Equine viral arteritis: in vivo and ex vivo ...

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Sabrina Vairo, Annelies Vandekerckhove, Lennert Steukers, Sarah Glorieux, Wim Van den Broeck, ...... Ruggiero E., Toschi E. and Federico M. (2011).
Equine viral arteritis: in vivo and ex vivo pathogenesis of a European isolate

Sabrina Vairo

Thesis for obtaining the degree of Doctor in Veterinary Sciences (Ph.D.)

2013

Promoters: Prof. H. Nauwynck Prof. A. Scagliarini

Laboratory of Virology Department of Virology, Parasitology and Immunology Faculty of Veterinary Medicine, Ghent University Salisburylaan 133, B-9820 Merelbeke

To the experimental animals, and in particular to my ten Shetland ponies, which sacrificed their life for science. I hope this PhD worthes your lives.

A mi abuela: la roca sobre la que fundé mi moral y mis principios. Te amo con todo mi corazón

TABLE OF CONTENTS I. LIST OF ABBREVIATIONS

1

II. INTRODUCTION

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1. EQUINE ARTERITIS VIRUS

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1.1. History

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1.2. Taxonomy

3

1.3. Morphology

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1.3.1. General structure of the virion

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1.3.2. Genomic organization

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1.3.3. Nucleocapsid protein

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1.3.4. Major envelope proteins

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1.3.5. Minor envelope proteins

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2. EQUINE ARTERITIS VIRUS - CELL INTERACTION: The replication cycle

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2.1. Virus attachment and entry

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2.2. Genome replication and gene expression

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2.2.1. Genome translation and processing of polyproteins in non-structural proteins

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2.2.2. Genome replication

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2.2.3. Subgenomic-length RNA transcription and expression of structural proteins

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2.3. Virus assembly, budding and release

3. EQUINE ARTERITIS VIRUS - ANIMAL INTERACTION

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3.1. Prevalence

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3.2. Pathogenesis of infection with North American EAV strains

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3.2.1. Routes of infection

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3.2.2. Pathogenesis of generalized infection following respiratory uptake of the virus

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3.2.3. Pathogenesis of abortion following respiratory infection

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3.2.4. Pathogenesis following venereal route

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3.2.5. Mechanism of persistence in stallions

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3.3. Clinical signs

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3.4. Pathology

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3.4.1. Gross lesions

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3.4.2. Histopathology

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3.5. Clinical signs and pathology of infection with European EAV strains

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3.6. Immunity

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3.7. Diagnosis

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3.7.1. Clinical diagnosis

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3.7.2. Detection of EAV virus, viral components or antibodies

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4. EQUINE ARTERITIS VIRUS - UPPER RESPIRATORY MUCOSA INTERACTION: EX VIVO MODEL

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4.1. Mucosa explants in research

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4.2. General characteristics of the respiratory mucosa

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4.3. Histology of the upper respiratory tract mucosa

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4.3.1. Respiratory epithelial components and their functions

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4.3.2. Cell-cell and cell-matrix adhesions

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4.3.3. Extracellular matrix

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4.3.3.1. The basement membrane

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4.3.3.2. The lamina propria

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4.4. Mucosal immune cells

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REFERENCES

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III. AIMS OF THE THESIS

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IV. Clinical and virological outcome of an infection with the Belgian equine arteritis virus strain 08P178

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V. Identification of target cells of a European equine arteritis virus strain in experimentally infected ponies

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VI. Development and use of a polarized equine upper respiratory tract mucosal explant system to study the early phase of pathogenesis of a European strain of equine arteritis virus

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VII. GENERAL DISCUSSION

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VIII. SUMMARY-SAMENVATTING

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IX. CURRICULUM VITAE

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X. ACKNOWLEDGEMENTS

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I. LIST OF ABBREVIATIONS aa:

Amino acid

BM:

Basement membrane

CD:

Cluster of differentiation

CF:

Complement fixation test

CoV:

Coronavirus

CPE:

Cytopathic effect

DC:

Dendritic cells

MALT:

Mucosa-associated lymphoid tissue

dpi:

days post inoculation

DRT:

Deep respiratory tract

EAV:

Equine arteritis virus

EDTA:

Ethylenediamine tetraacetic acid

EHV:

Equine herpesvirus

ELISA:

Enzyme-linked immunosorbent assay

ER:

Endoplasmic reticulum

EVA:

Equine viral arteritis

FAE:

Follicle-associated epithelium

GP:

Glycoprotein

HIV:

Human immunodeficiency virus

hpi:

hours post inoculation

Ig:

Immunoglobulin

IL:

Interleukin

Kb:

Kilobase

kDa:

Kilodalton

LDV:

Lactate dehydrogenase-elevating virus

mAbs:

Monoclonal antibodies

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mRNA:

Messenger ribonucleic acid

N protein:

Nucleocapsid protein

nm:

Nanometer

nsp:

Non-structural protein

ORF:

Open reading frame

PBMC:

Peripheral blood mononuclear cell

PCR:

Polymerase Chain Reaction

pp:

Polyprotein

PRRSV:

Porcine reproductive and respiratory syndrome virus

RdRp:

RNA-dependent RNA-polymerase

RK13:

Rabbit kidney 13

RNA:

Ribonucleic acid

SARS:

Severe acute respiratory syndrome

sg RNA:

Subgenomic ribonucleic acid

SHFV:

Simian hemorrhagic fever virus

SN:

Serum neutralizing

TCID50:

Tissue culture infectious dose with 50% endpoint

URT:

