (KV, also known as inactivated) vaccines, and (2) subunit and recombinant vaccines. Both MLV and KV vaccines serve to partially protect horses against

EHV-1-induced respiratory disease and abortion and are available on the market, while subunit and recombinant vaccines for the same purpose are in development and are not yet available 143. Despite the widespread vaccination practices, no currently registered

EHV-1 vaccine is licensed to be completely effective against preventing EHV-1 infection or disease, and no EHV-1 vaccine claims to provide any level of protection against the neurologic form of EHV-1 infection 25.

Rhinomune (Boehringer Ingelheim, St. Joseph, MO) and Prevaccinol (MSD, Munich,

Germany) are the only two currently available licensed MLV vaccines in North America and

Europe, respectively 143. Both vaccines are based on an attenuated avirulent EHV-1 RacH strain, which contains a deletion in the IR6 protein that causes the strain's attenuation 144.

Both vaccines are considered to trigger a more effective immune response than KV vaccines, as administration causes both humoral and cell-mediated immunity to be robustly activated 143. While with MLV vaccines, safety is always a concern, these vaccines have an exceptional safety record and are able to protect horses against respiratory disease; however, the efficacy of viremia, abortion, and neurologic disease prevention has been unclear 145. In a 2012 study, it was shown that there were no differences in abortion rates between horses vaccinated with an MLV vaccine (Prevaccinol, MSD) that does not claim to protect against EHV-1 induced abortions and inactivated combination vaccine

(Duvaxyn® EHV 1,4, Zoetis) that does claim to protect against EHV-1 induced abortions 146.

Important of note is that recent evidence of protection against neurologic disease

(experimental conditions) has been shown 147. For the neurologic disease study, three groups of five female horses, aged 3-10 years, in each group received two intramuscular shots of MLV vaccine (Rhinomune), inactivated vaccine (Fluvac Innovator 6 combination

16 vaccine, Fort Dodge), or placebo (control), at 30-day intervals. Horses were then challenged with neurologic EHV-1 strain Ohio 2003, 59 days after the first treatment. All five horses from the MLV group showed no neurologic anomalies over the course of the experiment

(14 days), comparing to the three out of five horses showing neurologic signs in both the inactivated vaccine group and the control group 147. High viral loads were detected in all three groups; however, MLV group recovered from the fever more quickly than inactivated and control groups, and had significantly lower levels of virus shedding 147.

KV vaccines are the dominant vaccines on the market, based almost exclusively on the preparations of inactivated virus, and the majority are combination therapies directed towards other equine pathogens 143. The only three vaccines that claim to protect against

EHV-1-induced respiratory disease and abortion are all KV and are labeled Pneumabort-

K® +1B, Duvaxyn® EHV 1,4 (both by Zoetis, Florham Park, NJ), and Prodigy® with

Havlogen® (Intervet Inc., Merck Animal Health, Summit, NJ). The basis of inactivated vaccine activity is the ability to induce high level of virus neutralizing antibodies, which reduces virus shedding through nasal secretions and reduces clinical symptoms, particularly the respiratory disease. In regards to abortion, it has been shown that humoral immunity is not the most effective method of protection against this EHV-1 infection sequela, as it has been observed in pregnant mares irrespective of high level of virus neutralizing antibodies 148.

The second group of vaccines in development, subunit and recombinant vaccines, hold promise and include new generation MLV, subunit/vector vaccines, and DNA vaccines

143.

New generation MLV vaccines are based on current strains, in comparison to conventional MLV vaccines, based on the RacH strain, which is genetically distant from epidemic EHV-1 strains. High genetic similarity of new generation MLV vaccines is an advantage; however, targeted genes, such as genes involved in immune evasion (UL56,

UL49.5, and gG), are not fully optimized to provide efficacy, and a number of virulence- associated genes are not attenuated to the right degree, which is a safety concern 143.

Subunit and vector vaccines are composed of mutant EHV-1 strains with deletions for distinct virulence-associated genes, such as those coding for viral glycoproteins, the immediate early gene, or genes responsible for immune evasion. While numerous studies undertaken have shown that this group of vaccines is safe, limited immune response induced is a disadvantage, as it only leads to partial protection of the equine host

56,143,149,150.

DNA vaccines, such as plasmids expressing glycoproteins gB, gC, or gD, or plasmids expressing viral genes (such as immediate early protein and early UL5 gene) have been shown safe and able to induce cell-mediated immunity, although poor antigenicity was found to be a concern. In a 2010 study, ponies of MHC-I haplotype A3/B2 treated with a recombinant modified vaccinia virus Ankara expressing EHV-1 IE gene and challenged with

EHV-1 have shown reduced clinical signs and viremia, despite low antibody titers before challenge 151.

It is clear that, although currently available therapies successfully induce immune response, no vaccine to date is able to induce all of the desired components of protective immunity in the horse. The strongest protection provided lies in the reduction of virus shedding and overall alleviation of severity of clinical signs. No vaccine has shown to

18 efficiently reduce viremia, which allows the virus to spread to either the endometrium of the uterus in a pregnant mare and cause abortion or to the endothelial cells of the central nervous system and lead to neurologic damage. Also, no vaccine claiming to protect against

EHV-1-induced neurologic disease is currently available 152. The ideal vaccine, in addition to being safe, would induce both high levels of virus neutralizing antibodies at mucosae, as well as robust and broad cytotoxic T lymphocyte immunity in equines with various antigenic backgrounds 143. Toward this end, MLV vaccines, provided they are safe, hold the most promise for providing complete protection for the horse against one of the most devastating equine viral diseases.

EHV-1 Antiviral Therapies

Antiviral drugs present an attractive alternative to vaccines against EHV-1 infections, as these compounds, being nucleoside and nucleotide analogues, affect virally encoded enzymes directly terminating viral DNA replication 17. These antivirals work to reduce viral replication and virus spread in infected horses, as well as prevent viral replication in in-contact horses. While many antiviral drugs are available for treating herpesvirus infections, many of these compounds mimic four natural deoxynucleosides: adenosine, guanosine, thymidine, and cytidine and act as viral DNA replication inhibitors 17.

Acyclovir, an analogue for guanosine, is the most abundantly prescribed drug against herpesvirus infections, in particular, HSV. The drug has low toxicity on healthy host cells while successfully inhibiting viral DNA replication in infected cells. The reason for the compound having low toxicity on healthy cells is that it is activated specifically by the virus-encoded enzymes; first, the virus-encoded thymidine kinase phosphorylates

19 acyclovir, followed by two additional cellular kinase phosphorylations 17. The activated drug is then able to be incorporated by the viral DNA polymerase into the growing viral

DNA chain 17. However, because acyclovir is deficient in a 3'-OH group, no further substrates may be added to the DNA chain, and DNA replication is thus halted. Naturally, herpesviruses have evolved to be acyclovir-resistant through modification of their enzymes. Such resistance occurs when the mutant viral thymidine kinase is unable to phosphorylate acyclovir, as well as when the DNA polymerase is unable to assimilate the activated drug into the growing DNA chain. Chemical modifications of acyclovir, such as valacyclovir, penciclovir, and famciclovir, act analogously to acyclovir, while allowing for more efficient uptake by the host after oral ingestion 17. Ganciclovir is also a guanosine analogue, resembling acyclovir.

Foscarnet is a pyrophosphate analogue simulating the pyrophosphate part of the nucleoside triphosphates. Like acyclovir, this compound acts against the viral DNA polymerase. Because the molecule is a non-nucleoside inhibitor of viral DNA replication, it is often used to treat acyclovir resistant mutants, although foscarnet-resistant mutant viruses have also emerged.

Regardless of the antiviral drug, all are geared towards reducing clinical signs and symptoms of the herpesvirus infection. However, no anti-herpesvirus drug is able to cure latent infection, neither is it able to modify the risk, frequency, or severity of recurring infections, once the drug administration is discontinued 17.

According to the American Quarter Horse Association 153, it is recommended to begin anti-EHV-1 therapy only once the fever is detected, due to high cost and questionable efficacy of anti-viral treatments when the horse has merely been potentially exposed to

EHV-1 infection. For horses who are at high risk of developing EHM, anti-viral drugs may aid in reducing the risk of developing the severe EHV-1 infection outcome; in this case, the drug of choice for prophylactic therapy is valacyclovir. Acyclovir, due to its failure to be absorbed efficiently from the gastrointestinal tract, is unlikely to carry a therapeutic value.

