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GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2013-2014

CHARACTERISTICS OF EQUINE HERPESVIRUS TYPE 1 (EHV-1)

ENTRY IN MONOCYTIC CELLS

Jolien VAN CLEEMPUT

Promoter: Prof. dr. Hans Nauwynck Research thesis in the context

Co-promoter: Kathlyn Laval of the Master’s dissertation

© 2014 Jolien Van Cleemput

PREFACE

The first and most important person I need to thank is my copromotor, Kathlyn Laval. Thank you

for all your guidance and for the time you spent helping me! If there is one person that is committed to

a subject, it would be you. Without your tremendous energy and thirst for perfection, this thesis would

have never reached its present quality. You also encouraged me to write in English and helped me

use the language in a correct way. For that I am grateful, since it gave me more experience in writing

scientific texts. Merci!

Prof. dr. Nauwynck is degene die mij de gelegenheid gaf om onder zijn vleugels onderzoek uit te

voeren op equine herpesvirus 1 (EHV-1). Vorig jaar heeft hij mij begeleid in het schrijven van mijn

literatuurstudie en bood mij daarna de kans om op EHV-1 te blijven werken. Ik wil daarom dank u

zeggen voor al het vertrouwen en de begeleiding die u mij gaf in mijn eigen stukje onderzoek. Dankzij

deze kennismaking met onderzoek en met EHV-1 ben ik gebeten om dieper in te gaan op zijn

pathogenese. Het is dan ook een hele eer om volgend jaar verder te mogen werken bij u op hetgene

dat mij boeit.

Natuurlijk moet ik ook mijn ouders en vrienden bedanken voor hun steun en geduld gedurende

het schrijven van deze masterproef.

TABLE OF CONTENTS

Dislaimer

Title

Preface

List of abbreviations ................................................................................................................................. 1

Summary ............................................................................................................................................... 2

Samenvatting ........................................................................................................................................... 3

Introduction .............................................................................................................................................. 4

Literature study ........................................................................................................................................ 4

1. THE VIRUS ...................................................................................................................................... 4

1.1. STRUCTURE ............................................................................................................................ 4

1.2. REPLICATION CYCLE ............................................................................................................. 5

2. THE PATHOGENESIS .................................................................................................................... 7

2.1. ENTRY ...................................................................................................................................... 7

2.2. VIREMIA .................................................................................................................................... 8

2.3. REPRODUCTIVE DISORDERS ............................................................................................... 8

2.4. NERVOUS SYSTEM DISORDERS .......................................................................................... 9

3. IMMUNITY OF THE HOST ............................................................................................................ 10

3.1. RESPIRATORY MUCOSA ...................................................................................................... 10

3.2. IMMUNE CELLS ..................................................................................................................... 10

4. IMMUNO-EVASION ....................................................................................................................... 12

5. VACCINATION .............................................................................................................................. 13

5.1. INTRODUCTION ..................................................................................................................... 13

5.2. ATTENUATED VACCINES ..................................................................................................... 13

5.3. INACTIVATED VACCINES ..................................................................................................... 14

5.4. DNA VACCINES ..................................................................................................................... 15

5.5. CONCLUSION ........................................................................................................................ 15

Research project ................................................................................................................................... 16

1. AIM ................................................................................................................................................. 16

2. MATERIALS AND METHODS ....................................................................................................... 17

2.1. CELLS ..................................................................................................................................... 17

2.2. VIRUS ..................................................................................................................................... 17

2.3. VIRUS PURIFICATION AND DIO-LABELLING ...................................................................... 18

2.4. VIABILITY AND INOCULATION WITH EHV-1 ....................................................................... 18

2.5. KINETICS OF EHV-1 ATTACHMENT .................................................................................... 19

2.6. KINETICS OF EHV-1 INTERNALISATION ............................................................................. 19

2.7. TREATMENT WITH LYSOSOMOTROPIC AGENTS ............................................................. 19

2.8. INDIRECT IMMUNOFLUORESCENCE STAINING OF EHV-1 PROTEINS .......................... 20

2.9. STATISTICAL ANALYSIS ....................................................................................................... 20

3. RESULTS....................................................................................................................................... 21

3.1. KINETICS OF EHV-1 BINDING .............................................................................................. 21

3.2. KINETICS OF EHV-1 INTERNALISATION ............................................................................. 24

3.3. THE EFFECT OF LYSOSOMOTROPIC AGENTS ON EHV-1 INFECTION .......................... 24

Discussion ............................................................................................................................................. 26

References ............................................................................................................................................ 31

1

LIST OF ABBREVIATIONS

APCs antigen presenting cells

CHO-K1 Chinese hamster ovary cells

CME clathrin-mediated endocytosis

CTLs cytotoxic T lymphocytes

DCs dendritic cells

DPBS Dulbecco’s phosphate-buffered saline

EBMECs equine blood microvascular endothelial cells

EC endothelial cells

ED equine dermis

EHM equine herpes myeloencephalopathy

EHV-1 equine herpesvirus type 1

FCS foetal calf serum

g glycoprotein

HSV herpes simplex virus

hpi hours post infection

ICAM intracellular adhesion molecule

IE immediate-early

IEP immediate-early protein

Ig immuglobulin

IL interleukin

MEM modified Eagle’s medium

MHC major histocompatibility complex

NH4Cl ammonium chloride

PBMC peripheral blood mononuclear cells

pi post infection

RK rabbit kidney

RPMI Roswell Park Memorial Institute

SuHV-1 suid herpesvirus type 1

2

SUMMARY

Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus that causes abortion and central

nervous disorders in horses. During acute infection, the virus replicates in the respiratory epithelium

and disseminates through the body via a cell-associated viremia in monocytic cells in order to reach

target organs such as pregnant uterus and/or central nervous system. Vaccination does not provide

full protection against severe symptoms, as the virus can spread via the blood in the presence of

neutralizing antibodies. Recently, it has been shown that only 4% of equine nasal mucosal and blood

CD172a+ monocytic cells, two targets cells of EHV-1, were susceptible to EHV-1 infection in vitro.

However, little is known about EHV-1 binding and entry in these target cells. In this thesis, EHV-1

kinetics of binding and route of entry were compared between blood CD172a+ monocytic cells and

rabbit kidney (RK)-13 cells, a cell line fully susceptible to EHV-1 infection. We found that EHV-1

efficiently binds to 80% of the RK-13 cells with an average maximum of 30 virus particles attached per

cell after 1h incubation period. In contrast, only 12% of the blood CD172a+ cells showed bound virus

with an average of 2 virus particles per cell. Besides, we demonstrated that treatment with endosomal

inhibitors of blood CD172a+ and RK-13 cells both decreased viral infection in a dose-dependent

manner. Taken together, these results showed that EHV-1 does not efficiently bind to CD172a+

monocytic cells but can enter these cells through endocytosis in a pH dependent manner. This

process may be part of an immune evasive strategy responsible for the restriction of EHV-1 replication

in monocytic cells and suggests the presence of specific cell surface receptors with restricted

expression patterns.

Keywords: EHV-1, CD172a+ cells, binding, internalisation, endocytosis

3

SAMENVATTING

Equine herpesvirus type 1 (EHV-1) is een alphaherpesvirus dat abortus en milde tot erge

zenuwsymptomen veroorzaakt bij paarden. Het virus vermeerdert ter hoogte van het respiratoir

epitheel en kan dankzij een cel-geassocieerde viremie in monocyten tot bij de drachtige baarmoeder

en het centraal zenuwstelsel geraken. Vaccinatie kan het paard niet volledig beschermen tegen

symptomen want het virus kan, ondanks de aanwezigheid van virusneutraliserende antistoffen, toch

spreiden via de bloedbaan. Recent werd aangetoond dat in vitro slechts 4% van de equine nasale

mucosa en bloed CD172a+ cellen (2 doelwitcellen) gevoelig zijn voor EHV-1 infectie. Over

virusbinding, internalisatie en virusreplicatie in deze doelwitcellen is echter nog weinig geweten. In

deze masterproef werd de kinetiek van EHV-1-binding en -intrede bestudeerd in bloed CD172a+

monocyten en in rabbit kidney (RK)-13 cellen, een cellijn die gevoelig is aan EHV-1 infectie. Eén uur

na inoculatie, bond EHV-1 efficiënt aan 80% van de RK-13 cellen met een gemiddeld maximum van

30 partikels per cel. In vergelijking hiermee kon het virus slechts aan 12% van de bloed CD172a+

cellen binden, met een gemiddelde van 2 partikels per cel. Daarnaast werd ook aangetoond dat na

behandeling van de cellen met endosomale inhibitoren, de infectie dosisafhankelijk werd verminderd.