Upper respiratory tract

2

INTRODUCTION

Introduction

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1. EQUINE ARTERITIS VIRUS 1.1. History Equine arteritis virus (EAV) is the etiological agent of the disease currently known as equine viral arteritis (EVA). EAV was first isolated in 1953 from an outbreak of respiratory disease and abortion on a Standardbred breeding farm near Bucyrus (Ohio, USA). However, descriptions of disease outbreaks that most likely were EVA, have first been published in the late 18 th and early 19th centuries and were called “pinkeye”, “infectious or epizootic cellulites”, “influenza erysipelatosa”, “pferdestaupe”, and “equine influenza” (Bergman et al., 1913 and Pottie et al., 1888). In 1957, the agent isolated from the Ohio outbreak was named as EAV because of the distinctive vascular lesions leading to arteritis (Doll et al., 1968). A first and important outbreak in Europe was reported in Bern (Switzerland) in 1964 involving over 400 remount horses (Bürki and Gerber, 1966). Recently, the number of EAV outbreaks, has drastically increased worldwide. EVA is an economically important viral disease of equids and its prevalence is increasing, possibly due to intensified transportation of horses and semen. Although deaths are very rare in infected adults, acute illness may occur, pregnant mares may abort and very young foals may die of a fulminating pneumonia and enteritis. Further, stallions may become carriers and transmit EAV during breeding (Timoney and McCollum, 1993a). Although the global dissemination and incidence of EAV have increased during the last decades, several aspects of its pathogenesis remain uninvestigated. A better understanding of EAV pathogenesis can lead to an effective control of the disease and a reduction of economic losses.

1.2. Taxonomy EAV is the prototype virus of the genus Arterivirus, family Arteriviridae, order of Nidovirales. Members of the order Nidovirales contain a positive-sense ssRNA genome within an external lipid bilayer (envelope) with associated proteins which encloses the internal nucleocapsid structure (Perlman et al., 2012). Virions of Nidovirales vary in morphology from spherical to bacilliform depending on the family to which they belong (Lai and Holmes, 2001). Based on phylogenetic analysis of the RNA-dependent RNA-polymerase (RdRp), the order of the Nidovirales was divided in three families: Arteriviridae (1 genus), Roniviridae (1 genus) and Coronaviridae (2 subfamilies: Coronavirinae and Torovirinae). Nidovirales cause important diseases in a broad range of hosts including humans, other mammals, birds, shrimps, and fishes (de Groot et al., 2012; Granzow et al., 2001; Siddell and Snijder, 2008 and Walker et al., 2005). Nidovirales are characterized by an extraordinary genetic complexity which allows them to expand the host range and to adapt rapidly to changing environmental conditions (Ziebuhr et al., 2000). The ORFs located in the 3’-part of the nidoviruses genome are expressed from a nested set of subgenomic (sg) mRNAs, a property that was reflected in the name of the virus order (nidus in Latin means nest) (Cavanagh, 1997). Since the multinuclear zinc-binding domain (contained in nsp10) and uridylate-specific endoribonuclease domain (contained in nsp11) have not been identified in other RNA virus families, they are used to discriminate between nidoviruses and other RNA viruses (Posthuma et al., 2006).

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Introduction

Besides EAV, the family of Arteriviridae contains 3 other members: lactate dehydrogenase-elevating virus (LDV; mice), porcine reproductive and respiratory syndrome virus (PRRSV; pigs) and simian hemorrhagic fever virus (SHFV; monkeys) (Snijder and Meulenberg, 1998). Arteriviruses have a highly restricted host range and may cause prolonged or lifelong infections in their natural host (Timoney and McCollum, 1993a). The ability to persist in their host suggests that they can escape to some extend from recognition and elimination by the host immunity. However, the immune-evasion mechanisms are largely unknown. Although only one neutralization serotype of EAV has been identified so far (Golnik et al., 1986), there is considerable genetic variation among EAV field strains as demonstrated by comparative sequence analysis of ORFs 2 to 7 (Hornyak et al., 2005). Since ORF 5 contains several variable regions, it became the main target for tracing the origin of EAV strains (Balasuriya et al., 1999 and 2004a). Phylogenetic analyses based on M (ORF 6) and N protein (ORF 7) genes confirmed EAV strain variation (Chirnside et al., 1994). Sequence analysis of the ORF 6 was used to separate distinct groups of EAV isolates from Europe and the USA. Comprehensive phylogenetic analyses have identified two phylogenetic groups: Group I consists of viruses originally isolated in North America and Group II consists of viruses originally isolated in Europe (Stadejek et al., 1999). In addition, several subgroups have been identified within each group. Particularly, the European group can be further divided into two subgroups: EU-1 and EU-2 (Zhang et al., 2010). More recently, North American lineage viruses have been isolated in Europe and vice versa, indicating interchange of viruses between the two continents. In South America, isolates of both EAV groups have been identified (González et al., 2003). EAV strains belonging to the North American group are present in Australia. This geographic exchange is most probably the outcome of the movement of carrier stallions and/or shipment of virus-contaminated semen. The nucleotide identity between North American and European isolates of EAV is about 85% (Balasuriya et al., 1995a and 2004a). A genetically very diverse strain of EAV has been isolated from the semen of a donkey in South Africa having only 60-70% of nucleotide identity with EAV strains isolated from North American and European horses and donkeys (Stadejek et al., 2006). Experimental infection of horses and donkeys with the South African asinine strain has demonstrated that it is poorly transmissible to horses. Although there is a widespread distribution of this South African strain among South African donkeys (Paweska et al., 1997), the number of seropositive horses is very low. Genetically, EAV remains rather stable during horizontal and vertical transmission in the course of a disease outbreak but genetic variants can emerge during persistent infections of stallions (Balasuriya et al., 1999 and 2004a). EAV isolates may markedly vary in their severity to induce clinical signs and in their abortigenic potential (Balasuriya and MacLachlan 2004). The genetic determinants of virulence have not yet been defined.

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Introduction

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1.3. Morphology 1.3.1. General structure of the virion EAV is a small, spherical virus with a diameter of 40-60 nm. EAV virions contain an icosahedral nucleocapsid core of 25 to 35 nm in diameter (Horzinek et al., 1971). This nucleocapsid consists of capsid proteins (N) that enclose the viral genome. Virions are enclosed in an envelope with tiny surface projections (Horzinek et al., 1971). Seven structural proteins have been identified in EAV virions: the 14-kDa phosphorylated nucleocapsid (N) protein and six envelope proteins: glycoprotein (GP) 2b (previously named Gs), envelope protein E, GP3, GP4, GP5 (previously named GL due to its larger dimensions compared to the other structural proteins) and M protein (Figure 1).