For horses who have already developed EHM, the efficacy of anti-viral drug therapy is questionable and administration of anti-inflammatory drugs, such as flunixin meglumine, is recommended. Should the anti-viral drugs be used, however, the use of intravenously delivered ganciclovir is recommended for its potentially greater efficacy against the disease, when compared to valacyclovir.

The main of goal of the proposed research was to gain a better understanding of

EHV-1 infection mechanics in the equine and to provide novel data that could be used for the development of effective therapies against severe outcome of EHV-1, EHM. As outlined in section 3 of the Introduction, currently available therapies do not protect the horse against the neurologic disease. The two major directions of this project aimed to address the gaps in current knowledge via (1) investigating potential differences between neurologic and non-neurologic EHV-1 strains in terms of viral replication and cell to cell spread, in equine endothelial (EE) cells, and (2) examining critical viral and cell factors that contribute to the efficient fusion of EHV-1 with host cell membranes, as well as aid in the efficient cell to cell spread. Viral factors studied comprised EHV-1 glycoproteins, while the cell factors examined included two major alphaherpesvirus entry receptors, nectin-1 and herpesvirus entry mediator (HVEM). Novel data from this research was aimed at contributing to the field of engineering of EHV-1 antiviral therapies that would target and

21 inhibit key virus-host interactions in the equine and, subsequently, reduce the risk of the development of EHM.

CHAPTER 1: CLINICAL ISOLATE ANALYSIS

MATERIALS AND METHODS

Cells and Viruses

Rabbit kidney (RK13) cells were provided by Dennis O'Callaghan (Louisiana State

University Health Sciences Center, Shreveport, LA) and equine cardiac artery endothelial

(EE) cells were provided by a Udeni Balasuriya (University of Kentucky Maxwell H. Gluck

Equine Research Center, Lexington, KY). All cells were grown in Dulbecco's Modified

Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2% of

10,000U/mL Penicillin G and 10,000µg/mL Streptomycin in normal saline (P/S) (JR

Scientific, Inc., Woodland, CA) ("complete DMEM"). Cells have been maintained at 37°C 5%

CO2.

A total of 9 neurologic and 8-non-neurologic EHV-1 strains were used in this study.

Neurologic EHV-1 strains include Ab4, OH03, T313, T954, T955, T956, T964, T967, and

T970. Non-neurologic EHV-1 strains include L11 (in the form of EHV-1 recombinant virus

L11ΔgIΔgE), KyA, T61, T75, T220, T493, T547, T572. L11ΔgIΔgE (L11) was described previously and contains a LacZ reporter gene in place of glycoproteins gI and gE 116. L11 is deficient in cell to cell spread as evident by a small plaque phenotype, but the virus does not different in entry efficiency compared to the wild-type RacL11, as shown in a one-step growth assay 116.

Generation of Virus Stocks

Except for strains Ab4, OH03, L11, and KyA, a stock of each virus had to be generated and viral yields calculated. A total of 300µL of each virus sample in 10mL DMEM was added to a confluent monolayer of RK13 cells in a 150mL tissue culture flask, and incubated at 37°C 5% CO2 until significant cytopathic effects (CPE) were observed. Total virus was harvested and viral yield determined via a standard plaque assay. To harvest the total virus, supernatant was collected from the flask and placed into a 15mL tube and then the tube was centrifuged at 10,000rpm for 10 minutes to pellet the cells. Supernatant was transferred to a new 15mL "supernatant" tube and stored on ice. The remaining cell pellet was resuspended in 100µL of the supernatant, and the sample was subject to three freeze- thaw cycles between -80°C and 37°C to release the viral particles from the cells. After three freeze-thaw cycles, the cell solution was recombined with the supernatant, and the resulting sample was centrifuged at 10,000rpm for 10 minutes. The supernatant was then passed through a 0.8µm filter, and virus aliquots were stored at -80°C.

For a standard plaque assay, a total of 4 x 105 RK13 cells were seeded in a 24-well plate (18 wells) and incubated for 24 hours at 37°C 5% CO2. Virus sample was thawed and subjected to a ten-fold dilution, from neat fraction to 10-5. For each dilution step, 100µL of the virus sample was diluted in 900µL of DMEM. One hundred microliters of each dilution was then added onto the monolayers of RK13 cells in a 24-well plate and incubated at 37°C

5% CO2 for 90 minutes, with plate rocked every 10 minutes to enhance viral entry.

Following the 90 minute incubation, cells were overlaid with 1mL of semi-solid complete

DMEM containing 1% methylcellulose. Cells were incubated at 37°C 5% CO2 for 3 days to allow for plaque formation. After 3 days of incubation, semi-solid media was aspirated off

23 and cells were stained with 1% crystal violet solution for 60 minutes. Crystal violet contains 50% methanol, which acts as a fixative. The stain was washed off with deionized water and cells allowed to dry. Cells were examined under a Motic inverted light microscope and plaques were counted in each of the three wells of the same dilution. Viral titer, expressed as plaque forming unit per mL (pfu/mL), was calculated as follows:

(average number of plaques in a well) x (dilution factor) x (fraction of 1mL added on to the cells), where dilution factor is equal to the serial dilution of the well (e.g., 10-5 is equal to

105) and fraction of 1mL added on to the cells is 10 (1000 µL/100µL).

Viral Replication Analysis of EHV-1 Isolates in EE Cells

To evaluate replication kinetics of EHV-1 strains, virus growth curve assays were performed. Confluent monolayers of EE cells (8 x 105 cells in a 24-well plate) were infected with each strain at MOI of 1 in triplicate and incubated at 37°C 5% CO2 for 24 hours.

Twenty-four hours p.i. total virus was harvested, using 300µL of trypsin and 700µL of

DMEM per well, and samples were centrifuged at 14,000rpm for 5 minutes to pellet the cells. Viral yields were calculated via a standard plaque assay, as described previously.

Plaque Phenotype Analysis of EHV-1 Isolates in EE Cells

In order to evaluate cell to cell spread efficiency of each EHV-1 isolate, a cell to cell spread assay was performed. Confluent monolayers of EE cells in a 6-well plate were infected with approximately 100 pfu of each strain at 37°C 5% CO2 for 2 hours. After two hours, cells were overlaid with 5mL of semi-solid complete DMEM containing 1% methylcellulose, and incubated for 4 days. After the 4 day incubation, semi-solid media was

24 aspirated off and the cells were stained with 1% crystal violet for 60 minutes. Stained cells were examined under a Motic inverted light microscope (Motic, Causeway Bay, Hong

Kong). Representative images (with a mean sample size of 45 plaques) were analyzed in

ImageJ (U. S. National Institutes of Health, Bethesda, Maryland, USA). Statistical analysis was performed using one-way ANOVA with a Tukey's multiple comparison post-hoc test to identify significant differences (p ˂ 0.05).

RESULTS

Generation of Virus Stocks

In order to perform assays for this study, a stock of each clinical EHV-1 isolate was generated and titered as described in Materials and Methods. A list of viral stocks along with a titer for each is shown in Table 1.

Viral Replication Analysis of EHV-1 Isolates in EE Cells

In order to compare replication rates of various neurologic and non-neurologic

EHV-1 isolates in equine endothelial cells, cells were infected in triplicate with MOI of 1 for

24 hours, virus harvested, and standard plaque assay was performed on collected samples, as described in Materials and Methods. A total of 9 neurologic and 6 non-neurologic strains have been analyzed (Table 2, Figure 2). No major differences have been observed in replication rates between the neurologic and non-neurologic virus group.

Plaque Phenotype Analysis of EHV-1 Isolates in EE Cells

In order to compare cell to cell spread ability of neurologic and non-neurologic EHV-

1 isolates in equine endothelial cells, cells were infected with approximately 100 pfu of each isolate, overlaid with complete DMEM containing 1% methylcellulose, and stained with 1% crystal violet after 4 day incubation. As shown in Figure 3, with the exception of

Ab4 and L11, which produced largest and smallest plaques respectively, the plaque sizes for the isolates analyzed were within the same size range. Generally, no significant differences in cell to cell spread between neurologic and non-neurologic virus group were observed.