Deze resultaten tonen aan dat EHV-1 niet efficiënt kan binden aan bloed CD172a+ cellen maar deze

cellen kan binnendringen via pH-afhankelijke endocytose. Dit kan, als onderdeel van een immuno-

evasieve strategie, verantwoordelijk zijn voor de restrictie in virusreplicatie in monocyten en wijst erop

dat het virus bindt aan een beperkt aantal specifieke celreceptoren.

Sleutelwoorden: EHV-1, CD172a+ cellen, binding, internalisatie, endocytose

4

INTRODUCTION

Equine Herpesvirus-1 (EHV-1) is classified as a member of the family Herpesviridae, subfamily

Alphaherpesvirinae, genus Varicellovirus (Roizman and Baines, 1991). EHV-1 shares this

classification with bovine herpesvirus type 1 (BoHV-1), suid herpesvirus type 1 (SuHV-1) and

varicella-zoster virus (VZV). It is one of nine herpesviruses known to infect horses which has also

been found in mules, donkeys, zebras, onagers, giraffes and gazelles (Borchers and Frolich, 1997;

Hoenerhoff et al., 2006; Ibrahim et al., 2007).

Dimonck and Edwards documented the virus for the first time in 1933 during an outbreak of

abortion. The virus was first known as ‘equine abortion virus’ and later as ‘equine rhinopneumonitis

virus’ (Allen and Bryans, 1986). The virus was isolated for the first time in 1966 (Saxegaard, 1966;

Jackson and Kendrick, 1971). EHV-1 is not only responsible for abortion and respiratory disorders but

also for neonatal death and nervous system disorders. Outbreaks keep occurring every year and the

virus causes serious economic losses in the horse industry worldwide.

LITERATURE STUDY

1. THE VIRUS

1.1. STRUCTURE

The structure of an herpesvirus consists of a double-stranded DNA molecule of approximately

150 kbp, an icosahedral capsid build up from 162 capsomers, the tegument and an envelope which

contains 12 different glycoproteins, as shown in Figure 1 (Telford et al., 1998).

The envelope is derived from the trans-Golgi network of the host cell and forms the outer layer of

the virus (Granzow et al., 2001). Several glycoproteins are embedded in the envelope and each one

has a specific role in virus attachment, entry, fusion, penetration, egress and cell-to-cell spread.

Glycoprotein (g) B, gC and gD are necessary for attachment (Neubauer et al., 1997;

Osterrieder, 1999; Csellner et al., 2000). Glycoproteins gB, gD, gM and gK help the virus in a process

called penetration, which is the release of naked nucleocapsids in the host cell cytoplasm (Osterrieder

et al., 1996a; Neubauer et al., 1997; Neubauer and Osterrieder, 2004). EHV-1 gB, gD, gE/gI, gK, gM

and gH assist the virus in cell-to-cell spread (Neubauer et al., 1997; Csellner et al., 2000; Flowers and

O'Callaghan, 1992; Matsumara et al., 1998; Neubauer and Osterrieder, 2004; Osterrieder et al.,

1996a; Azab et al., 2012). Glycoprotein G can modulate the inflammation process by binding

chemokines such as interleukin (IL) 8 (Van de Walle et al., 2007; Thormann et al., 2012). Gp2 is

somewhat of an outsider because it does not have a positional or structural counterpart in

herpesviruses of other species. It was proposed that this glycoprotein plays a role in the modulation of

the host’s immune system (Smith et al., 2005).

5

Glycoprotein H not only has a function in cell-to-cell spread but it was hypothesised that it also

plays a role in determination of EHV-1’s entry pathway (Azab et al., 2013). Until present, the function

of gL remains unclear.

1.2. REPLICATION CYCLE

A schematic overview of EHV-1 replication cycle is given in Figure 2.

The initial step in EHV-1 replication is the attachment of the virus to the cell. EHV-1 interacts with

heparan sulphate on the cell surface via gB and gC (Neubauer et al., 1997; Osterrieder et al., 1999).

This is followed by an interaction between the viral glycoprotein gD and a cellular receptor to stabilise

the binding (Csellner et al., 2000). Previous studies have shown that major histocompatibility complex

class I (MHC I) acts as a putative receptor (Kurtz et al., 2010; Sasiki et al., 2011).

After binding, EHV-1 has to get into the cell. The virus is known to enter cells through two

different pathways. The virus enters equine endothelial cells (EC), equine dermis (ED) cells and rabbit

kidney (RK-13) cells via fusion, while EHV-1 enters peripheral blood mononuclear cells (PBMC) and

Chinese hamster ovary cells (CHO-K1) via endocytosis (Frampton et al., 2007; Van de Walle et al.,

2008). It was shown that the endocytic pathway is mediated by the interaction between cellular V

integrins and a RSD motif present in EHV-1 gD (Van de Walle et al., 2008).

Both entry pathways require the activation of a cell signalling pathway by activating Rho-

associated coiled-coil kinase (ROCK1) (Frampton et al., 2007). After entry into the cell, the virus

releases a naked nucleocapsid in the cytoplasm, which travels along the microtubule network. At the

nuclear membrane, the virus injects its DNA via the nuclear pores (Frampton et al., 2010).

Transcription of viral DNA occurs in a cascade-like manner. The immediate-early (IE) gene is first

synthesized and encodes for a major IE polypeptide (IEP) that functions as a regulatory protein. IEP

expression is necessary for the expression of early (E) genes and late (L) genes (Gray et al., 1987).

The E genes encode proteins required for DNA replication, such as DNA polymerase and thymidine

kinase. L genes encode multiple viral structural proteins such as capsid and tegument proteins and

envelop glycoproteins (Vandekerckhove, 2011).

Figure 1 Structure of an EHV-1 virion. (a) Schematic representation. (b) Transmission electron

microscopic image. The bar represents 100 nm (adapted from Vandekerckhove, 2011).

6

Figure 2 A schematic representation of the various steps of the EHV-1 replication cycle, from

Vandekerckhove (2011). The first step in the replication cycle is the attachment of free virions to the

surface of the target cell (1). The binding is initially unstable and includes interaction of viral envelope

glycoproteins gB and gC with heparan sulfate glycosaminoglycan moieties of the cell surface (2a).

Subsequently, EHV-1 can enter cells via 2 pathways, depending on the infected cell type. In equine

endothelial, equine dermal and rabbit kidney cells, fusion of the viral envelope with the plasma

membrane occurs through interaction of gD with an entry receptor, possibly MHC-1 (2b). In equine

PBMCs and CHO-K1 cells, entry occurs via a nonclassical endocytic pathway mediated by the

interaction between cellular αV integrins and an RSD motif present in EHV-1 gD (3). The nucleocapsid is

released into the cytoplasm (4) and transported to the nucleus along microtubules (5). In the nucleus,

DNA replication (6) and transcription (7) occur. The genome is transcribed in a cascade-like manner

with first the immediate-early (IE) genes, then the early (E) genes and finally the late (L) genes. RNA

molecules are transported to the cytoplasm and translated to proteins (8). Capsid proteins are

redirected into the nucleus where assembly of the capsid occurs (9). Subsequently, DNA is pulled into

the newly formed capsids and hence nucleocapsids are assembled (10). The nucleocapsids leave the

nucleus via budding through the inner leaflet of the nuclear membrane (11), hereby requiring their

primary envelope (12). This results in the entry of naked nucleocapsids into the cytoplasm (13). The

nucleocapsids acquire their secondary envelope at the Golgi apparatus (14) and then leave the cell via

vesicle-mediated exocytosis (15, 16, 17).