Figure 1: Electron photomicrograph (a) and schematic representation (b) of EAV morphology. The nucleocapsid core consists of a capsid that encloses the viral genome. The nucleocapsid, composed of nucleocapsid protein (N), is surrounded by an envelope that contains 6 (glyco)proteins: envelope protein E, GP2b, GP3, GP4, GP5 and matrix protein M. GP5 and M proteins are present in the envelope as heterodimers and are the backbone of the envelope. GP2b, 3 and 4 are connected and form heterotrimers. Protein E is suggested to interact with the GP2b/GP3/GP4 heterotrimer on the one hand and with the GP5/M heterodimer and/or the nucleocapsid on the other hand.

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Introduction

1.3.2. Genomic organization The EAV genome consists of an approximately 12.7 kilobases (Kb) single-stranded positive-sense RNA molecule and is divided in 9 open reading frames (ORFs): 2 large replicase ORFs and 7 smaller ORFs coding for structural proteins. Besides the coding regions, the genome contains a 5’ non-coding region which carries a cap at its 5’ end and a 3’ non-coding region to which a poly-A tail is attached (Figures 1 and 2). The two largest 5’ ORFs, ORF 1a and ORF 1b, occupy three-quarters of the genome and overlap each other in a small area containing a ribosomal frameshift signal (Figure 2b). ORF 1a and 1b are processed in 13 nonstructural proteins (nsps), including the RNA-dependent RNA-polymerase (RdRp; nsp9). The RNA sequence downstream of ORF 1a and 1b contains 7 overlapping ORFs: 2a, 2b, 3, 4, 5, 6, and 7 encoding respectively the structural proteins E, GP2b, GP3, GP4, GP5, M, and N (Figure 2) (de Vries et al., 1992). A detailed computational analysis revealed an additional ORF which overlaps the 5’ end of ORF 5, named ORF 5a, that is conserved in all arterivirus species. The ORF 5a is predicted to be a type III membrane protein (59aa) and is thought to be expressed from the same subgenomic mRNA (sg mRNA5). The function of this protein is yet to be characterized but studies, using reverse genetics, suggested that it is the eighth structural protein of arteriviruses and may be important for arterivirus infection (Firth et al., 2011).

Figure 2: Schematic diagram of the genome organization and expression of EAV (a) and of the EAV ORF1a/1b frameshift-directing signals: the ‘shifty’ codons (5’ GUUAAAC 3’) and RNA pseudoknot structure (b). Adapted from Snijder and Meulenberg, 1998.

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Introduction

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1.3.3. Nucleocapsid protein The nucleocapsid (N) protein is a small protein, encoded by ORF 7, with a high content of basic amino acids and a hydrophilic nature (de Vries et al., 1992). The N protein is phosphorylated and present in the virion as a monomer (Snijder and Meulenberg, 1998). The N protein is expressed abundantly in infected cells and constitutes about 35 to 40% of the protein moiety in the virion (de Vries et al., 1992). The nucleus normally contains only trace amounts of EAV N protein, which targets to the nucleus immediately after translation. The EAV N protein is localized in the nucleus as foci, most likely in nucleoli in view of the data obtained with PRRSV (Rowland et al., 1999). Although only small quantities of the N protein are normally present in the nucleus, all N protein molecules are initially transported to the nucleus. Genome replication and mRNA synthesis can continue with the N protein trapped in the nucleus and, thus EAV N protein is dispensable for viral RNA synthesis in the cytoplasm (Molenkamp et al., 2000a). However, since it plays an important role in encapsidating the viral genomic RNA and in interacting with envelope proteins during virus assembly, N protein is necessary for virus assembly and for production of infectious virus particles (Wieringa et al., 2004). Since both nucleocapsid formation and budding of arteriviruses are assumed to be strictly cytoplasmic events (Snijder and Meulenberg, 1998) the N protein has to be shuttled back to the cytoplasm to fulfill its role in the virion biogenesis. The function of the nucleo-cytoplasmic shuttling of the arterivirus N protein is still unclear. Firstly, nuclear shuttling frequently involves protein phosphorylation and this pathway could thus be used to achieve an essential post-translational modification. Secondly, since nucleoli are implicated in a variety of host cell processes (Olson et al., 2000), the nuclear/nucleolar localization of the N protein may be part of a strategy to modulate host cell functions. Studies on Coronaviridae have also revealed nuclear and nucleolar import of the N protein, a process that was postulated to disrupt host cell division (Wurm et al., 2001). These observations suggest that the nuclear import of the N protein is important for a mechanism common to other nidoviruses.

1.3.4. Major envelope proteins The non-glycosylated membrane protein (M) and the large envelope glycoprotein GP5 are the two major envelope proteins and are encoded by ORF 6 and 5, respectively (de Vries et al., 1992). The M protein (16-kDa) is assumed to span the viral envelope three times with its internal trans-membrane segments, leaving a short stretch of 10-18aa exposed at the outside of the virion (ectodomain) and an approximately 72-residue buried at the inside (endodomain). In EAV infected cells, disulfide-linked M protein homodimers are also observed but they are not incorporated into virions (Snijder and Meulenberg, 1998). The GP5 protein (30 to 42-kDa; 255aa) is a heterogeneously glycosylated protein with an ectodomain (19116aa), three membrane-spanning domains and an endodomain of about 64 amino acids (Balasuriya and MacLachlan, 2004). The GP5 protein expresses the neutralization determinants of the virus, all located on the ectodomain of the GP5 protein (Balasuriya et al., 1997 and 2004b).

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Introduction

M and GP5 proteins are present in virions in equimolar amounts. When expressed individually, GP5 and M proteins are retained only in the ER. In contrast, when co-expressed, the M protein localizes both in ER and the Golgi complex and the GP5 protein consistently co-localizes with the M protein in the Golgi complex showing that their transport is dependent upon the formation of a GP5/M heterodimer. The ectodomain of GP5 forms a disulfide bond with the M ectodomain, which mediates GP5/M heterodimerization (Snijder et al., 2003). The GP5/M heterodimers constitute the basic protein matrix of the envelope (de Vries et al., 1992 and 1995). Further, GP5 and M proteins are indispensable for both virus assembly and the production of infectious virus particles (Wieringa et al., 2004).