DISCUSSION

The main goal of this study was to determine whether neurologic and non- neurologic EHV-1 strains differ in viral replication and cell to cell spread when infecting equine endothelial cells. We hypothesized that neurologic strains would produce higher viral yields, as well as show an enhanced cell to cell spread phenotype, compared to non- neurologic strains. However, no trends in significant differences in either replication or cell to cell spread have been found. Overall, the findings of this study suggest that the severity of disease in the equine associated with infection with neurologic strains is attributed to factors other than replication and cell to cell spread.

Infection with neurologic EHV-1 strains, classified as neurologic based on a point mutation in ORF 30 encoding for viral DNA polymerase, has been found to cause an earlier

PBMC-associated viremia that is of greater magnitude and longer duration comparing to the infection with non-neurologic strains 136. While EHV-1 is able to infect three different

26 cell types (namely, respiratory epithelium, PBMCs, and endothelium), damage to the endothelial cells is considered to predispose the equine host to the greatest risk for developing the most severe clinical manifestations of EHV-1 infection including abortion and EHM. Multiple studies have shown that EHV-1 infection of the endothelium triggers inflammation, ischemic thrombosis and vasculitis, which leads to indirect damage of neuronal tissue, as shown by swollen axons and hindered vesicle transport 134,135,139,154.

These host inflammatory responses to EHV-1 are greatly responsible for induction of abortion and EHM. Therefore, our group chose to investigate patterns of EHV-1 replication and cell to cell spread mechanics in equine endothelial cells.

Because of the positive correlation between the increased viral load and development of EHM, we first analyzed replication rates of various EHV-1 clinical isolates

(9 neurologic and 8 non-neurologic). Due to the labor-intensive procedure of collecting and processing viral samples at a specific time point post-infection, collecting viral yield replication data at 24 hours post-infection was chosen as a fitting starting point to focus on in this study. Due to the initial relatively low titer of two non-neurologic strains, T547 and

T572, they were temporarily excluded from the study, leaving 9 neurologic and 7 non- neurologic strains for the virus growth assay. Equine endothelial cells were infected with each strain in three replicates for 24 hours, virus harvested, and titered on RK13 cells. Titer data for three replicates were averaged, and standard deviation calculated. No consistent or significant differences were observed in 24 hour viral replication yields between neurologic and non-neurologic EHV-1 strain groups.

The likely cause of lack of significant replication differences between the two EHV-1 strain groups may be that all isolates analyzed share a high homology between

27 glycoproteins essential for viral infection. This would provide no selective advantage to either neurologic or non-neurologic strains in terms of attachment, entry, and cell to cell spread and would therefore lead to all isolates falling within the same range of data for the three factors.

Due to the limited span of this study, it would be made more comprehensive if the viral yield was investigated at additional time points, such as at 6, 12, and 48 hours post- infection. Viral replication could also be quantitatively analyzed by using real-time quantitative PCR with primers for IE gene, where higher levels of the gene produced would be indicative of selective advantage in replication for that isolate. Another approach for analyzing replication rate, more likely to be addressed over an extended research period, is to selectively engineer a number of neurologic and non-neurologic EHV-1 strains, such that each includes a reporter gene, such as LacZ in L11, which would allow a facile assessment of EHV-1 infection rate qualitatively using X-gal staining or using MTS and ONPG colorimetric assays for quantitative assessment of infection.

As previously indicated, a part of the reason for neurologic EHV-1 strains leading to a more severe disease outcome could be a selective advantage in attachment to the host cell; however, due to the time constraint of performing a virus binding assay on a panel of all strains used in the study, the attachment rate would only be investigated if replication rate results demonstrated significant differences. Because there were no major differences, it may be inferred that the strains do not differ in the degree of attachment to the host cell.

Another reason for neurologic strains being more aggressive could stem from these strains having a higher potential for entry through MHC-I, or perhaps the ability to utilize additional, presently unidentified, entry receptors on various cell types. To investigate the

28 entry ability of EHV-1 strains through MHC-I on equine endothelial cells, a plaque reduction assay would be performed. In this assay, MHC-I would be blocked with anti- equine MHC-I neutralizing antibody, and cells would be infected with EHV-1 to determine whether infection is inhibited in a dose-dependent manner. Plaque reduction assay could also provide pertinent data as to whether various EHV-1 isolates are able to enter equine endothelial cells when the entry receptor, MHC-I, is blocked.

In addition to replication rate, cell to cell spread is another imperative factor in viral infectivity. In order to determine whether neurologic and non-neurologic EHV-1 strains differ in cell to cell spread, the plaque phenotype of various isolates was characterized.

Equine endothelial cells were infected with approximately 100 plaque forming units of each virus for 4 days, stained, and plaque sizes were qualitatively assessed. Preliminary data of five neurologic and five non-neurologic strains suggests that there are no consistently major differences in cell to cell spread between the two EHV-1 groups. It is significant that the exclusively large plaque size phenotype was observed with neurologic strain Ab4, in two independent replicates (data shown for one replicate). Despite average replication rate, the data may potentially suggest that Ab4 possesses an advantage unlike the other analyzed EHV-1 strains in spreading within cells. This data is also consistent with severe clinical outcomes of infection with Ab4. For a more comprehensive, quantitative analysis of cell to cell spread of various EHV-1 isolates, more isolates need to be analyzed and the sizes of at least 50 plaques for each isolate need to be analyzed via Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD).

Because EHV-1 glycoproteins play an essential role in the viral life cycle and may be responsible for major differences observed in either attachment, entry, or cell to cell

29 spread, the analysis of neurologic and non-neurologic strains would be enhanced by sequence data of the essential glycoproteins from each strain. The major target for sequencing would be glycoprotein gD, as it is crucial for viral entry and cell to cell spread.

Differences in specific amino acid residues within glycoprotein gD, or another essential

EHV-1 glycoprotein, could be associated with increased virulence.

CHAPTER 2: CORE FUSOGENIC GLYCOPROTEIN SET

MATERIALS AND METHODS

Cells

Murine melanoma B78H1 cells were provided by Gary Cohen and Roselyn Eisenberg

(University of Pennsylvania, Philadelphia, PA). B78H1-C10 (human nectin-1) cells were a gift from Claude Krummenacher (University of Pennsylvania School of Veterinary Medicine,

Philadelphia, PA) and is the cell line stably expressing human nectin-1, an entry receptor for HSV. B78H1-C2 (equine MHC-I) cells were described previously and they stably express

EHV-1 entry receptor, MHC-I (50 Kurtz). All cells were maintained in complete DMEM at

37°C 5% CO2.

Generation of EHV-1 Glycoprotein Expression Plasmids

A set of glycoproteins gB, gD, gH and gL was to be generated from two neurologic

(Ab4, OH03) and two non-neurologic strains (L11, KyA).

A total of 1 x 106 RK13 cells were seeded in two 60mm dishes, one served as an experimental control and one served as a negative control. The cells were incubated at

37°C 5% CO2 for 24 hours and then inflected or mock-infected with each EHV-1 strain at multiplicity of infection (MOI) of 3 for 24 hours at 37°C 5% CO2.

Genomic DNA from infected or mock-infected RK13 cells was isolated using a

Wizard® SV Genomic DNA Purification System (Promega, Madison , WI) following the manufacturer's instructions. Each of the four glycoproteins was amplified via polymerase chain reaction (PCR) using specific primers (Integrated DNA Technologies, Coralville, IA). gB was isolated using primers: forward 5’-GCGGCATTTACATAACCTACG-3’ and reverse 5’-

GAGGTCACACTTTGAGTACG-3’; gD - forward 5’-CGAAACCAGGCCAGGCGGAC-3’ and reverse

5’-AGCGTAGGCGAGTCAAGCCG-3’; gH - forward: 5’-GCCGCGGTGTGGCCTATTGA-3’ and reverse 5’-CGGCCAGACGCGCAACAATG-3’; gL - forward 5’-CCGGTCGTTCGGTTGAGCAAGT-3’ and reverse 5’-AGGCGGTTTATGCGCTGCTGG-3’. Primers and the genomic target sequences are summarized in Table 3.