7

Assembly of the nucleocapsid occurs in the nucleus. The virus acquires its primary envelop from

the inner nuclear membrane but loses this one when it exits the perinuclear space. This step results in

the release of a naked nucleocapsid in the cytoplasm. The final envelopment takes place in the trans-

Golgi complex and the virion leaves its host cell through vesicle-mediated exocytosis (Granzow et al.,

2001). Electron microscopy images of primary and secondary envelopment are shown in Figure 3.

2. THE PATHOGENESIS

2.1. ENTRY

Horses are infected through the upper respiratory tract after inhalation of virus-loaded aerosol

droplets, originating from direct contact with other infected horses or from the air (Patel et al., 1982;

Kydd et al., 1994). The virus comes into contact with the mucus and the epithelium lining the nasal

septum, the turbinates, the nasopharynx, the soft palate and the trachea.

During primary infection, EHV-1 infects epithelial cells in a plaquewise manner and causes cell

destruction. This destruction leads to clinical symptoms consisting of fever, anorexia, depression,

nasal discharge, swelling of the submandibular and retropharyngeal lymph nodes and sometimes

conjunctivitis with ocular discharge (Patel et al., 1982; Allen and Bryans, 1986). The incubation period

varies from 2 to 10 days (Allen and Bryans, 1986). The virus can be detected in nasal discharge from

day 1 post infection (pi) up until 14 days pi (Edington et al., 1986; Gryspeerdt et al., 2010).

Figure 3 Exit of nucleocapsids from the nucleus. Enveloped EHV-1 particles in the perinuclear cisterna

(left). Secondary envelopment and virus egress: envelopment of EHV-1 at the membranes of the trans-

Golgi area (right). The bars represent 150 nm. N designates the nucleus. (from Granzow et al., 2001).

8

2.2. VIREMIA

EHV- 1 can only cross the basement membrane via single infected cells, in contrast to some

other herpesviruses, which drill holes through the basement membrane. Upon infection of diapeding

leucocytes, the virus can cross the basement membrane and enter the lymph nodes and the

bloodstream. This cell-associated viremia enables the virus to reach the pregnant uterus and the

central nervous system (Kydd et al., 1994). Infected leucocytes can be found in the bloodstream from

1 day pi but mostly between day 4 and 10 pi (Gryspeerdt et al., 2010). These infected leucocytes have

been identified mainly as CD172a+ cells, followed by CD5

+ T-lymphocytes and B-lymphocytes

(Gryspeerdt et al., 2010). CD172a+ cells originate from the myeloid lineage and interestingly, after in

vitro EHV-1 infection, these cells show a hampered expression of some late EHV-1 glycoproteins (van

der Meulen et al., 2003; Gryspeerdt et al., 2012). This suggests that the virus uses these cells to

spread through the body. At the target organs, EHV-1 initiates a secondary replication cycle in

endothelial cells lining the blood vessels of the pregnant uterus or the central nervous system.

2.3. REPRODUCTIVE DISORDERS

EHV-1 infection is the main cause of abortion in horses and has been first documented by

Dimonck and Edwards in 1933. In the 70’s, a first attempt was made to discover the pathogenesis of

this EHV-1 induced abortion. Bryans and Prickett found out that fluid between the allantochorion and

the endometrium causes them to detach (Prickett, 1969; Bryans and Prickett, 1970). Edington et al.

(1991) confirmed this hypothesis and demonstrated that the virus can infect endothelial cells, which

can cause vasculitis and thrombosis and subsequently leads to avascular necrosis and edema of the

endometrium. Smith et al. (2001 and 2002) showed that activation of adhesion molecules in

Figure 4 White necrotic spots on the liver of an EHV-1 aborted foetus (from Laugier

et al., 2011)

9

endothelial cells of the nasal mucosa and the reproductive tract is a key step in transferring virus from

infected leukocytes to endothelial cells. EHV-1 then switches its replication cycle back on and infects

its target cell. Some of these adhesion molecules have been identified as E- and P-selectin and

intracellular adhesion molecule (ICAM) (Smith et al., 2001). These surface molecules could be

induced by 17-oestradiol, equine chorionic gonadotropin (eCG), IL-2 en lipopolysaccharides (Smith et

al., 2002).

Endometrial vascular damage causes detachment of the foetal membranes and the foetus, thus

leading to abortion of a virus negative foetus. Less extensive endometrial vascular damage allows

EHV-1 to invade the foetus through the umbilical blood vessels, where endothelial cells are also

targeted (Edington et al., 1991). The viral aborted foetus can show following lesions: multifocal hepatic

necrosis (see Figure 4), subcutaneous edema, pleural fluid accumulation, pulmonary edema and

splenic enlargement (Corner et al., 1963; Machida et al., 1997).

EHV-1 induced abortion typically occurs in the last trimester of pregnancy (Allen and Bryans,

1986), although one case of abortion has been reported in the fourth month of gestation (Prickett,

1969). When a mare gets infected or the virus reactivates late in gestation or at full term, a live foal

can be delivered. However, this foal almost always shows signs of weakness, jaundice and respiratory

distress and usually dies within 7 days (Murray et al., 1998). This time dependent manner of viral

transfer has been a subject of investigation for several years. It was suggested that these surface

adhesion molecules are increasingly expressed at the end of gestation under the influence of

cytokines and hormones (Smith et al., 2001). In addition, the immune system of the mare is

compromised during that time because of the high concentrations of cortisone, progesterone and

oestrogens (Smith et al.,1996).

2.4. NERVOUS SYSTEM DISORDERS

EHV-1 can also replicate in vessel endothelia at the level of the nervous system (Edington et al.,

1986). However, the virus is endotheliotropic and does not cause encephalitis by replication in

neurons. Endothelial damage subsequently leads to vasculitis, thrombosis and edema in a similar

manner as in the pregnant uterus. The lack of nutrients and oxygen causes the neurons to degenerate

and eventually leads to equine herpes myeloencephalopathy (EHM).

Neurological symptoms go from ataxia to a complete fore and hind limb paralysis. Other clinical

signs comprise faecal and/or urinary incontinence, head tilting, tail paralysis, distal limb edema,

edema of testes, penis prolapse and even blindness (Jackson et al., 1977; van Maanen et al., 2001;

Borchers et al., 2006).

Neurovirulent strains are more likely to cause central nervous system disorders, not due to the

fact that they are neurotropic but because they initiate a higher and longer viremia (Allen and

Breathnach, 2006). It has been shown that a point mutation in the virus DNA polymerase determines

whether the virus is neurovirulent or non-neurovirulent (Nugent et al., 2006; Goodman et al., 2007).

10

3. IMMUNITY OF THE HOST

3.1. RESPIRATORY MUCOSA

Upon infection, the respiratory mucosa and the overlining mucus blanket are the first defence

mechanisms to eliminate the virus.

The mucus forms two layers, a top viscous layer which captures pathogens from the air and a

basal serous layer which surrounds the cilia (Harkema et al., 2006). This conformation is crucial for

the mucociliary clearance mechanism to work. Synchronized beating of the cilia pushes the mucus

with entrapped materials towards the oropharynx where it is then swallowed. An in vitro experiment

demonstrated that mucus entraps SuHV-1 in the mucoprotein network by the interaction of opposite

charges on the virus and mucoproteins (Yang et al., 2012). The mucus layer also contains

immunoglobulins, especially secretory immunoglobulin A (sIgA) (Marriot et al., 1990). The purpose of

this Ig is to capture pathogens in the respiratory airway, thus preventing them to attach to the host

cells.

The tunica mucosa of the upper airway tract consists of an epithelium with a basement

membrane, a connective tissue layer called the lamina propria and a layer of smooth muscles called

the lamina muscularis. First of all, the tunica mucosa forms a physical barrier against pathogens due

to the tight cell-cell and cell-matrix adhesions. Second, intruders are countered by the different

immune cells, which release their enzymes upon activation (Vandekerckhove, 2011).

3.2. IMMUNE CELLS

Figure 5 shows the different immune cells present in the respiratory mucosa that play a major role

in the defence against EHV-1. During infection, PBMC have been shown to be target cells of the virus.

PBMC have a round nucleus and consist of monocytes, macrophages and lymphocytes. The

lymphocyte population consists of T lymphocytes, B lymphocytes and natural killer (NK) cells. On the

opposite, blood cells that have a lobed nucleus (neutrophils and eosinophils) are called

polymorphonuclear cells.