1.3.5. Minor envelope proteins Besides the three major structural proteins, the EAV virion contains four minor envelope proteins: the GP4 (28-kDa), the GP3 (36 to 42-kDa), the GP2b (25-kDa), and the protein E (8-kDa). GP4 and GP2b proteins are encoded by ORF 4 and 2b, respectively and are type I integral membrane proteins with a 22-24aa residue cleaved off during transport through the ER (Wieringa et al., 2002). The GP3 protein is encoded by ORF 3 and is a heavily glycosylated integral membrane protein with hydrophobic sequences. Hydrophobic terminal domains anchor the GP3 protein to the membrane and no part of its structure is detectably exposed cytoplasmically (Wieringa et al., 2002). The protein E is a small (67aa) unglycosylated integral membrane protein encoded by ORF 2a with a central hydrophobic domain (40aa). The E protein does not form covalently linked multimers and associates with intracellular membranes (both the ER and Golgi complex) (Snijder et al., 1999). GP2b, GP3, and GP4 are abundantly expressed in EAV-infected cells, but only a small fraction of them is assembled into the virion (Wieringa et al., 2003a). GP2b, GP3 and GP4 form heterotrimers (Snijder et al., 2003). It has been postulated that the GP2b and GP4 protein first form a heterodimer which then interacts with GP3 protein. The GP2b/GP4/GP3 complex is finally assembled into the virion (Wieringa et al., 2003a and 2003b). When one of the GP2b, GP3, or GP4 proteins is missing, incorporation of the remaining proteins is blocked. Further, since absence of the E protein entirely prevents incorporation of the GP2b, GP3 and GP4 proteins into the virion, the existence of a GP2b/GP4/GP3/(E) complex was suggested (Wieringa et al., 2004). The E protein is thought to be the component, which interacts with the GP2b/GP4/GP3 heterotrimer on the one hand and interacts with the GP5/M heterodimer and/or the nucleocapsid, on the other hand. It has been shown that the E, GP2b, GP3, and GP4 proteins are dispensable for the formation of virus-like particles while they are essential for the production of infectious virus particles suggesting that the GP2b/GP4/GP3/(E) complex may be involved in the virus attachment and cell entry process (Molenkamp et al., 2000a).

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Introduction

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2. EQUINE ARTERITIS VIRUS - CELL INTERACTION: The replication cycle EAV can be propagated in a variety of primary cell cultures such as equine macrophages (Moore et al., 2003a), equine endothelial cells (Moore et al., 2003b), equine kidney cells (McCollum et al., 1961), and hamster kidney cells (Wilson et al., 1962). The virus can also replicate efficiently in several continuous cell lines such as rabbit kidney (RK13) (McCollum et al., 1962), baby hamster kidney (BHK-21) (Hyllseth, 1969), and African green monkey kidney (Vero) (Konishi et al., 1975). EAV infection of primary cells and continuous cell lines is highly cytocidal. The cytopathic effect (CPE) is characterized by rounding of cells and cell detachment from the culture plate surface (McCollum et al., 1962). In primary equine cells, one replication cycle takes 4 to 6 h and maximum virus yield is obtained at 36 hours post inoculation (Moore et al., 2002). The replication of positive-stranded RNA (+RNA) viruses of eukaryotes is schematically presented in Figure 3 and depends on a unique process of cytoplasmic RNA-dependent RNA synthesis. A common feature is the involvement of host cell membranes, which are often modified to accommodate the +RNA virus replication complex (Pedersen et al., 1999). Like other viruses, EAV infection of cells involves virus attachment (Figure 3.1), entry (Figure 3.2), viral genome replication (Figure 3.3 and 3.4), mRNA transcription (Figure 3.5), viral protein synthesis (Figure 3.6 a-b-c), virus assembly (Figure 3.7), budding (Figure 3.8 and 3.9), and release (Figure 3.10).

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Introduction

Figure 3: Schematic representation of EAV replication cycle. (3.1): attachment; (3.2): entry and translation; (3.3 and 3.4): viral genome replication; (3.5): mRNA transcription; (3.6 a-b): viral protein synthesis; (3.6 c): formation of the nucleocapsid; (3.7): virus assembly; (3.8 and 3.9): budding; and (3.10): release.

2.1. Virus attachment and entry The first steps of the EAV replication cycle are viral attachment (Figure 3.1) to a specific receptor on a susceptible cell and internalization (Figure 3.2). Virus attachment molecule(s), specific cell receptor(s) or mechanism of fusion between EAV envelope and endosomal membrane have not yet been identified. It is still unknown how many receptors or co-receptors are needed for EAV attachment, whether EAV utilizes the same receptor(s) in different cells or whether different EAV strains use the same receptor(s). Therefore, the attachment and entry processes of EAV still need further study. In analogy with many other animal RNA viruses and in view of its recognition by neutralizing antibodies, the EAV GP5 protein has been postulated to serve as the virus attachment protein and to mediate receptor recognition. However, exchange of the ectodomain of the EAV GP5 protein with that of PRRSV or LDV did not alter the cell tropism of the mutant virus (Dobbe et al., 2001). Similarly, exchange of the ectodomain of the PRRSV M protein with that of EAV or LDV still retained their ability to infect porcine alveolar macrophages and did not acquire tropism for cells susceptible to the respective viruses from which the foreign ectodomains were derived (Verheije et al.,