PCR reaction mix was made using 12.5µL of EconoTaq® PLUS GREEN 2X Master Mix

(Lucigen, Middleton, WI), 1µL of the forward primer, 1µL of the reverse primer, 1µL of the

DNA template, and 9.5µL of nuclease-free water to bring the total volume of the mix to

25µL. The thermal cycler was programmed to run as follows: initial denaturation for 5 minutes at 95°C followed by 35 cycles, where each cycle consists of denaturation for 15 seconds at 94°C, annealing for 30 seconds at 55°C, extension for 3 minutes at 72°C, and concluding with a final extension at 72°C for 7 minutes. Each PCR product was electrophoresed through a 1% agarose gel containing 12.5% Ethidium Bromide at 100V along with a 1Kb Plus DNA Ladder (Life Technologies, Carlsbad, CA), excised and purified using a Gel/PCR DNA Fragments Extraction kit (IBI Scientific, Peosta, IA) following the

31 manufacturer’s instructions. The concentration of the isolated DNA was determined using a

NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

Each glycoprotein PCR product was then ligated into the pcDNATM3.3-TOPO® mammalian expression plasmid (Life Technologies, Carlsbad, CA). For each ligation, 100ng of the amplified glycoprotein DNA, 1µL of pcDNATM3.3-TOPO® plasmid vector, and 1µL of salt solution were combined, and then the mixture was brought up to a total of 6µL by adding nuclease-free water. The reaction mix was incubated at room temperature for 30 minutes. Following that, One Shot® TOP10 chemically competent E. coli cells (Life

Technologies, Carlsbad, CA) were transformed with 2µL of the cloning mix and incubated for 30 minutes on ice. Incubation was followed by a 30 second heat shock in a dry bath set at 42°C and a subsequent 2 minute incubation on ice. To the transformed E. coli, 250µL of super optimal broth with catabolite repression (SOC) medium was added and the cells were incubated on a horizontally shaking platform set at 225rpm for 1 hour in a 37°C incubator. Afterwards, various volumes of the transformed cell mixture were evenly spread on pre-warmed selective yeast-tryptone (2xYT) agar plates containing 0.1mg/mL ampicillin. Plates were incubated at 37°C overnight to allow the ampicillin-resistant cells to form colonies.

To screen colonies for a successful clone, each colony was amplified in liquid 2xYT media containing 0.1mg/mL ampicillin. The neck of each culture tube was sealed with aluminum foil and culture tubes were incubated overnight on a horizontally shaking platform set at 225rpm in a 37°C incubator.

Plasmid DNA from each culture was extracted and purified using E.Z.N.A.® Plasmid

Mini Kit I (Omega Bio-Tek, Norcross, GA) following the manufacturer’s instructions and the

32 concentration of the isolated DNA plasmids were quantified using a NanoDrop 2000 UV-Vis spectrophotometer.

The insertion and orientation of each gene was verified via restriction digest analysis. Each plasmid was digested with a specific restriction enzyme (New England

Biolabs, Ipswich, MA), using 10 units of enzyme, 500ng DNA, and a minimum 1 hour incubation period at the enzyme's optimal temperature. For each set of samples, an undigested sample was included as a control. Enzyme choices and expected DNA fragment sizes are listed in Table 4. Additionally, restriction maps for each of the glycoproteins, along with a representative gel electrophoresis picture for each of the digested glycoprotein clones, are shown in Figures 4-7.

Following the minimum 1 hour incubation period, to each sample 2µL of 6X gel loading dye (New England Biolabs, Ipswich, MA) was added and samples were electrophoresed through a 1% agarose gel containing 12.5% Ethidium Bromide at 100V along with a 1Kb Plus DNA Ladder. The gel was viewed under the UV light to identify clones in the forward orientation.

Cultures of each correctly oriented expression plasmid were amplified by combining in an Erlenmeyer flask 150mL of 2xYT media containing 0.1mg/mL ampicillin and 1mL of the successfully generated glycoprotein plasmid culture. The neck of the flask was sealed with aluminum foil and the flask was incubated overnight on a horizontally shaking platform set at 225rpm in a 37°C incubator to allow ampicillin-resistant cells to grow. DNA was extracted from the amplified culture using E.Z.N.A.® Plasmid Maxi Kit (Omega Bio-Tek,

Norcross, GA) following the manufacturer’s instructions. The concentrations of the isolated

33 plasmids were quantified using a NanoDrop 2000 UV-Vis spectrophotometer and each clone was further verified by restriction digest analysis.

PCR Analysis of the Glycoprotein mRNA Transcripts from B78H1 Cells Transfected with

Glycoprotein Expression Plasmids

A total of 3 x 105 B78H1 cells were seeded in a 12-well plate and incubated overnight at 37°C 5% CO2. Cells were transfected or mock transfected with the glycoprotein plasmids using LipofectamineTM 2000 (Life Technologies, Carlsbad, CA) following manufacturer's instructions. Specifically, for every well, a mixture of 75µL of serum-free

DMEM and 4.5µL of LipofectamineTM 2000 was made. For every glycoprotein plasmid to be transfected, a mixture of 350µL of serum-free DMEM and 7µg of glycoprotein plasmid was made. For a mock well, 350µL of serum-free DMEM was prepared. Afterwards, contents of each DMEM/glycoprotein or DMEM/mock tube were combined with the contents of the corresponding DMEM/Lipofectamine tube at 1:1 ratio (75µL + 75µL). Solutions were mixed and incubated for 20 minutes to allow for the lipophilic agent to coat the DNA. After 20 minutes, media was aspirated off each B78H1 well to be transfected and washed once with serum-free DMEM. To each well, corresponding Lipofectamine/glycoprotein or

Lipofectamine/mock mixture was added for a total of 1250ng of DNA per well

(approximately 125µL of the solution). Volume in each well was brought up to 1mL with serum-free DMEM and the cells were incubated at 37°C 5% CO2 for 3 hours. After 3 hours, serum-free DMEM was replaced with complete DMEM. The cells were then incubated at

37°C 5% CO2 for a total of 48 hours.

After 48 hours, total RNA was harvested from transfected and mock-transfected cells using E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, Norcross, GA) following the manufacturer's instructions. The isolated RNA was quantified using NanoDrop 2000 UV-Vis spectrophotometer. RNA (2 µg) was reverse-transcribed (RT-PCR) into a complimentary

DNA (cDNA) using Cloned AMV First Strand cDNA Synthesis Kit (Life Technologies,

Carlsbad, CA) following the manufacturer's instructions. Tubes with the reaction mix were placed in a thermal cycler set to run as follows: 55 minutes at 50°C, 5 minutes at 85°C.

Generated cDNA was used as a template for PCR, as described previously, with full-length glycoprotein primers used. PCR products were run on a 1% agarose gel and visualized under the UV light.

Additionally, once the expression plasmids for glycoproteins gB, gD, gH and gL from one isolate were generated, 1.75µg of each glycoprotein plasmid was co-transfected into a confluent monolayer of B78H1 cells following the transfection protocol described above to confirm the co-expression of all four expression plasmids, at 24 hour post-transfection.

Elucidation of the Core Set of EHV-1 Fusogenic Glycoproteins

A total of 8 x 105 B78H1 cells were seeded in a 6-well plate and incubated for 24 hours at 37°C 5% CO2. Four wells were seeded per EHV-1 isolate and four wells per HSV positive control - two for co-transfection with four glycoprotein plasmids, and two for mock transfection. In addition, 3 x 105 B78H1-C2 cells and 3 x 105 B78H1-C10 cells were seeded in a 12-well plate for overlaying the corresponding transfected cells.

B78H1 cells were co-transfected or mock co-transfected with 3.5µg of each of EHV-1 expression plasmid for glycoprotein gB, gD, gH, and gL from the same isolate, as described

35 previously. Analogously, B78H1 were co-transfected or mock co-transfected with 3.5µg of

HSV gB, gD, gH, and gL. Twenty-four hours post transfection, B78H1-C2 and B78H1-C10 were harvested using 500µL of trypsin and 1000µL complete DMEM. Monolayers of B78H1 transfected and mock-transfected with EHV-1 glycoproteins were overlaid with 500µL of

B78H1-C2 (equine MHC-I) cell solution. Similarly, monolayers of B78H1 transfected and mock-transfected with HSV glycoproteins were overlaid with 500µL of B78H1-C10 (human nectin-1) cell solution. All cells were incubated at 37°C 5% CO2 for 24-48, and observed for fusion daily. To aid in visualizing the fused cells (syncytia), 24 and 48 hours after combining cells expressing the glycoproteins with cells expressing the corresponding entry receptors, cells were stained with KaryoMAX® Giemsa stain (Life Technologies, Carlsbad,

CA) for 5 minutes, following two washes with phosphate buffered saline (1xPBS), and rehydration with 1xPBS. KaryoMAX® Giemsa stains nuclei a deep purple, and contains methanol, which acts as a fixative. Cells were examined under Motic inverted light microscope, and representative images taken with Moticam.