Monocytes, macrophages and dendritic cells (DCs) belong to the myeloid lineage CD172a+.

Monocytes are mainly phagocytes and play an essential role as cells of the first line of defence

against pathogens. They originate from the bone marrow and are released into the peripheral blood,

where they circulate for several days before entering tissues to renew tissue macrophage and

dendritic cell populations (Auffray et al., 2009; Geissmann et al., 2010). Macrophages are resident

cells in lymphoid and non-lymphoid tissues, where they mainly function as phagocytes and are

responsible for the clearance of apoptotic and infected cells. Macrophages also act as antigen

presenting cells (APCs). Finally, they can activate the inflammatory pathway by producing tumor

necrosis factor alpha (TNF-α), IL-1 and IL-6 (Geissmann et al., 2010). Dendritic cells (DCs), in the

respiratory mucosa known as Langerhans cells, are specialized as APCs. Upon activation, they

migrate from tissues to the draining lymph nodes, where they regulate T cell responses (Fokkens et

al., 1989). Differentiation of monocytes into DCs is mostly observed in inflammatory conditions and is

11

dependent on the cytokine and hematopoietic growth factor receptor Csf1r, also known as CD115

(Auffray et al., 2009; Geissmann et al., 2010).

Figure 5 A schematic overview of the different immune cells present in the equine respiratory mucosa

(from Vandekerckhove, 2011).

12

Once activated by an antigen and a helper T cell, B Lymphocytes differentiate into plasma cells.

Plasma cells lay embedded in the lamina propria and secrete the main important antiviral antibody in

the respiratory epithelium: immunoglobulin A (IgA) (Fallgreengebauer et al., 1993). This antibody

works in an antiviral way by neutralising viral particles in the respiratory lumen. Immunoglobulins G

also neutralise viral particles and are mainly present in the bloodstream. However, IgG cannot protect

the horse against cell-associated viremia and thus EHM and abortion. Indeed, the expression of gD

and gC is hampered in EHV-1 infected PBMC, which does not allow antibody-dependent cell lysis.

T Lymphocytes are found in the epithelium, the lamina propria and the bloodstream. CD8+ T

cells express the glycoprotein CD8 in combination with their T cell receptor (TCR) and are also known

as cytotoxic T lymphocytes (CTLs). Their function is to kill infected cells that express foreign proteins

on their MHC I receptor. These T cells are rather found in the epithelium, in contrast to CD4+ T cells,

which are mainly localised in the lamina propria (Vandekerckhove, 2011). CD4+ T cells provide the

necessary signals to B cells for their maturation and to CD8+ T cells and macrophages for their

activation after binding with their MHC II receptor.

Natural Killer cells (NK cells) keep viral infections under control until the adaptive immunity can

take over. Viruses can hamper expression of MHC I class molecules at the cell surface in order to

escape the host’s immune system. NK cells, however, can recognise these infected cells due to a

down regulation of MHC I class molecules and then work in a similar way as CTLs. Upon activation

they release their cytotoxic granules on the target cell and induce programmed cell death through

effector proteins (Murphy, 2008).

4. IMMUNO-EVASION

EHV-1 has developed several strategies to escape the host immune system. Firstly, the virus

hampers expression of its late glycoproteins gD and gC in PBMC. As a result, exposure of viral

proteins at the plasma membrane is declined and this protects the virus against antibody-dependent

lysis and allows EHV-1 to remain silent in the cell (Van der Meulen et al., 2003; Gryspeerdt et al.,

2012).

One of the IEP or EP is responsible for the down regulation of MHC I class molecules, which are

necessary for CTLs to recognize and kill infected cells (Rappocciola et al., 2003; Van der Meulen et

al., 2006). The host NK cells recognize the lack of MHC I expression at the plasma membrane.

However, some infected cells still show some MHC I expression, thus EHV-1 is also avoiding this

immune mechanism.

Direct cell-to-cell spread is another major strategy for EHV-1 to bypass the extracellular

environment, containing fagocytes, antibodies and complement (van der Meulen et al., 2002;

Nauwynck et al., 2007).

13

5. VACCINATION

5.1. INTRODUCTION

In case of EHV-1 infection, the main purpose of vaccination is to reduce viral replication in the

upper respiratory tract and thus minimize viral shedding and respiratory symptoms. Besides,

vaccination should also prevent the cell-associated viremia and subsequent spread of EHV-1 to

internal organs, such as the pregnant uterus and/or the central nervous system (Kydd et al., 2006; van

der Meulen et al., 2007). The elicited immunity needs two arms in order to fully protect a horse (Kydd

et al., 2006). On the one hand, a mucosal immune response consisting of local IgA producing plasma

cells and diapeding memory CTLs is needed. On the other hand, the systemic immune response

should comprise IgG antibodies, which neutralise viral particles, and CTLs, which kill infected cells.

Commercially available vaccines can minimize respiratory symptoms and nasal shedding but they

do not guarantee a full protection against viremia and secondary symptoms. Indeed, clinical signs can

occur despite the presence of neutralizing antibodies (Kydd et al., 2006).

5.2. ATTENUATED VACCINES

A live attenuated virus strain can be obtained by serial passages on heterologous cells or at

different temperatures. The biggest advantage of a live attenuated vaccine is its ability to stimulate

CTLs, because these vaccines can actually infect cells. However, they represent a potential danger as

they can mutate to the original virus strain and cause serious symptoms. Another possibility to

produce an attenuated vaccine is to remove genes encoding for virulent EHV-1 proteins. These

vaccines are only experimental until present.

Commercial vaccines 5.2.1.

Attenuation of RacH strain on porcine embryonic kidney cells formed the base of the current

commercial vaccine Prevaccinol. Further passage on RK cells and ED cells led to the origination of

Rhinomune (Kydd et al., 2006). The commercial vaccines are both safe because they lack certain

proteins necessary for full replication at body temperature (Osterrieder et al., 1996b; Osterrieder et al.,

1998). In trials with the commercial vaccines, viremia could not be prevented (Burkï et al., 1990;

Goodman et al., 2006). However, in the field, a decline in abortion cases was observed after

vaccination with Prevaccinol (Frymus et al., 1986; Becker, 1988; Bresgen et al., 2012). For

Rhinomune, this could not be concluded (Dutta and Shipley, 1975; Neeley and Hawkins, 1978;

Mitchell et al., 1978). But surprisingly, the vaccine gave protection against EHM in a later experiment

(Goodman et al., 2006). It could also reduce nasal shedding and clinical symptoms in infected ponies

(Goehring et al., 2010)

14

Experimental vaccines 5.2.2.

Vaccination with the Kentucky (KyA) strain, adapted to mouse fibroblast cells, could reduce the

duration of viremia (Matsumara et al., 1996). Upon vaccination with the C147 strain, restricted for

growth at temperatures above normal body temperature, fewer horses became viremic after infection.

A decline in the percentage of abortion could also be observed (Patel et al., 2003a; Patel et al.,

2003b).

A thymidine kinase-deficient (TK-) mutant could ascertain a decreased infection rate of PBMC

after vaccination (Slater et al., 1993). But inoculation with the TK- strain or with the gE/gI deficient

(gE-/gI

-) strain could not guarantee a decline in percentage of viremic horses, nor abbreviate the

duration of viremia (Slater et al., 1993; Matsumara et al.,1998). A later vaccination trial with the gE-

strain in a group of ponies showed a reduction in infected PBMC upon infection (Tsujimura et al.,

2009). From a more recent circulating EHV-1 strain, NY03, the attenuated vaccine strain

rNY03ΔIR6/1gp2S was produced. Horses that received the vaccine did not develop viremia upon

infection compared to the mock-vaccinated horses (Van de Walle et al., 2010). These promising

results led to the development of a NY03ΔIR6/1gp2S_H3 strain, which could not only elicit antibodies

against EHV-1, but also against equine influenza virus (Van de Walle et al., 2010).

5.3. INACTIVATED VACCINES

These vaccines either contain inactivated whole virus or viral envelope glycoproteins in

combination with an adjuvant. A recombinant vector virus is created by engineering DNA from EHV-1

into another virus, such as vaccinia virus or canarapox virus. Then, cell cultures can be infected with

this virus, after which the specific glycoproteins are synthetized. Finally, the cells are collected,

washed and the virus is inactivated. The suspension can finally be used as a vaccine.