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Introduction

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2002). This suggests that, in case of arteriviruses, GP5 and M proteins are not responsible for receptor binding. Since E, GP2b, GP3, and GP4 proteins are not required for the formation of EAV particles but are essential for ensuring that the virus particles are infectious (Wieringa et al., 2004), it was proposed that the GP2b/GP4/GP3/(E) complex might be involved in the EAV attachment/entry process. However, it is possible that EAV, similarly to PRRSV, utilizes more than one attachment molecule for binding to cells and that, as PRRSV (Delputte and Nauwynck, 2004), EAV uses different attachment molecules to attach to different cells. Asagoe et al. (1997) showed that heparin can reduce EAV infection of RK13 cells and that this inhibition was due to the direct interaction between heparin and EAV rather than to the interaction between heparin and RK13 cells. Furthermore, treatment of RK13 cells with heparinase before virus inoculation decreased EAV infection of the cells. These data suggested that, similarly to PRRSV (Delputte et al., 2002), a heparin-like molecule on the surface of RK13 cells might serve as a cell receptor for EAV. However, heparinase treatment of RK13 cells could not reduce EAV infection below the 13% even in the presence of a very high concentration of heparin (Asagoe et al., 1997). This implies that, as demonstrated for PRRSV (Delputte et al., 2002), other molecules on the cell surface might serve as EAV receptors. Little is known on the mechanism(s) used by EAV to enter cells and to uncoat the envelope. In analogy with PRRSV which uses a mechanism of clathrin-dependent-receptor-mediated endocytosis to enter cells (Nauwynck et al., 1999), EAV is assumed to use a process of receptor-mediated endocytosis. Further, since for PRRSV it was demonstrated that low pH is necessary for the fusion between the endosomal membrane and the viral envelope and subsequent virus uncoating (Nauwynck et al., 1999), it is believed that, to uncoat the envelope, EAV needs similar conditions.

2.2. Genome replication and gene expression Once the viral RNA is released into the cytoplasm, arteriviruses start their replication cycle in the cell. The EAV replication cycle commences with translation of the replicase polyproteins (RdRp) from the genome, followed by genome replication and transcription and translation of structural proteins from subgenomic (sg) mRNAs (Figure 3.3) (Snijder and Meulenberg, 1998).

2.2.1. Genome translation and processing of polyproteins in non-structural proteins EAV genome translation is initiated via a cap-dependent mechanism. The EAV replicase (RdRp) is expressed directly from the viral genome in the form of polyprotein (pp) 1a and 1ab. The pp1a is translated directly from ORF 1a while ORF 1b translation requires a ribosomal frame-shift just before ORF 1a translation is terminated (den Boon et al., 1991). Two RNA structures, a slippery sequence (7 nucleotides) located upstream of the ORF 1a stop codon and a pseudo-knot structure downstream of the slippery sequence, are considered to be essential for efficient ribosomal frame-shift (Snijder and Meulenberg, 1998) (Figure 2). Once pp1a and pp1ab are synthesized, they are cleaved 7 and 11 times, respectively by three different viral proteases localized in non-structural proteins (nsps) 1, 2 and 4. In total, 13 end-products (1, 2,

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Introduction

3, 4, 5, 6, 7α, 7β, 8, 9, 10, 11, 12 nsps) and multiple processing intermediates are generated (van Aken et al., 2006). In particular, the nsps 1 to 8 are encoded by ORF 1a and nsps 9 to 12 by ORF 1b (van Dinten et al., 1999). Besides its proteolytic role, nsp 2, together with nsp 3 is necessary and sufficient to induce the formation of virus-induced host cell-derived double-membrane vesicles (DMVs) from paired endoplasmic reticulum membranes (Snijder et al., 2001). Once DMVs are created, the nsps 2, 4, 7, 7-8, 8, 9, and 10 assemble on them creating the viral replication complex (Figure 3.3c). Afterwards, the hydrophobic domains located in nsp2, nsp3 and nsp5 mediate the association between the viral replication complex and the intracellular membrane (Figure 3.3d) (van der Meer et al., 1998). As result of this association, the membrane-bound scaffold, which will direct the replication and the transcription of both viral genome and sg RNAs, is formed (Figure 3.4 and 3.5) (Snijder et al., 2001). The formation of paired membranes and, consequently, DMVs in the perinuclear region is a typical feature of EAV and other arterivirus infections (Pol et al., 1997).

2.2.2. Genome replication EAV RdRp is the only viral protein required for genome replication (Molenkamp et al., 2000b). However it is still uncertain whether other host cell proteins are involved in genomic replication. The RdRp copies (+) genomic strands into full-length (-) genomic strands (Figure 3.4) and then utilizes (-) genomic strands as templates to synthesize (+) genomic strands (Figure 3.5). EAV RNA synthesis is directed by the viral replication complex assembled on the membrane of DMVs and associated with the intracellular membrane as described in the previous section.

2.2.3. Subgenomic-lenght RNA transcription and expression of structural proteins Structural proteins are derived through the expression of subgenomic-length RNAs (sg mRNAs). As for genomic replication, synthesis of sg mRNAs is also directed by the RdRp complex (Molenkamp et al., 2000b). The sg mRNAs transcription occurs at the same intracellular location and follows the same principles of RNA synthesis (den Boon et al., 1996 and Godeny et al., 1998). The only difference is that genomic replication is a continuous process while sg mRNA transcription involves a discontinuous mechanism where the RdRp has to stop transcription at one site and reinitiate transcription at another site (den Boon et al., 1995). As a result, 17 small transcription-regulating sequences (5’UCAAC3’) are created of which only 6 will attach to sg mRNAs directing the expression of the structural proteins. Structural proteins E, GP2b, GP3, GP4, GP5, M, and N, are derived through the expression of 2a, 2b, 3, 4, 5, 6 and 7 sg mRNAs, respectively (Figure 3.6 a-b-c) (den Boon et al., 1991).