RESULTS

Generation of EHV-1 Glycoprotein Expression Plasmids

In order to generate each glycoprotein expression plasmid (gB, gD, gH, and gL), corresponding glycoprotein sequence was first amplified from each of Ab4, Ohio 2003, KyA, and L11 EHV-1 strains and ligated into pcDNATM3.3-TOPO® plasmid, as described in

Materials and Methods. Each clone was digested with a corresponding restriction enzyme, as shown in Table 4, producing specific fragment sizes for the forward-facing clone, as shown in Figures 4-7. As shown in Table 5 and Figure 8 of successfully generated

36 expression plasmids, a full set of gB, gD, gH, and gL has been obtained for the Ohio 2003 strain. For both Ab4 and L11 strains, gB, gD, gH, and gL have also been obtained. For KyA, gB, gD, and gL have been obtained.

PCR Analysis of the Glycoprotein mRNA Transcripts from B78H1 Cells

Transfected with Glycoprotein Expression Plasmids

In order to confirm the expression of each glycoprotein, B78H1 cells were transfected with each expression plasmid, total RNA was harvested, cDNA generated, and

PCR performed, all as described in Materials and Methods. Successful expression and co- expression at 24 and 48 hours post transfection of Ohio 2003 gB, gD, gH, and gL genes has been confirmed, as shown in Figure 9. Although a full set of gB, gD, gH, and gL plasmids has been obtained from Ab4 and L11 strains, gB transcripts were not detected after transfection of expression plasmids containing Ab4 gB or L11 gB. In addition, gB transcript was also not detected in cells transfected with the KyA gB plasmid (Figure 10).

Elucidation of the Core Set of EHV-1 Fusogenic Glycoproteins

After a set of Ohio 2003 expression plasmids gB, gD, gH, and gL were confirmed for co-expression in B78H1 cells, the set was transfected into B78H1 to be used as a proxy for the virus. Naturally, the virus structure is more complex, containing numerous proteins embedded in the viral envelope; however, for the purposes of successful cell to cell fusion, the determinant viral factor is the correct set of glycoproteins expressed on the cell surface.

As long as B78H1 cells express gB, gD, gH, and gL, this cell line is an appropriate substitute for the virus, in cell to cell fusion assay.

As described in Materials and Methods, 24 hours post transfection the cells expressing gB, gD, gH, and gL from Ohio 2003 were overlaid with B78H1-C2 cells, which stably express the EHV-1 entry receptor, MHC-I. As a positive control, B78H1 cells co- transfected with gB, gD, gH, and gL from HSV-1 were overlaid with B78H1-C10, a cell line that stably expresses the HSV-1 entry receptor, nectin-1. Cells were stained with

KaryoMAX® Giemsa 24 and 48 hours after combining the two cell types.

As shown in Figure 11, the positive control yielded multinucleated cells; however, no such phenotype was observed in the Ohio 2003 experimental control. B78H1-C2, instead of B78H1, were also transfected with gB, gD, gH, and gL and stained 24 hours post- transfection and again no multinucleated cells were observed (Figure 12). Finally, B78H1 expressing gB, gD, gH, and gL from Ohio 2003, were overlaid with RK13 cells susceptible to

EHV-1 infection, and this resulted in no multinucleated cells (data not shown). Infection of

B78H1-C2 with L11 at MOI 3 for 24 hours showed complete susceptibility of the cells to

EHV-1 (Figure 13).

DISCUSSION

In this study, the minimum subset of EHV-1 glycoproteins necessary and sufficient for EHV-1 fusion with cell membranes was found to likely extend beyond glycoproteins gB, gD, gH, and gL. Glycoprotein set from Ohio 2003, when expressed in B78H1 cells and combined with B78H1-C2 cells stably expressing the EHV-1 entry receptor MHC-I, did not yield multinucleated cells, an indicator of the membrane fusion event. The same glycoprotein set transfected into B78H1-C2 directly did not yield fusion, and neither has fusion been observed when the glycoprotein set expressed on B78H1 cells was combined

38 with RK13 cells, which express an entry receptor for EHV-1. Overall, the findings of this study suggest that more cell or viral factors may be required for EHV-1 to fuse with host cell membranes.

It is well established that, in terms of viral factors, alphaherpesviruses require at the minimum functional glycoproteins gB, gD, and the gH-gL heterodimer to successfully infect the host cell, as this set is required for viral growth in tissue culture. Among specific functions of each glycoprotein, such as gB facilitating binding to the host cell glycosaminoglycans and gD mediating binding to the entry receptor, each of these glycoproteins plays a crucial role in viral attachment, entry, and cell to cell spread. At least with HSV-1, it has been found that cell surface heparan sulfates are not required for successful fusion of cell membranes, which makes conduction of tissue culture cell to cell fusion studies, such as this one, more feasible 91.

A number of successful alphaherpesvirus cell to cell fusion studies 91,155–157 have been previously performed, evident of the validity of applying the results of the tissue culture study to the behavior of the virus in the host. Naturally, in vivo, it may be easier for the virus to overcome the pressure of the host cell membrane before fusion occurs, as well as certain components on the surface of the host cell, such as entry receptors, may be recruited to the virus due to dynamics of the cell membrane in vivo. However, the fusion is largely determined by the interactions between the correct viral glycoprotein set and the corresponding entry receptor on the host cell. When the cell line expressing the glycoproteins necessary for the fusion event is overlaid with a cell line stably expressing the entry receptor, the mechanical interaction will cause the formation of multinucleated

39 cells. Albeit they may be of a different phenotype and frequency than in the host, for this study, the occurrence in itself would be the most essential observation.

Results from this study suggest that EHV-1 may require more than glycoproteins gB, gD, gH, and gL, as well as the entry receptor, in order to fuse with cell membranes and thereby gain access to the host cell. Granted, only a set from one strain, Ohio 2003, has been tested; therefore, cell to cell fusion assays with sets from additional strains need to be performed for more conclusive results. Additionally, there is a possibility that, despite the messenger RNA transcripts for the glycoproteins generated in the cell, one or more of the glycoproteins may not be expressed on the cell surface. In order to rule out the possibility that gB, gD, gH, and gL from Ohio 2003 are not sufficient to cause fusion, it would be critical to examine the cell surface expression of each glycoprotein. One of the approaches to assess the cell surface expression is via fluorescent microscopy using antibodies directed against each of the glycoproteins.

To confirm that the Ohio 2003 gB, gD, gH, and gL are functional in B78H1 cells, the glycoproteins were tested for expression at 24 hours post-transfection, which is slightly longer than the 16 hour time-point of choice for overlaying transfected cells with cell line expressing an entry receptor in a successful 2000 cell to cell fusion study by Muggeridge

155. Confirmed expression of each of the four glycoproteins eliminated the possibility that lack of fusion was brought upon by the lack of expression of one or more glycoproteins after transfection. The fact that susceptible cell types are successfully infected with the

Ohio 2003 strain also corroborates the functionality of the glycoproteins. To determine whether MHC-I expression in the B78H1-C2 cell line has not been compromised due to continuous passaging, the cells were infected with a LacZ reporter virus, L11. Robust

40 infection indicated that MHC-I is abundantly expressed. Therefore, we eliminated the possibility that the fusion event did not occur due to non-functional MHC-I on the surface of

B78H1-C2. Allowing for the fusion between Ohio 2003 glycoproteins and MHC-I to occur for 24 and 48 hours after the combining event, which is a standard period in related studies, has not yielded the formation of multinucleated cells 91,155. Considering numerous replicates of the experiment were performed, as well as the success of the positive control replicates, we have inferred that, for Ohio 2003, an additional glycoprotein is likely required for the fusion event to occur. One of the possible choices may be glycoprotein gK, as it is considered to be "most likely essential" for viral replication, and numerous studies, addressed in the Introduction, have found the significance of glycoprotein gK in viral entry and cell to cell spread 18.

For conclusive results, the cell to cell fusion experiment will need to be performed with glycoprotein sets from additional strains. However, due to the laborious task it has been proven for our group to generate glycoprotein gB expression plasmids alone, as well as to confirm their expression in B78H1, it is valid to consider using glycoprotein gB from

Ohio 2003 with glycoproteins gD, gH, and gL from strains Ab4 and L11. While it is ideal to use a complete set from each strain, in the meantime, the approach of using one glycoprotein from a different strain is scientifically appropriate and warranted for the advancement of this study.