The biggest disadvantage of inactivated vaccines is their inability to activate CTLs, which are

necessary to kill infected cells (Minke et al., 2004).

Commercial vaccines 5.3.1.

The first studies to determine the protection of inactivated vaccines were done with Pneumabort

K, an inactivated whole virus and oil-adjuvanted vaccine containing EHV-1, in pregnant mares,

yearlings and two-year-old ponies (Burrows et al., 1984; Bürki et al., 1990). The results showed that

the vaccine could not prevent, nor decline viremia.

A decade later, a vaccination/challenge study with Duvaxyn EHV1,4 was performed in pregnant

mares and in naïve foals (Heldens et al., 2001). Duvaxyn EHV1,4 is a whole virus inactivated and

carbomer-adjuvanted vaccine containing EHV-1 and EHV-4. In pregnant mares no reduction in viremic

horses, nor in duration of viremia was observed. In contrast, only 30% of the vaccinated foals became

viremic upon challenge, compared to 80% of the control foals. This could be due to the use of a less

virulent strain to challenge the foals (Vandermeulen et al., 2007).

In 2006, a trial with Flu-Vac Innovator 6 also showed that inactivated vaccines could not reduce

the number of viremic horses, nor decline duration of viremia (Goodman et al., 2006).

15

Some field studies did however show a reduction in percentage of abortion upon vaccination with

Pneumabort K or Duvaxyn EHV1,4 (Bryans and Allen, 1982; Bresgen et al., 2012).

Experimental vaccines 5.3.2.

Experimental vaccines, like gD/gB, gB/gC/gD and IE cannot reduce the level, nor the duration of

viremia (Foote et al., 2006; Minke et al., 2006; Paillot et al., 2006; Soboll et al., 2010). Only the

immune stimulating complexes (ISCOMs) vaccine, which contains all the important EHV-1

glycoproteins, reduced the duration of viremia upon infection (Hannant et al., 1993). ISCOM is an

adjuvant with an open cage-like structure and a diameter of about 40 nm. They are built up by

cholesterol, lipid, immunogen, and saponins. The biggest advantage of ISCOM adjuvant is that, in

contrast to most other adjuvants, it can induce cellular immunity (Sjölander et al., 1998).

5.4. DNA VACCINES

DNA vaccines consist of plasmids that contain genes encoding glycoproteins against which

immunity has to be elicited, flanked by a promotor (Gurunathan et al., 2000). The vaccine is

administered intramuscular or intradermal. Once host cells take up the plasmid, viral proteins are

expressed, transported into the endoplasmatic reticulum and cleaved into peptides by cellular

proteases. These peptides are subsequently bound to MHC I molecules in the endoplasmatic

reticulum. Through these molecules, the viral peptides are expressed at the cellular surface and

immunity is elicited.

The main advantage of DNA vaccines is that, next to a humoral response, they can induce

cellular immunity by activating CTLs. Despite this promising fact, vaccination with the experimental

gB/gC/gD could not significantly reduce viremia upon challenge. Neither this was the case for the

IE/Unique Long (UL)-DNA vaccine (Minke et al., 2006; Soboll et al., 2006).

5.5. CONCLUSION

Outbreaks with EHV-1 keep occurring every year so there is a growing need for a proper

treatment or an efficient vaccine. Our current vaccines (Prevaccinol, Rhinomune, Pneumabort K,

Duvaxyn EHV1,4 and Flu-Vac Innovator 6) do not stimulate the cellular and/or humoral immunity in a

way to garantee a 100% protection (Burrows et al., 1984; Bürki et al., 1990; Heldens et al., 2001;

Goodman et al., 2006; Goehring et al., 2010). In order to develop efficient vaccines and actual

therapies, a better understanding of the pathogenesis of EHV-1 is urgently required.

16

RESEARCH PROJECT

1. AIM

EHV-1 induced endothelial damage is the main cause of placentitis with subsequent abortion in

horses and EHM forces the vet to euthanize the horse in most cases. Currently, commercial vaccines

do not give full protection against EHV-1 infection. Despite the presence of a strong immune

response, clinical signs are still being observed (Burrows et al., 1984; Heldens et al., 2001; Goodman

et al., 2006; Sellon and Long, 2007).

Previous studies on the pathogenesis of EHV-1 showed that cells, originating from the myeloid

lineage, are the main target cells of EHV-1 (Van der Meulen et al., 2003; Gryspeerdt et al., 2010). This

suggests that these cells are the source of the cell-associated viremia. Preliminary research revealed

that EHV-1 infection is restricted and EHV-1 replication is delayed in CD172a+ cells, originating from

blood and nasal mucosa (Laval et al., 2014 unpublished data). This delay in replication is an example

of an immune evasive strategy and clearly indicates that the virus is using CD172a+ cells as ‘Trojan

horses' to facilitate its spread within the host and delay immunosurveillance. However, little is known

about the regulation of monocytic cell tropism and susceptibility to EHV-1. We propose that an early

block in the replication cycle of the virus in CD172a+ cells may be responsible for the restricted

replication in those cells. Thus in the present project, we investigated the initial interaction between

EHV-1 and blood CD172a+ cells, which consist of binding and subsequent fusion and/or internalisation

of the virus.

17

2. MATERIALS AND METHODS

2.1. CELLS

Isolation of equine blood CD172a+ cells 2.1.1.

Healthy horses, between 8 to 10 years old were used as blood donors. The collection of blood

was approved by the ethical committee of Ghent University (EC2013/17). Blood was collected by

jugular venipuncture on heparin (15U/ml) (Leo) and diluted in an equal volume of Dulbecco's

phosphate-buffered saline (DPBS) without calcium and magnesium (Gibco). PBMC were isolated by

density centrifugation on Ficoll-Paque (d=1.077g/ml) (GE Healthcare, Life Sciences) at 800 xg for 30

minutes at 18°C. The interphase cells, containing PBMC, were collected and washed three times with

DPBS. Cells were resuspended in leukocyte medium based on Roswell Park Memorial Institute

(RPMI, Gibco) supplemented with 5% fetal calf serum (FCS) (Grainer), 100U/ml penicillin, 0.1mg/ml

streptomycin, 0.5µg/ml gentamycin (Gibco). Afterwards, cells were seeded in 24-well plates (Nunc

A/S) at a concentration of 106cells ml and cultivated at 37 with 2. After 12h, non-adhering

lymphocytes were removed by washing cells three times with RPMI-1640. The adherent cells

consisted of >90% monocytic cells, as assessed by flow cytometry after indirect immunofluorescence

staining with a mouse monoclonal (mAb) anti-CD172a+ (VMRD, clone DH59B, 1:100, IgG1) directed

against cells from myeloid lineage, followed by goat anti-mouse IgG FITC (Molecular probes, 1:100).

Rabbit kidney epithelial (RK-13) cells 2.1.2.

RK-13 cells were used as a control for the blood and nasal CD172a+ cells in this study and were

maintained in modified Eagle's medium (MEM) supplemented with antibiotics and 5% FCS.

2.2. VIRUS

The Belgian EHV-1 non-neurovirulent strain 97P70 first isolated in 1997 from the lungs of an

aborted foetus was used in this study (Van der Meulen et al., 2000). Virus stocks used for inoculation

were at the 6th passage; 5 passages in equine embryonic lung cells (EEL) and 1 subsequent passage

in RK-13.

18

2.3. VIRUS PURIFICATION AND DIO-LABELLING

Four batches of culture fluids of EHV-1-infected (97P70) RK-13 cells were clarified by

centrifugation at 4000 xg for 20 min at 4°C. Virus was collected by centrifugation at 63 000 xg for 2h in

a Beckham T35 rotor. The pellet was resuspended in 500 l of cold PBS. The concentrated virus was

pooled and purified on a 60% iodixanol gradient at 100 000 xg for 2.5h at 4°C in a Beckham SW41

rotor. The 60% iodixanol gradient was prepared as described by Dantas-Lima et al. (2013). Briefly, the

gradient was prepared by underlayering 2.5 ml of iodixanol (Optiprep, Axis-Shield, UK) fractions at

different concentrations. The fractions were loaded in 15 ml non-pyrogenic polypropylene centrifuge

tubes (Sarstedt, Germany) using 2 ml syringes and 20G (0.9 × 70 mm) needles. The gradient

concentration profiles (from the top to the bottom of the tube) were: 10%, 15%, 20%, 25% and 30%.