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Introduction

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2.3. Virus assembly, budding and release EAV acquires its envelope from internal membranes of the infected cell rather than from the plasma membrane and its assembly takes place at the cytoplasmic faces of the ER and/or the Golgi complex (Magnusson et al., 1970). All seven EAV structural proteins (E, GP2b, GP3, GP4, GP5, M, and N proteins) are indispensable for the production of infectious progeny virus (Snijder et al., 1999 and 2003) but only the structural proteins GP5, M, and N are essential for the formation of virus-like particles. Besides the GP5, M and N proteins, additional unknown factors are involved in EAV particle formation as demonstrated by the unsuccessful attempts to produce virus-like particles by co-transfection of cells with expression plasmids encoding the EAV GP5, M and N proteins (Wieringa et al., 2004). It is generally thought that disulfidelinked GP5/M heterodimers constitute the basic protein matrix of the envelope and, since transport of EAV GP5 and M proteins from ER to the Golgi complex is dependent upon the formation of a GP5/M heterodimer, the latter is a prerequisite for virus assembly (Snijder et al., 2003). The cytoplasmically exposed domains of the GP5/M heterodimers may interact with the synthesized nucleocapsid. Thus, the EAV nucleocapsid buds into the lumen of ER or Golgi network and acquire a lipid membrane carrying viral envelope proteins. Since the E, GP2b, GP3, and GP4 proteins are also integral membrane proteins and are anchored to the lipid membrane of ER or Golgi complex, the EAV nucleocapsid also acquires these envelope proteins forming virus particles. Afterwards, virus particles are transported from the intracellular compartments to the plasma membrane where they are released via exocytosis. After the release of virus particles, the non-covalent linkage between the GP3 protein and the GP2b/GP4 heterodimer becomes covalently linked forming the GP2b/GP4/GP3 heterotrimers (Figure 3.7 to 3.10) (Wieringa et al., 2003a).

13

Introduction

3. EQUINE ARTERITIS VIRUS - ANIMAL INTERACTION 3.1. Prevalence EVA is an infectious disease of Equidae. Antibodies to EAV have been reported in horses, ponies, donkeys and mules. Illness occurs mainly among horses and ponies but clinical signs have also been reported in experimentally infected donkeys (McCollum et al., 1995 and Timoney and McCollum, 1993a). EAVspecific neutralizing antibodies were detected in 51 zebras (24%) of the Burchell’s zebra population in the Serengeti National Park (Borchers et al., 2005) but not in free-ranging or captive zebra species in southern Africa (Paweska et al., 1997). There is only one reported EAV infection in alpaca through the detection of EAV nucleic acid from aborted fetal tissues by reverse-transcription polymerase chain reaction (RT-PCR) and the demonstration of neutralization antibodies in a high percentage of its cohorts (Weber et al., 2006). Retrospective serological investigations revealed that the virus was present in nearly all countries before the disease was recognized. Antibodies to EAV have been found in most countries where sero-surveillance has been carried out. Seropositive horses have been reported in North and South America, Europe, Asia, Africa and Australia. This virus has not been reported in Iceland and Japan. In the Netherlands, examination of sera collected from horses between 1963 and 1966 and from 1972 onwards showed an incidence of EAV infection of 14%. Equine sera collected in Europe between 1966 and 1976 demonstrated that, in Austria, England and France 59%, 14% and 14% of the horses were seropositive, respectively. In Africa, the rate of infection was high in Morocco (52.5%), average in Egypt (14%) and low in Ethiopia and Senegal (7.7% and 2.5%, respectively) (Moraillon and Moraillon, 1978). Further, examination of stored sera demonstrated that EAV has been present in Australia since at least 1975 (Huntington et al.,1990). EAV (European strain) was introduced in South Africa in 1981 by importing a Lipizzaner stallion from Yugoslavia (Guthrie et al., 2003). A schematic representation of EVA distribution is given in Figure 4. The seroprevalence of EAV infection varies not only among countries but also among equine breeds. In the USA, the infection is particularly common among Standardbreds. In a survey done in the USA in 2001, 85% of Standardbreds, 5% of Thoroughbreds, 0.6% of Quarter horses, and 3.6% of Warmblood horses had antibodies to this virus (Hullinger et al., 2001). In Europe, 55 to 93% of Austrian Warmblood stallions are positive for antibodies to EAV (Newton et al., 1999). Breed-related differences in seroprevalence might be due to genetic differences, but they are more likely to be caused by different management practices. In experimentally infected horses, the breed has no apparent effect on susceptibility to infection or on the establishment of carrier’s state. In 1964 a very large EVA outbreak occurred in Switzerland where over 400 horses showed clinical signs (Bürki and Gerber, 1966). In 1984, a widespread EVA outbreak occurred in 41 Thoroughbred breeding farms in Kentucky (Timoney, 1984). As a result, concerns about EVA and its economic impact in equine industry increased. In 1986 and 1995, EAV was isolated from an outbreak of epidemic abortion of mares in Germany (Eichhorn et al., 1995 and Golnik et al., 1986). During 1992, a widespread epidemic occurred in a riding center in Spain with a total of 31 out of 186 horses showing severe clinical signs (Monreal, et al.,

14

Introduction

_

1995). In 1993, six premises and around 100 horses were infected in UK (Wood et al., 1995). Between 1997 and 1999, three different fatal outbreaks resulted in three dead foals and an aborted fetus in Denmark (Larsen et al., 2001). Between 1998 and 2000, eight cases of abortion in Hungary in six different herds were attributed to EAV infection (Szeredi et al., 2005). In 2006, outbreaks occurred in a breeding farm of Quarter Horses in New Mexico in the USA and subsequently extended to premises in five other states (Kansas, Montana, Oklahoma, Utah, and Alabama) where a quarantine period of 7 months was subsequently imposed. In France, an outbreak was registered in the summer of 2007. Both incidences were associated with artificial insemination of cool-shipped semen (Holyoak et al., 2008). The global dissemination and rising incidence of EAV probably reflects the intensified national and international movement of horses for competition and breeding. In Belgium, a first serological survey carried out between 1997 and 1998 showed that 16 out of 165 sera (10%) tested positive for antibodies against EAV (Lauwers, 1999; University of Ghent, unpublished data). Equine sera collected between 2009 and 2010 demonstrated that the seroprevalence of EAV had increased to 29% (Lauwers, 2011; University of Ghent, unpublished data). The first disease outbreak of EVA abortion occurred in Belgium in an Arabian stud farm in 2001 (Van der Meulen et al., 2001). A second outbreak in Belgium occurred in 2008 (Gryspeerdt et al., 2009) and started with one foal that suffered from acute dyspnea and died four days after birth. Three weeks later, another foal, born on the same farm in a healthy condition but with placental edema, showed acute respiratory distress, severe dyspnea at the age of 10 days and died shortly thereafter. Post-mortem examination of both foals revealed consolidated diaphragmatic lung lobes with compensatory emphysema. Histologic lesions consisted of a mild acute interstitial pneumonia. Microscopic examination of the allantochorion of the second foal showed a focal necrotizing vasculitis, and immunolabeling demonstrated the presence of EAV-antigens in chorionic vascular endothelial cells and macrophages.