When sufficiently advanced, the findings of this comprehensive study will fill the gap in knowledge on the specifics of EHV-1 interaction with the host cell on the molecular level, leading to successful entry. The data on the minimum set of glycoproteins required

41 for EHV-1 to enter the host cell will aid in the development of successful anti-viral therapies that could target and disrupt essential virus-host cell interactions.

CHAPTER 3: ALTERNATIVE EHV-1 ENTRY RECEPTORS

MATERIALS AND METHODS

Cells

Murine melanoma B78H1 cells were provided by Gary Cohen and Roselyn Eisenberg

(University of Pennsylvania, Philadelphia, PA). B78H1 were grown in complete DMEM at

37°C 5% CO2.

Generation of Equine Nectin-1 and HVEM Expression Plasmids

RNA was harvested from equine endothelial cells and reverse-transcribed into cDNA, as described previously. cDNA was used as a template for PCR, also as described previously. For nectin-1, the following primers were used: forward 5'-

CTCCTCGCGACCGCTCTCGCCGGAGC-3' and reverse 5'-

CGCAGACAGACGCTGTGGAGAGGACG-3'. For HVEM, the following primers will be used: forward 5'-GTGCTAAAGCTCGTCCTGCTGGCAGG-3' and reverse 5'-

CTGTCTCTGCTGGATGGAGTCTGAAGC-3'. PCR products of nectin-1 and HVEM were electrophoresed through a 1% agarose gel and isolated, as described previously.

Receptor genes were cloned into pcDNATM3.3-TOPO® plasmid vector, as described previously. One Shot® TOP10 chemically competent E. coli were transformed with 2µL of the ligation mix, as described previously. Various mixtures of transformed E. coli were

42 spread on pre-warmed 2xYT plates containing 0.1mg/mL ampicillin and incubated at 37°C overnight to allow ampicillin-resistant cells to form colonies.

Colonies were screened, as described previously. Diagnostic digest enzyme choices for nectin-1 and HVEM, along with the expected DNA fragment sizes, are listed in Table 6.

Successful expression plasmids were amplified and DNA extracted using E.Z.N.A.® Plasmid

Maxi Kit following the manufacturer’s instructions, as described previously. The concentration of the isolated plasmid DNA was quantified using a NanoDrop 2000 UV-Vis spectrophotometer.

Generation of Stable Nectin-1 and HVEM Receptor Lines in B78H1 Cells

A total of 3 x 105 B78H1 cells were seeded in a 12-well plate and incubated for 24 hours at 37°C 5% CO2. Nectin-1 and HVEM expression plasmids (7µg each) were transfected into separate B78H1 cell monolayers using LipofectamineTM 2000, following the transfection procedure described previously. Hygromycin-resistant cells were grown and maintained in the presence of Hygromycin B antibiotic (0.1mg/mL).

RESULTS

Generation of Equine Nectin-1 and HVEM Expression Plasmids

In order to generate nectin-1 and HVEM expression plasmids, both genes were PCR amplified from the equine endothelial cell cDNA template and ligated into the pcDNATM3.3-

TOPO® mammalian expression plasmid, as described in Materials and Methods. Clonal populations were subjected to restriction digest analysis with specific enzymes, as shown in Table 6.

The obtained nectin-1 expression plasmids have been consistently ligated in reverse orientation, unsuitable for use in this study (Figure 14, Figure 15). As a result of the unsuccessful ligation into the TOPO plasmid, nectin-1 has been attempted to be directionally ligated into a different plasmid, pcDNA3.1/Hygro(+) plasmid (Life

Technologies, Carlsbad, CA); however, this more time-consuming process yielded only re- ligated plasmids. The HVEM expression plasmid has been successfully generated (Figure

16). HVEM plasmid was also confirmed for expression in B78H1 cells (data not shown).

Generation of Stable Nectin-1 and HVEM Receptor Lines in B78H1 Cells

Due to low success with generating nectin-1 expression plasmid, B78H1-nectin-1 cell line is yet to be generated. Since functional HVEM plasmid has been obtained, a B78H1 cell line stably expressing HVEM is in the process of being generated.

DISCUSSION

The major aim of this study was to identify and characterize additional cell receptors used by EHV-1 for entry and cell to cell spread. We hypothesized that EHV-1 is able to use equine homologues of the two major alphaherpesvirus entry receptors, nectin-1 and HVEM, for entry and cell to cell spread. The work is currently underway to generate cell lines that would stably express equine nectin-1 and equine HVEM. Further work, as described below, is required in order to investigate the entry ability of equine nectin-1 and

HVEM for EHV-1.

Over the course of this project, it has been consistently challenging to ligate nectin-1 into pcDNATM3.3-TOPO® plasmid, as the only clones obtained were in a reverse

44 orientation, unsuitable for the experiment. The ligation was made more demanding when

PCR amplification of nectin-1 out of equine endothelial DNA began to consistently yield

DNA concentrations inadequately low for use in ligation, and, in particular, when no gene was amplified at all. The fact that the same primer set used to result in abundant nectin-1

DNA was puzzling. In addition, although the working primer stock has been re-generated in an attempt to increase the nectin-1 product yields, this approach has been unsuccessful. In order to obtain sufficient amount of nectin-1 DNA for ligation, primer optimization will be required. While our lab has the B78H1-C10 (human nectin-1) cell line, using it for the purposes of this study may not be accurate due to nectin-1 being of human origin, and thus this human form of the nectin-1 receptor may not interact with EHV-1. However, sequence analysis suggests that cross-reactivity may be possible. According to NCBI BLAST alignment of the protein sequences for the equine form of nectin-1 (66,079 amino acids in length; NCBI accession number NC_009150.2) and the human form of nectin-1 (90,628 amino acids in length; NCBI accession number NG_013083.1), 76% of the human nectin-1 produced significant alignments with the equine form of nectin-1. Therefore, B78H1-C10 cell line may be considered an initial alternative to the equine nectin-1 cell line for this study.

Because the HVEM expression plasmid was successfully generated, generation of the

B78H1-HVEM cell line has been initiated.

Once the B78H1 cell lines stably expressing nectin-1 and HVEM will be generated, cell surface expression of nectin-1 and HVEM will need to be confirmed. This will be achieved via immunofluorescence analysis using antibodies directed against equine nectin-

1 and HVEM.

To assess the EHV-1 entry potential of nectin-1 and HVEM receptors, generated

B78H1 cell lines stably expressing nectin-1 and HVEM will be infected with L11 for 6 hours, and then total RNA will be harvested. The RNA will be reverse-transcribed into cDNA, and the cDNA will be used as a template for PCR using IE gene primer set. IE gene is the first to be produced during the viral replication and the presence of the transcript is the marker of infection. To further quantify the IE gene production, quantitative PCR may be performed to compare the efficacy of EHV-1 entry through nectin-1 and HVEM receptors.

In order to examine whether, and to what extent, the EHV-1 is able to use nectin-1 and HVEM for cell to cell spread, plaque phenotype assay will be performed. B78H1 cell lines stably expressing nectin-1 and HVEM will be infected with L11 for 24 hours, and then fixed and stained with X-gal. Additionally, the cell lines may also be infected with various clinical isolates for 2 hours, overlaid with complete media containing 1% methylcellulose, and incubated for 4 days, until staining with 1% crystal violet solution.

Should EHV-1 is able to enter B78H1 through nectin-1 and/or HVEM, antibody blocking assay will be performed to test the specificity of virus-receptor interaction. B78H1 cell lines expressing the receptors will be treated with various dilutions of the antibodies directed against equine nectin-1 and equine HVEM, and infected with L11 for 24 hours.

Then, ONPG assay will be performed to examine whether infection can be inhibited in a dose-dependent manner.

The knowledge of additional EHV-1 entry receptors will be an invaluable asset for understanding the virus-host interaction, as in the period of over twenty years that EHV-1 has been officially recognized, the only known entry receptor for the virus is MHC-I 90.

Additionally, the knowledge will provide evidence for how homologous EHV-1 is related to

46 alphaherpesviruses, such as HSV-1 and HSV-2, that are able to utilize nectin-1 and HVEM for entry. Finally, expanded knowledge of EHV-1 entry receptors will allow for development of antiviral therapies that could target these receptors and potentially prevent viral entry into a specific cell type that could in turn reduce the risk of developing severe EHV-1 induced disease.