The iodixanol solutions were prepared by diluting the stock solution (60% iodixanol) in DPBS. After

centrifugation, the virus band was harvested at the interface of the 15 and 20% layers. The purified

virus was aliquoted and stored at -80°C. Viral infectivity was determined before and after purification

by a standard virus titration on RK-13 cells. The titer of the resulting virus preparation was

approximately 108.8

TCID50/ml compared to 107.5

TCID50/ml prior to virus purification.

To ensure an efficient Dio labelling, we first exchanged buffer into HNE buffer (50 mM NaCl, 5mM

EDTA, 10 mM Tris-HCl, pH 7.4) by the use of a 100-kDa filter device (Millipore). Approximately 108.8

TCID50/ml of purified EHV-1 were added to the filter and concentrated to an end volume of 50 l. While

vortexing, 1 l (=2 nM) of a 2 mM of Dio solution prepared in DMSO (Molecular probes) was added to

the virus and incubated for 20 min at RT. To remove unbound dye, the Dio-labelled virus was filtered

by the use of a Sepharex G-50 fine column (Pharmacia) and centrifuged for 2 min at 300 xg. HNE

buffer was used to equilibrate the column. The virus goes through the column as a blue band

(unicorporated Dio stays on top of the column). As a negative control, unlabelled virus was eluted

through the column. Different fractions were collected, stored at 4°C and used within 2 days. Dio-

labelling did not decrease the virus infectivity significantly.

2.4. VIABILITY AND INOCULATION WITH EHV-1

Cell viability was determined by flow cytometry, using 1 µg/ml propidium iodide, prior to virus

inoculation and was > 90% in all cell populations.

For the endosomal acidification inhibition assay, cell populations were inoculated in vitro with

EHV-1 strain 97P70 at a multiplicity of infection (m.o.i.) of 5 in 200 µl leukocyte medium for 1h at 37°C

with 5% CO2. Cells were gently washed twice with RPMI to remove the inoculum and further

incubated with fresh medium. At 1, 3, 5, 7, 9, 12 and 24 hours post-inoculation (hpi), cells were

collected for quantification of EHV-1 infected cells, viral production and production of infectious EHV-1

by immunofluorescence staining, virus titration and cocultivation assays, respectively.

19

2.5. KINETICS OF EHV-1 ATTACHMENT

To characterize the attachment of EHV-1 to RK-13 and blood CD172a+ cells, direct virus-binding

studies were carried out with Dio-labelled EHV-1-particles. Cells were chilled on ice for 15 min prior to

inoculation and washed with ice-cold RPMI-1640. Fluorescence-labelled EHV-1 particles were added

to RK-13 cells at m.o.i. of 5 only and to CD172a+ cells at a m.o.i. of 5 and 50. Cells were incubated for

0, 5, 10, 15, 30 and 60 min on ice. Cells were washed twice to remove unbound particles with cold-

PBS and cells were fixed with 1% PFA diluted in PBS for 10 min at room temperature. Cell nuclei were

stained with Hoechst 33342 (10 µg/ml; Molecular Probes) for 10 min at 37°C. Coverslips were

mounted on microscopy slides. The quantification of fluorescent EHV-1 particles attached to the cell

membrane was performed by confocal microscopy. The percentage of EHV-1 positive cells was

calculated based on the proportion of cells with viral particles bound on the plasma membrane of 300

random cells. The percentage of virus particles attached per cell was calculated based on the

number of particles attached at the plasma membrane of 5 random EHV-1 positive cells. For each cell,

the entire plasma membrane was screened for the presence of virus particles by making a Z-stack at

the confocal microscope (Leica TCS SP2 Laser scanning spectral confocal system, Leica

microsystems GmbH, Germany). For the Z-stack, confocal images were taken at 5 different levels

within the cell and merged together. In this way, a 3D view of the infected cell was obtained.

2.6. KINETICS OF EHV-1 INTERNALISATION

Cells were chilled on ice (4°C) for 15 min prior to inoculation with fluorescence-labelled EHV-1

particles at a m.o.i. of 5 for 1h at 4°C. Next, cells were washed with PBS to remove unbound virus

particles and transferred at 37°C to enable virus uptake. The cells were incubated for 0 (control), 15,

30, 60, 90, 120, 180 and 210 min. At these time points, cells were fixed in 100% methanol at -20°C for

20 minutes. Internalised virus particles were analysed by immunofluorescence staining. Phalloidin-

TexasRed was used to stain F-actin and thus, to distinguish the inside from the outside of the cell.

Samples were subsequently analysed by confocal microscopy.

2.7. TREATMENT WITH LYSOSOMOTROPIC AGENTS

In order to investigate the entry route of EHV-1 and a possible role of endosomal acidification in

EHV-1 infection of CD172a+ cells, two weak bases (ammonium chloride (NH4Cl) and chloroquine) and

a carboxylic ionophore (monensin) were used in this study. CD172a+ cells were pre-incubated with

various concentrations of NH4Cl (0, 10, 20 or 30 mM), chloroquine (0, 1, 10 or 50 µM) or monensin (0,

1, 10 or 50 µM) for 2h at 37°C. Control cells were incubated in leukocyte medium for 2h at 37°C.

Control and treated cells were inoculated with EHV-1 at a m.o.i. of 5 for 1h at 37°C in 200l leukocyte

medium containing various concentrations of different drugs. After inoculation, cells were washed

twice with medium. Cells were further incubated in presence of the drugs for 12h at 37°C. Cells were

then fixed with 1 ml methanol for 20 min at -20°C.

The concentrations of the inhibitors that were used were based on concentrations described in

literature, providing that they were non-toxic for the cells (Melinda et al., 2005; Nicola et al., 2003;

20

Fredericksen et al., 2002). Cell viability was determined by incubating cells with different

concentrations of inhibitors for 12h and is described in 2.4

2.8. INDIRECT IMMUNOFLUORESCENCE STAINING OF EHV-1 PROTEINS

To determine the infectivity of EHV-1 in cells treated with endosomal inhibitors, an

immunofluorescence staining was performed on cells fixed in 100% methanol at -20°C for 20 minutes.

Cells were incubated for 1 hour at 37°C with a rabbit polyclonal Ab anti-IEP (1:1000) to detect

immediate early protein (IEP) expression, following by incubation with secondary goat anti-rabbit IgG

FITC (1:100) (Molecular probes) for 50 min at 37°C. The IEP antibody was kindly provided by Dr.

’ allaghan (USA). These antibodies were diluted in DPBS. The nuclei were counterstained with

Hoechst 33342 for 10 minutes at 37°C. As negative control, mock-inoculated cells were stained

following the aforementioned protocols. The percentage of viral antigen positive cells was calculated

based on three hundred cells counted in distinct fields. Samples were analysed by confocal

microscopy.

2.9. STATISTICAL ANALYSIS

Data were analysed with GraphPad Prism 5 software (GraphPad software Inc., San Diego, CA,

USA). Analysed data for statistical significance were subjected to a two-way analysis of variance

(ANOVA). All results shown represent means and standard deviation (SD) of three independent

experiments. Results with P-value ≤ 0.0 were considered statistically significant.

21

Figure 6 Kinetics of EHV-1 binding to RK-13 cells. Time-course of EHV-1 particles binding per cell.

3. RESULTS

3.1. KINETICS OF EHV-1 BINDING

EHV-1 binds efficiently to RK-13 cells 3.1.1.

In a first experiment, we determined the efficiency of EHV-1 particles to bind to RK-13 cells, a

control cell line. Cells were incubated with EHV-1 particles at a m.o.i. of 5 on ice for different durations

and bound virus was visualized on the plasma membrane by confocal microscopy.