15

Introduction

a

b

Figure 4: Schematic representation of EAV cases reported in the World Organization for Animal Health (OIE) manual of 2012 (a) worldwide and (b) in Europe.

16

Introduction

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3.2. Pathogenesis of infection with North American EAV strains 3.2.1. Routes of infection A schematic representation of the routes of infection is given in Figure 5. The two major routes are the respiratory and venereal ones. Aerosol spreading is the most important and main route of EAV spreading during an outbreak of the disease. Throughout the acute phase of infection, significant amounts of virus are shed in the respiratory tract fluids. Direct and close contact appears to be necessary for aerosol transmission (Timoney, 1988). Sexual transmission is another important route of virus infection. The virus can be transmitted venereally by mares and stallions not only during the acute phase but also through persistently infected stallions (Timoney et al., 1986 and 1987). From 30 to 70% of infected stallions become persistently infected and constantly shed the virus in their semen. The virus is associated with the sperm-rich and not the pre-ejaculatory fluid fraction of semen. Duration of virus persistence can vary, carrier stallions can stop shedding virus in their semen weeks to years after infection with no evidence of reversion to a shedding status later. Frequency of the carrier state varies between different groups of stallions but no breed predisposition was demonstrated (Timoney et al., 1986 and 1987). EAV can efficiently spread through artificial insemination and the use of fresh or frozen semen. Approximately 85 to 100% of seronegative mares become infected when they are bred to persistently infected stallions or artificially inseminated with semen containing virus. The venereal infection and transmission is most relevant from an epidemiological point of view. Reciprocal venereal transmission from an acutely infected mare to a seronegative stallion, though plausible, has not been documented (Timoney et al., 1987). The viral shedding in semen has also been demonstrated in donkeys (Paweska et al., 1996). A carrier state has never been reported in mares, geldings or sexually immature colts. Genetic variants of EAV can emerge during the persistence of the infection in stallions (Balasuriya et al., 2004a) giving a potential hazard for new EVA outbreaks. Therefore, carrier stallions may occupy a special niche in the epidemiology of EAV infection since they are not only a natural virus reservoir, but can also be a natural source of genetic diversity of EAV. Outbreaks of EAV occur when one of these variants is transmitted to a susceptible cohort. Thus, the percentage of actively shedding carrier stallions likely determines the prevalence of EAV infection in horse breeds (Balasuriya et al., 2004a). Acutely infected horses also shed virus in their urine, feces, vaginal and other body secretions, although in smaller amounts (Timoney and McCollum, 1993a). Virus can be detected from 3 to 14 days post infection (dpi) in respiratory secretions and from 5 to 19dpi in urine (McCollum et al., 1971). EAV can also be found in aborted fetuses, fetal membranes, placenta, and fluids of a mare that has aborted (Timoney and McCollum, 1993b). These sources of virus may contribute to aerosol transmission but also to indirect transmission via fomites. EAV can also, but less commonly, be transmitted by other means such as indirect contact with viruscontaminated fomites or by an infected teaser stallion or by a nurse mare (Timoney and McCollum, 1996). A recent study by Broaddus et al. (2011) demonstrated that there could also be a risk of EAV transmission

17

Introduction

resulting from in vivo embryo transfer from a donor mare inseminated with EAV infective semen (Broaddus et al. 2011 and Timoney et al., 1987). Finally, a vertical trans-placental transmission was also documented (Vaala et al.,1992).

Figure 5: Schematic representation of routes of infection and transmission of EAV. Carrier stallions (a) may transmit EAV to mares through infected semen. Acutely infected mares (b) shed the virus in respiratory secretions infecting animals of the same cohort. As a result, newly infected animals shed virus through the respiratory route, disseminating the infection (c). Further, naïve stallions can become carrier (d) and infect mares through the venereal route. If a pregnant mare is acutely infected, beside shedding the virus in respiratory secretions, she can abort (e). Aborted fetuses and fetal membranes contain high quantities of EAV and, therefore, they can contribute to the EAV epidemiology (f).

3.2.2. Pathogenesis of generalized infection following respiratory uptake of the virus The pathogenesis of North American EAV strains was studied following the distribution of viral antigens and lesions in horses experimentally infected with virulent EAV strains via the respiratory route (Crawford and Henson, 1972 and Del Piero, 2000). The first cells to be infected at the site of entry are not yet characterized. At 24 hours post infection (hpi), the virus infects the alveolar macrophages and pneumocytes with viral-antigens localized within their cytoplasm (Wilkins et al., 1995). At 48hpi, the virus can be found in the satellite lymph nodes, especially in the bronchial lymph nodes. EAV-antigens are contained within stromal dendrite-like cells and within the macrophages of the lymph node sinuses (Jones et al., 1957). Viraemia starts from 2 to 3dpi depending on the animal and the virus strain. Therefore, at 3dpi, the virus replicates in broncho-pulmonary lymph nodes, endothelium and circulating monocytes. Different authors described a cell-associated EAV viraemia with sporadic isolations of the virus from the non-cellular fraction.

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Introduction

_

It was noticed that the highest EAV titers were associated with platelets although significant virus titers were also present in red blood cells and PBMC fraction. EAV can be harvested from the buffy coat from 1 to 19dpi while serum can yield the virus from 1 to 9dpi (MacLachlan et al., 1996). Experimental infections with EAV can cause a cell-associated viraemia that lasts several weeks after serum SN antibodies become detectable (Fukunaga et al., 1981 and Neu et al., 1987). However, the systemic distribution of the virus through viraemia results in infection of macrophages and dendritic cells of associated lymphoid tissues of several organ systems. Approximately from 6 to 8dpi, the virus localizes within endothelium and myocytes of blood vessels and mesothelium (McCollum et al., 1971). At 10dpi, EAV-antigens localize in endothelium, myocytes and pericytes of blood vessels. Marginating macrophages containing intracytoplasmic EAVantigens may be seen, occasionally associated with infected endothelial cells. Apparently, the last site to be invaded is the renal tubular epithelium. Abundant intracytoplasmic viral-antigens can be found within morphologically intact and necrotic tubular epithelial cells, intratubular cellular hyaline casts, glomerular endothelium and stellate and fusiform cells located in the renal interstitium (Del Piero, 2006). After 10dpi, EAV-antigens have decreased in all the locations except the tunica media of small muscular arteries. Infectious EAV is no longer detectable in most tissues after 28dpi, with the exception of the reproductive tract of some stallions (Del Piero, 2000).