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TABLES

EHV-1 Strains Virus Collection Date Titer (pfu/mL) Neurologic T313 5/23/13 4.73 x 107 T954 5/31/13 3.07 x 107 T955 5/30/13 1.60 x 108 T956 6/6/13 5.27 x 107 T964 6/6/13 5.53 x 107 T967 6/10/13 1.73 x 108 T970 6/10/13 2.90 x 108 Ab4 9/20/13 4.27 x 107 Ohio 2003 9/20/13 1.03 x 107 Non-neurologic T61 6/14/13 5.43 x 107 T75 6/14/13 4.23 x 107 T220 6/17/13 4.93 x 107 T493 6/19/13 1.70 x 108 T547 9/16/13 3.23 x 107 T572 9/16/13 3.13 x 106 KyA 1/13/11 3.27 x 108 L11 7/13/13 2.70 x 108 L11 2/23/14 5.76 x 107

Table 1. Titers of stock neurologic and non-neurologic EHV-1 strains, obtained by infecting RK13 cells.

EHV-1 Titer of each sample (pfu/mL) Mean Std deviation Std error Strains #1 #2 #3 Neurologic T313 2.10 x 107 3.70 x 107 2.50 x 107 2.77 x 107 0.83 x 107 0.48 x 107 T954 2.40 x 107 2.37 x 107 5.43 x 107 3.40 x 107 1.76 x 107 1.02 x 107 T955 2.17 x 107 2.33 x 107 1.13 x 107 1.88 x 107 0.65 x 107 0.38 x 107 T956 3.23 x 107 2.10 x 107 1.90 x 107 2.41 x 107 0.71 x 107 0.41 x 107 T964 2.83 x 107 3.27 x 107 1.90 x 107 2.67 x 107 0.70 x 107 0.40 x 107 T967 3.03 x 107 4.20 x 107 2.50 x 106 2.49 x 107 2.03 x 107 1.17 x 107 T970 1.20 x 107 6.37 x 107 5.90 x 107 4.49 x 107 2.86 x 107 1.65 x 107 Ab4 9.67 x 106 2.13 x 107 2.00 x 107 1.70 x 107 0.64 x 107 0.37 x 107 Ohio 2003 1.10 x 107 8.66 x 106 8.00 x 106 0.92 x 107 0.16 x 107 0.91 x 106 Non-neurologic T61 3.90 x 107 1.80 x 107 3.07 x 106 2.00 x 107 1.81 x 107 1.04 x 107 T75 2.00 x 107 3.06 x 107 1.70 x 106 1.74 x 107 1.46 x 107 0.84 x 107 T220 1.93 x 107 2.83 x 106 1.50 x 107 1.24 x 107 0.85 x 107 0.49 x 107 T493 1.47 x 107 1.60 x 107 1.23 x 107 1.43 x 107 0.19 x 107 0.11 x 107 KyA 8.66 x 106 2.80 x 106 4.43 x 106 5.30 x 106 3.02 x 106 1.75 x 106 L11 8.57 x 107 8.50 x 107 8.43 x 107 8.50 x 107 0.70 x 106 0.40 x 106

Table 2. Twenty-four hour replication yields of neurologic and non-neurologic EHV-1 strains. Equine endothelial cells were infected with each strain at MOI 1 in triplicate for 24 hours, virus was harvested and titered on RK13 cells.

EHV-1 Forward Primer Reverse Primer EHV-1 Genome Glycoproteins Sequence gB 5’-GCGGCATTTACATAACCTACG-3’ 5’-GAGGTCACACTTTGAGTACG-3’ 61,432 - 64,388 gD 5’-CGAAACCAGGCCAGGCGGAC-3’ 5’-AGCGTAGGCGAGTCAAGCCG-3’ 131,521 - 132,781 gH 5’-GCCGCGGTGTGGCCTATTGA-3’ 5’-CGGCCAGACGCGCAACAATG-3’ 71,192 - 73,738 gL 5’-CCGGTCGTTCGGTTGAGCAAGT-3’ 5’-AGGCGGTTTATGCGCTGCTGG-3’ 108,147 - 108,704

Table 3. Primers used for PCR amplification of gB, gD, gH, and gL, along with the amplified target sequence length. Nucleotide sequences are numbered according to GenBank accession number NC_001491.2.

EHV-1 Restriction Expected Fragments Sizes, bp Glycoproteins Enzyme Forward Clone Reverse Clone (5' → 3') (3' → 5') gB StuI 1420, 7000 3800, 4300 gD NdeI 1250, 5480 950, 5780 gH SmaI 2930, 5240 2090, 6080 XmnI 1960, 2660, 3380 3130, 2660, 3380 gL PvuII 600, 1070, 1100, 3420 830, 1070, 1100, 3190

Table 4. Restriction digest enzyme choices for the glycoprotein expression plasmids. Approximate expected DNA fragment sizes for forward and reverse orientation of each of the glycoproteins within pcDNATM3.3-TOPO® expression plasmids is shown.

EHV-1 gB gD gH gL Glycoproteins EHV-1 Strain Ab4 E* E E E OH03 E E E E L11 E* E E E KyA E* E X E

Table 5. Overview of generated pcDNATM3.3-TOPO® expression plasmids of glycoproteins gB, gD, gH, and gL. Shown are (E) generated plasmids that have been confirmed to express their respective glycoproteins in B78H1 cells, (E*) plasmids that are yet to be confirmed to show expression of their glycoprotein, and (X) plasmids that remain to be generated.

Gene Restriction Expected Fragments Sizes For Enzymes the Forward Clone (5' → 3'), bp

Nectin-1 PvuII 422, 1071, 1096, 4433 HVEM StuI 1898, 4590

Table 6. Restriction digest enzymes and corresponding expected DNA fragment sizes for equine nectin-1 and HVEM cloned into pcDNATM3.3-TOPO® plasmid vector.

FIGURES

Figure 1. Structure of the Varicellovirus virion. Linear, double-stranded DNA is enclosed in a T=16 icosahedral capsid, surrounded by an amorphous tegument layer. This structure is in turn contained in a lipid envelope that contains host cell-derived glycoproteins. Image reproduced with the permission of the publisher (ViralZone, Swiss Institute of Bioinformatics).

Figure 2. Virus growth assay in equine endothelial cells. Neurologic and non-neurologic EHV-1 strain virus yield obtained by infecting equine endothelial cells at MOI 1 in triplicate for 24 hours. Virus was harvested and titered on RK13 cells. Each bar represents the average of nine data points (three wells per replicate, total of three replicates). Error bars + 1 SEM.

Ab4 Ohio 2003

T967 T970

Figure 3. Plaque size of neurologic and non-neurologic EHV-1 strains. Equine endothelial cells were infected with approximately 100 plaque forming units of select EHV- 1 strains for 2 hours, overlaid with complete DMEM containing 1% methylcellulose, and stained after 4 days with 1% crystal violet. (A) Representative images of plaque phenotypes of Ab4 and (B) L11. (C) Quantified summary of the plaque phenotype data. Error bars + 1 SEM.

Expected DNA fragment sizes for forward-facing gB in TOPO 1bp 2699 3015bp digested with StuI (bp):

1418, 7004 741 742bp

PCMV MCS bp Cut Uncut 1842 gB-pcDNA3.3-TOPO 4000 - + 3000 - 2000 - StuI 1650 - 1000 -

gB-Ab4

Figure 4. Restriction map of pcDNATM3.3-TOPO® glycoprotein gB expression plasmid digested with restriction enzyme StuI, along with the representative gel electrophoresis image. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents gB cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for StuI within both gB and the plasmid. The table includes the number and length of the fragments resulting from the restriction digest.

Expected DNA fragment sizes for forward-facing gD in 1bp 780 1259bp TOPO digested with NdeI (bp):

299 741 742bp 1221, 5445

PCMV MCS

bp Cut Uncut gD-pcDNA3.3-TOPO + NdeI 5000 -

2000 - 1650 -

1000 - gD-KyA

Figure 5. Restriction map of pcDNATM3.3-TOPO® glycoprotein gD expression plasmid digested with restriction enzyme NdeI, along with the representative gel electrophoresis image. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents gD cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for NdeI within both gD and the plasmid. The table includes the number and length of the fragments resulting from the restriction digest.