As shown in Figure 6, EHV-1 particles were first detected as early as 1 min incubation time on the

plasma membrane of RK-13 cells. After 5 min incubation, 30% of the cells showed bound virus

particles with an average of 13 particles bound per cell. The binding of EHV-1 particles to RK-13 cells

increased gradually in the initial 1-30 min and reached a maximum of 30 particles attached per cell

within 30 min. The percentage of cells carrying virus particles also increased overtime and reached a

plateau of 80% after 1h incubation. Longer incubation time did not increase the amount of virus

particles bound per cell or the percentage of infected cells. As shown in Figure 7, EHV-1 particle size

and distribution were homogenous at early incubation time but an increase in virus particle size was

observed starting from 30 min.

22

EHV-1 does not bind efficiently to blood CD172a+ cells 3.1.2.

Based on the ability of EHV-1 to bind to RK-13 control cells, we next evaluated the kinetics of

attachment of EHV-1 on blood CD172a+ cells, one of the main targets cells of EHV-1.

When cells were incubated with purified EHV-1 at a m.o.i. of 5, only 1 virus particle was bound

per cell after 15 min and a maximum of 2 virus particles bound per cell was observed after 60 min

incubation, as shown in Figure 8. A maximum of 12% of CD172a+ cells showed virus bound on the

plasma membrane after 1h incubation. The use of a higher m.o.i. (50) did not significantly increase the

percentage of cells with attached virus (14%). However, a higher number of virus particles (4) bound

per cell was observed at 60 min only. Z-stack pictures, shown in Figure 9, indicate that EHV-1

particles were not homogenously distributed on the cell surface. Instead, they were rather found

localised on the central part of the cell and not on peripheral areas.

Figure 7 Kinetics of EHV-1 binding to RK-13 cells. Confocal images of EHV-1 particles bound to RK-13

cells at 1, 15 and 30 min incubation time. Green dots represent virus particles and the nucleus is

counterstained with Hoechst (blue).

23

Figure 8 Kinetics of EHV-1 binding to blood CD172a+

cells. Comparison of the time-course of EHV-1

particles binding per cell at a m.o.i. of 5 and 50. Experiments were performed with cells from three

different horses and data are represented as means ± standard deviation (SD). * denotes a P-value < 0.05

between CD172a+ cells inoculated at a m.o.i. of 5 and 50.

Figure 9 Kinetics of EHV-1 binding to blood CD172a+

cells. Confocal images of EHV-1 particles bound to

blood CD172a+

cells at 0, 15 and 60 min incubation time. Green dots represent virus particles and the

nucleus is counterstained with Hoechst (blue). The scale bar represents 10 µm.

*

24

3.2. KINETICS OF EHV-1 INTERNALISATION

To determine whether EHV-1 binding was followed by internalisation into CD172a+ cells, cells

were incubated with EHV-1 particles for 1h on ice until maximal attachment was reached. Then, plates

were transferred to 37°C for different durations and fixed with methanol. Virus internalised into cells

was visualized by confocal microscopy.

After 15 min incubation, we observed single virus particles attached to the cell and virus particles

internalised in the form of particle aggregates or as individuals (Figure 10). The number of internalised

virus particles increases overtime as well as the formation of aggregates, as shown after 30 min

incubation at 37°C. Due to the formation of virus particle aggregates, it was impossible to quantify the

number of single virus particles internalised into CD172a+ cells.

3.3. THE EFFECT OF LYSOSOMOTROPIC AGENTS ON EHV-1 INFECTION

Inhibition of acidification of endosomes decreases the infectivity of EHV-1 in RK-13 and 3.3.1.

blood CD172a+ cells

To determine whether a pH drop was a crucial step in the infectious entry of EHV-1 in RK-13 and

blood CD172a+ cells, we examined the effect of lysosomotropic weak bases (NH4Cl and chloroquine)

and carboxylic ionophore (monensin) on EHV-1 infection. The number of IEP-positive cells was

quantified at 12 hpi in RK-13 and in blood CD172a+ cells treated with these agents.

Figure 10 Kinetics of internalisation of EHV-1 in CD172a+ cells. Confocal images of EHV-1 particles

internalised in CD172a+ cells after 15 and 30 min incubation time. Green dots represent virus particles

and the F-actin is stained red by phalloidin-TexasRed. Single virus particles attached at the plasma

membrane are marked by a blue arrow, while aggregates of internalised particles are marked by a

yellow arrow. The bars represent 10 µm.

25

Figure 11 Inhibitors of endosomal/lysosomal acidification decrease the infectivity of EHV-1 in blood

CD172a+ and RK-13 cells. The effects of ammonium chloride, chloroquine and monensin were quantified as

the percentage of cells expressing IEP at 12 hpi. The infectivity in the presence of acidotropic agents is

shown as a percentage of control. The number of IEP-positive cells when no inhibitors was present was set

to 100% in order to calculate the percentage of control. Error bars show ± SD.

Endosomal-lysosomal acidification inhibitors did not affect the viability of RK-13 and blood

CD172a+ cells, even at the highest concentration and after 14h of continuous treatment. The degree of

EHV-1 infection inhibition was dose-dependent, as shown in Figure 11. Treatment of RK-13 cells with

inhibitors reduced EHV-1 infection maximally to 15.6% ± 7.5% at 30 mM NH4Cl and 32.6% ± 7.3% at

50 µM chloroquine and 14.5% ± 7.3% at 50 µM monensin. No significant difference in the reduction of

EHV-1 infection was observed in blood CD172a+ cells treated with both weak bases. However,

treatment of blood CD172a+ cells with 1 µM monensin significantly reduced EHV-1 infection to 49.3%

± 25% compared to 33% ± 11.7% in RK-13 treated cells. EHV-1 replication was completely blocked in

blood CD172a+ cells treated with 50 µM monensin A schematic overview of the effect of the different

lysosomotropic agents on EHV-1 infection of RK-13 and blood CD172a+ cells is given in Figure 11.

26

DISCUSSION

In order to infect cells, EHV-1 has to properly bind to the cell surface. As expected, we found that

virus particles bound on almost all RK-13 cells, a cell line known to be susceptible to EHV-1 infection.

Besides, a homogenous distribution on the plasma membrane was observed, suggesting that cell

surface receptors are homogeneously distributed at the plasma membrane. Interestingly, we also

observed an increase in particle size, suggesting that the viral particles fuse together and/or the

receptors migrate and accumulate on certain areas of the plasma membrane. Epithelial cells in vivo

are polarized, featuring distinct 'apical', 'lateral' and 'basal' plasma membrane domains. The specific

proteins and lipids give each domain a different function, such as secretion, absorption and formation

of junctional complexes. This distinct composition also allows directional transport of molecules across

the epithelial sheet (Shuck and Simons, 2004). As the viral particles were found randomly around the

cell, we concluded that the receptor could be rearranged within the plasma membrane. Indeed,

compound transport from one side of the cell to the other requires flexible receptors that rearrange

around the cell.

Preliminary work showed that EHV-1 replication was restricted to 4% of nasal mucosal and blood

CD172a+ cells (Laval et al., 2014, unpublished data). In order to investigate whether a low efficiency of

binding of EHV-1 on CD172a+ cells may be responsible for the restriction of EHV-1 replication in these

cells, we analysed the kinetics of EHV-1 binding to CD172a+ cells. After 1h incubation with purified

and labelled EHV-1, 12-14% of CD172a+ cells showed virus particles attached at their plasma

membrane, independently of the m.o.i. used. This indicates that EHV-1 particles bind to a saturable

number of receptors on the surface of CD172a+

cells, which cannot be increased by using a higher

m.o.i. (50) or by incubating cells with virus for a longer period (120 min). This confirms that a block at

the level of virus binding to CD172a+

cells is partially responsible for the restriction of EHV-1

replication in these cells and may involve the presence of specific cell surface receptors with restricted

expression patterns.

A certain number of receptors mediating virus entry have already been described for

alphaherpesviruses (Spear and Longnecker, 2003). Herpesvirus entry mediator (HVEM), herpesvirus

entry mediator C (HveC), also known as nectin-1, HveB, also known as nectin-2 and paired

immunoglobulin-like type 2 receptor alpha (PILR alpha) are known to play a major role in herpes

simplex virus (HSV)-1 binding and subsequent entry in HeLa cells, CHO-K1 cells and human

conjuctival epithelium (Nicola et al., 2003; Akhtar et al., 2008; Arii et al., 2009). Moreover, SuHV-1

uses gC to interact with heparan sulfate (HS) linked to proteoglycans (HSPGs) on the cell surface for

attachment (Rue and Ryan, 2013). In addition, porcine HveC, also known as nectin-1, has been

shown to interact with SuHV-1 gD in order to enter the cell (Ono et al., 2004; Yoon and Spear, 2002).

In case of EHV-1 infection, it is known that the virus uses its glycoproteins gD and gB to interact with

heparan sulphate glycosaminoglycan moieties of the plasma membrane of the cell. Previous studies

identified MHC type I as a putative entry receptor in CHO-K1 cells (Kurtz et al., 2010; Sasiki et al.,

27

2011). However, MHC I is expressed on all cells and EHV-1 binds to a small fraction of CD172a+ cells.

So, it suggests that MHC I receptor is not sufficient for EHV-1-CD172a+ binding and that this

interaction requires more than one receptor as described for other viruses, such as human

immunodeficiency virus (HIV), hepatitis C virus (HCV) and HSV-1/2.

Interestingly, we found that virus particles gather together on top of CD172a+ cells, which is in

contrast to the homogenous distribution of EHV-1 particles observed in RK-13 cells. It is possible that

a certain number of receptors are only present on top of the cells and that binding of the virus to the

cell triggers receptor clustering. Indeed, it is known that individual virus-cell interactions may be weak,

but multivalent binding makes the avidity high and leads to receptor clustering, which in turn may

result in the association with lipid domains and eventually activation of signalling pathways (Mercer et

al., 2010). A differentiation can be made between attachment factors and virus receptors. The first

group help to concentrate viruses on the cell surface, such as heparan sulphate and sialic acids. On

the other hand, virus receptors trigger changes in the virus and induce cellular signalling or

penetration. Transferrin and low-density lipoprotein (LDL) receptors for example are known to trigger

the endocytic pathway, as described fot parvo- and rhinovirus infection (Cotmore and Tattersall, 2007;

Fuchs and Blaas, 2010). It could be that CD172a+ cells bear similar receptors for EHV-1 and that

complementary activation of these receptors upon EHV-1 binding is necessary in order to infect the

cell.

After binding of EHV-1 to blood CD172a+ cells, we investigated the route of EHV-1 entry in these

cells. The formation of virus particle aggregates inside the cell was the principal limitation of this work

and rendered the quantification of internalised single virus particles difficult. The use of an antibody

directed against the capsid protein would have been more convenient to analyse virus particles within

the cells, as we could not exclude the fact that virus particles may enter the cell via fusion by only the

use of labelled virus. Unfortunately, the only available antibody directed against the capsid protein was

designed for western blot analysis and did not work for immunofluorescence staining.

28

Endocytosis is a mechanism that allows the cell to engulf different molecules from the outside of

the plasma membrane to the inner of the cell.

Figure 12 Overview of the endocytic pathway. In general, substances are first internalised within primary

endocytic vesicles, which fuse with early endosomes (EE). From the EE, molecules can be expelled again by

recycling endosomes (RE) or can be further transported to the center by maturing endosomes (ME), which fuse

with late endosomes (LE). Delivery to the trans-golgi network results in secretion of the latter molecules and

the system reaches a dead end at the lysosomes, where molecules are degraded.

29

The endosome system, as shown in Figure 12 is tightly connected with the degradation pathway

through lysosomes, to the secretory pathway through the trans-golgi network and to the excretory

pathway through recycling endosomes. The best studied endocytic pathways are clathrin-mediated

endocytosis (CME), macropinocytosis, caveolar/raft-dependent endocytosis and phagocytosis (Ehrlich

et al., 2004; Nicola et al., 2003; Pelkmans et al., 2001,Ghigo et al., 2008). Endocytosis offers the virus

several advantages, such as virus delivery to the perinuclear area of the host cell. In this way, the

virus can bypass most part of the cytoplasm. By being hidden within an endosome, viruses can also

evade the immune system, as shown for HSV-1 and HIV (Lozach et al., 2011). During this transport,

the environment (pH and redox potential) gradually changes and this allows the virus to sense its

location within the cell (see Figure 12). Endocytic environment may also provide the necessary

proteolytic activation of certain viruses (Mothes et al., 2000; Kaletsky et al., 2007). However, some

viruses can enter a cell via endocytosis in a pH-independent mechanism, such as murine leukemia

virus (MLV), Ebola virus and rhesus rotavirus (Kamiyama et al., 2011; Chemello et al., 2002). Instead,

cathepsin proteases are activated without endosome acidification. For example, caveolar endocytosis

is considered to occur in a pH-neutral setting and has been shown to be an entry pathway for EHV-1

in equine blood microvascular endothelial cells (EBMECs) (Hasebe et al., 2009).

It is known that EHV-1 enters ED, EC and RK-13 cells through fusion and CHO-K1 and PBMC

cells through endocytosis (Frampton et al., 2007; Van de Walle et al., 2008). In this work, we wanted

to determine the entry pathway of EHV-1 in its target cells (CD172a+ cells). We hypothesized that

EHV-1 enters CD172a+ cells via endocytosis in a pH-dependent manner, as this process takes more

time than fusion and could explain why IEP is only detected at 5 hpi in the nucleus of CD172a+ cells,

as shown by Laval et al. (2014, unpublished data). Besides, endocytosis can lead to a non-productive

infection, as reported for HIV infection (Fredericksen et al., 2002). This may explain why only 4% of

the cells expressed nuclear IEP while 12% of the cells have virus particles attached to their cell

surface. Indeed, it is possible that virus particles enter the cell via endocytosis but remain somehow

trapped in the endocytic machinery (8% of the cells) and are further degraded by lysosome

acidification.

In this experiment we observed a decrease in EHV-1 infection by endosomal pH neutralisation.

This supports the hypothesis that EHV-1 enters blood CD172a+ cells via a low-pH dependent

mechanism and an acidic environment is required for transport of the virus through the cytosol. Next

step would be to determine whether the virus uses the traditional caveolar/raft pathway, by using

inhibitors that cause cholesterol depletion, such as nystatin. Chlorpromazine, which prevents the

assembly of clathrin-coated pits, can also be used to inhibit clathrin-mediated uptake of viruses.

Frampton et al. (2007) showed that EHV-1 entry into CHO-K1 cells does not require clathrin or

caveolae after treatment with chlorpromazine and nystatin. It would be interesting to see whether

similar results can be obtained in CD172a+ cells. Besides, the alphaherpesviruses HSV-1 and HSV-8

are known to use the macropinocytosis pathway (Nicola et al., 2003; Raghu et al., 2009; Hadigal and

Shukla, 2013).

30

Complementary approaches could also be used to study virus entry, such as an infectious virus

recovery assay, electron microscopy and live-cell imaging, where both virus and cell components can

be tagged with fluorescent molecules.

Surprisingly, a similar decrease in viral infectivity in RK-13 cells was observed compared to

CD172a+

cells, upon treatment of the cells with endosomal inhibitors. This suggests that EHV-1 enters

RK-13 cells via endocytosis in a pH-dependent mechanism. This is not in agreement with Frampton et

al. (2007), who demonstrated that EHV-1 enters RK-13 cells via fusion. This difference could be

attributed to the virus strain used. Indeed, Frampton et al., (2007) used the L11∆gI∆gE strain, which

lacks gI and gE. This is in contrast to the non-neurovirulent strain 97P70, used in our experiment. It

could also be that EHV-1 enters RK-13 cells both via endocytosis and fusion because it has been

shown that EHV-1 can enter different cell types in different ways. In comparison, Hasebe et al. (2009)

found that ED cells are infected through endocytosis in a pH-dependent manner, which is also in

contrast to the results obtained by Frampton et al. (2007).

In the present research project, we found that EHV-1 does not efficiently infect CD172a+ cells,

which is partially caused by a block at the level of binding. It was found that EHV-1 binds to a limited

number of receptors and that subsequently, this binding triggers receptor clustering. After binding,

EHV-1 enters CD172a+ cells via endocytosis in a pH-dependent manner. However, not all EHV-1

particles may be transported to the nucleus and some may be directed towards the degradation

pathway through lysosomes. These new insights in EHV-1 pathogenesis will contribute to the

development of new antiviral strategies.

31

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