Expected DNA fragment sizes for forward-facing gH in TOPO 1bp 711 2597bp digested with XmnI (bp):

1960, 2661, 3383 741 742bp MCS PCMV 1515

bp gH-pcDNA3.3-TOPO Cut Uncut

4898 + XmnI 4000 - 3000 -

2000 - 1650 - 1000 - gH-Ab4

Figure 6. Restriction map of pcDNATM3.3-TOPO® glycoprotein gH expression plasmid digested with restriction enzyme XmnI, along with the representative gel electrophoresis image. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents gH cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for XmnI within both gH and the plasmid. The table includes the number and length of the fragments resulting from the restriction digest.

Expected DNA fragment sizes for forward-facing gL in TOPO digested with PvuII (bp):

1bp 459 706bp 570, 1071, 1096, 3376

741 742bp 1063 MCS PCMV

bp Cut Uncut gL-pcDNA3.3-TOPO + PvuII 2159 4000 - 3000 - 2000 -

1000 - 850 - 3230 650 - 500 -

gL-Ab4

Figure 7. Restriction map of pcDNATM3.3-TOPO® glycoprotein gL expression plasmid digested with restriction enzyme PvuII, along with the representative gel electrophoresis image. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents gL cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for PvuII within both gL and the plasmid. The table includes the number and length of the fragments resulting from the restriction digest.

bp 1A 1B 1C 1D

5000 - 3000 - 2000 - 1650 - 1000 -

bp 2A 2B 2C 2D 3A 3B 3C 5000 - 3000 - 2000 - 1650 - 1000 -

bp 4A 4B 4C 4D

4000 - 3000 - 2000 - 1650 - 1000 - 850 - 650 - 500 -

Figure 8. Generated gB, gD, gH, and gL pcDNATM3.3-TOPO® expression plasmids. Each glycoprotein from EHV-1 strains Ab4, Ohio 2003, KyA, and L11 was ligated into TOPO plasmid and clonal populations screened with specific restriction enzymes for the genes ligated in the correct, forward orientation. (1 A-D) gB-L11, gB-Ohio 2003, gB-Ab4, gB-KyA - digested with StuI (expected DNA fragments, bp: 1418, 7002); (2 A-D) gD-Ab4, gD-Ohio 2003, gD-L11, gD-KyA - digested with NdeI (expected DNA fragments, bp: 1221, 5445); (3 A-C) gH-Ab4, gH-Ohio 2003, gH-L11 - digested with XmnI (expected DNA fragments, bp: 1960, 2661, 3383); (4 A-D) gL-Ab4, gL-Ohio 2003, gL-L11, gL-KyA - digested with PvuII (expected DNA fragments, bp: 570, 1071, 1096, 3376). Unlabeled DNA is the corresponding undigested glycoprotein plasmid.

1 2A 2B 2C 3A 3B 3C 4A 4B 4C 1 5A 5B 5C bp

3000 - 2000 - 1650 - 1000 - 700 -

gB gD gH gL

( 3015bp) (1259bp) (2597bp) (706bp)

Figure 9. Glycoproteins gB, gD, gH, and gL from Ohio 2003 expressed in B78H1 24 hours post-transfection. Glycoproteins were co-transfected into B78H1 cells, total RNA harvested at 24 hours post-transfection, cDNA generated and used as a template for PCR. (1) 1Kb Plus DNA ladder, (A) glycoprotein amplified from a cDNA template based on transfected B78H1, (B) corresponding glycoprotein amplified from a cDNA template based on B78H1 infected with EHV-1 (positive control), (C) corresponding glycoprotein amplified from a cDNA template based on mock-transfected B78H1. (2) gB, (3) gD, (4) gH, (5) gL.

1 2 3 4 5 6 bp

4000 - 3000 - 2000 -

1000 - 700 - 500 -

Figure 10. Glycoprotein gB from Ab4, L11, and KyA not expressed in B78H1 48 hours post-transfection. Glycoproteins were transfected into B78H1 cells, total RNA harvested at 48 hours post-transfection, cDNA generated and used as a template for PCR. (1) 1 Kb Plus DNA ladder, (2) Ab4 gB, (3) L11 gB, (4) KyA gB, (5) gB amplified from a cDNA template based on B78H1 infected with EHV-1 (positive control), (6) mock-transfected B78H1 (negative control). Expected gB DNA fragment size is 3015bp.

Figure 11A. Comparative overview of cell to cell fusion assay of HSV and Ohio 2003 glycoproteins gB, gD, gH, and gL. Twenty four hours post transfection, cell line expressing the glycoproteins was overlaid with the cell line expressing entry receptor and incubated for 48 hours. Shown are glycoprotein sets of (A) HSV combined with B78H1-C10 (human nectin-1) cell line and (B) Ohio 2003 combined with B78H1-C2 (equine MHC-I). Images taken at 40X total magnification.

Figure 11B. Comparative overview of cell to cell fusion assay of HSV and Ohio 2003 glycoproteins gB, gD, gH, and gL. Shown are glycoprotein sets of (C) HSV combined with B78H1-C10 (human nectin-1) cell line and (D) Ohio 2003 combined with B78H1-C2 (equine MHC-I). Images taken at 100X total magnification.

E G

F H

Figure 11C. Comparative overview of cell to cell fusion assay of HSV and Ohio 2003 glycoproteins gB, gD, gH, and gL. Shown are B78H1 cells mock-transfected with glycoprotein sets of (E, F) HSV, and combined with B78H1-C10 (human nectin-1) cell line and (G, H) Ohio 2003 combined with B78H1-C2 (equine MHC-I). Images taken at (E, G) 40X and (F, H) 100X total magnification.

A C

B D

Figure 12. Cell to cell fusion assay of Ohio 2003 glycoproteins gB, gD, gH, and gL. Shown are B78H1-C2 (equine MHC-I) cells (A, B) mock-transfected and (C, D) transfected with glycoprotein set of Ohio 2003. Images taken at (A, C) 40X and (B, D) 100X total magnification.

Ab4 Ohio 2003

T967 T970

B78H1 B78H1 -C2

B78H1 B78H1-C2 B78H1-C2 B78H1-C2

Figure 13. Diagnostic EHV-1 infection of B78H1-C2 cell line. B78H1-C2 and B78H1 (negative control) were infected with L11 at MOI 3 for 24 hours. Cells were stained with X- gal, marking infected cells blue. Top images are taken at 40X total magnification.

gB-Ab4

1bp 1516 1615bp

Expected DNA fragment sizes 741 742bp 1063 for forward- and reverse- facing nectin-1 in TOPO P MCS CMV digested with PvuII (bp): Forward:

nectin-1-pcDNA3.3- 422, 1071, 1096, 4433

TOPO Reverse: + 2159 1071, 1096, 1839, 3016 PvuII

3230 gB-Ab4

Figure 14. Restriction map of pcDNATM3.3-TOPO® nectin-1 expression plasmid digested with restriction enzyme PvuII. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents nectin-1 cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for PvuII within both nectin-1 and the plasmid. The table includes the number and length of the DNA fragments resulting from the restriction digest analysis.

bp Cut Uncut 3000 - 2000 - 1650 - 1000 -

Cut Uncut

Figure 15. Generated nectin-1 pcDNATM3.3-TOPO® expression plasmids. Equine nectin-1 was ligated into TOPO plasmid and clonal populations screened with PvuII for the genes ligated in the correct, forward orientation. All nectin-1 plasmids were ligated in reverse orientation (corresponding DNA fragment sizes (bp): 1071, 1096, 1839, 3016).

Expected DNA fragment sizes for forward-facing HVEM in TOPO digested with StuI (bp): 267 1bp 1081bp 1898, 4590

741 742bp bp Cut Uncut PCMV MCS 5000 - 4000 - 3000 - HVEM-pcDNA3.3- 1824

TOPO 2000 - + 1650 - StuI

HVEMgB-Ab4

Figure 16. Restriction map of pcDNATM3.3-TOPO® HVEM expression plasmid digested with restriction enzyme StuI. PCMV represents the promoter region of the plasmid. MCS is a multiple-cloning site between the 741st and the 742nd base pair of the plasmid. Blue line represents HVEM cloned in the correct forward orientation into the plasmid cloning site; length (in base pairs) is indicated. Triangles indicate restriction sites for StuI within both HVEM and the plasmid. The table includes the number and length of the fragments resulting from the restriction digest.