3.2.3. Pathogenesis of abortion following respiratory infection Timoney and McCollum (1993a) showed that transplacental infection, although rare, can occur when seronegative pregnant mares are exposed to EAV through the respiratory route. If the transplacental transmission occurs in the first months of gestation, it will result in abortion. In the case a pregnant mare is exposed to the virus in the last third of gestation, the foal will be congenitally infected but no abortion will occur (Vaala et al., 1992). EAV-antigens are inconsistently detectable within tissues of aborted fetuses and when present, they are localized within the cytoplasm of the trophoblast, allantochorionic mesenchyma, thymus epithelium, splenic reticular cells, endothelium of visceral blood vessels, and enterocytes (Johnson et al., 1991). Coignoul and Cheville (1984) attributed EAV-induced abortion to decreased blood supply to the fetus as a consequence of blood vessel compression by endometrial edema, alteration of vascular tone by various inflammatory mediators and/or virus-induced injury to the myometrium rather than to any direct effect of the virus on the fetus itself. Further, Del Piero (2000) reported that the progesterone levels in the mare’s serum constantly diminish from 48 to 6 hours before abortion. The decreased production of progesterone, due to hypoxia in the placenta, combined with a local release of prostaglandins, may trigger chorionic detachment. In addition, ischemia induced by vasculitis and thrombosis may also play a role. As a consequence of chorionic detachment and ischemia, expulsion of an infected or uninfected fetus may follow. Furthermore, Coignoul and Cheville (1984) indicated that virus in the fetus may reflect only contamination attributable to increased permeability of the placenta. However, considerably higher EAV titers were found in fetal than in maternal blood and a relative abundance of viral antigens was detected in fetal tissues, as compared to those of the dam (MacLachlan et al, 1996) indicating that the presence of EAV in fetal tissues

19

Introduction

does not simply reflect contamination but rather points towards EAV infection of the fetus itself. Fetal infection and pathology can activate the normal process of parturition. In addition, fetal death itself can also release the inhibitory effects of pregnancy on the myometrium, which may result in abortion (Norwitz et al., 1999).

3.2.4. Pathogenesis following venereal route Up to now, there is no recorded study on the pathogenesis of EAV following venereal exposure to the virus. However, it is presumed that the virus is taken up from vaginal mucosa and is transported to the local lymph glands where it likely replicates and is released into the bloodstream and the lymphatic circulation (Timoney, personal communication).

3.2.5. Mechanism of persistence in stallions EAV reaches the male reproductive tract through viraemia resulting in an acute infection and/or in a carrier state. Two situations have been demonstrated in the stallion: a short-term virus excretion period lasting 4-5 weeks and a long-term carrier state after clinical recovery persisting for years to life long (Timoney et al., 1986 and 1987). In carrier stallions, EAV persists exclusively in the reproductive tract and not in other sites of the body. The ampulla of the vas deferens and other accessory sex glands have been identified as the main sites of viral persistence (Neu et al., 1987). The virus is associated with the sperm-rich fraction of the ejaculate and the virus is not present in the pre-sperm fraction of semen (Timoney and McCollum, 2000). The detailed mechanisms of this virus persistence are not clearly understood. It has been proposed that viral persistence may be the result of two essential ingredients: the first could be that the virus has a unique strategy of viral replication by which, instead of killing its host cell, it causes little to no damage and thus can reside in some infected cells; the second is that the immune response does not or insufficiently reach the virus in the host or, for unknown reasons, is unable to eliminate it (Oldstone, 1989 and 1991). Since EAV in the reproductive tract of carrier stallions can be venereally transmitted to susceptible mares, it is clear that EAV has not acquired a restricted tropism for the reproductive tract of stallions (McCollum et al., 1988). It appears that humoral immunity does not prevent the establishment and the maintenance of EAV infection in the reproductive tract of stallions. In fact, it was observed that carrier stallions have moderate to high titers of serum neutralizing antibodies (Timoney and McCollum, 1993a) and that the sera of persistently infected stallions consistently recognize the GP5, N and M viral proteins while sera of mares, geldings and nonpersistently infected stallions only recognize the M viral proteins (MacLachlan et al., 1998). Comparison between sequential isolates recovered at regular intervals from the same naturally infected stallions, revealed ongoing oligonucleotide variation in the virus, which may be another mechanism for the establishment of the carrier state (Murphy et al., 1992). Also, down-regulation of viral antigens presented on the host-cell surface could mask EAV for antibodies allowing the virus to persist. However, since the virus variants emerging in carrier stallions can still be neutralized by polyclonal neutralizing antibodies, it seems unlikely that immune escape of viral mutants plays a significant role (Balasuriya and MacLachlan, 2004).

20

Introduction

_

Another hypothesis put forward to explain virus persistence may be related to the immunologically privileged site of the male reproductive tract. It may be that EAV in the male reproductive tract is inaccessible to circulating neutralizing antibodies or that neutralizing antibodies can only partially reduce virus replication. There is convincing evidence that establishment and maintenance of the carrier state in the stallion is testosterone-dependent. When persistently infected stallions are castrated and treated with testosterone, they continue to shed virus into the semen while castrated stallions given a placebo cease to shed virus (Little et al., 1991). Investigation of infected prepuberal and peripuberal colts showed that the virus, after clinical recovery, continues to replicate in the reproductive tract in a significant proportion of these colts for a variable period of time. This occurs as well in the absence of circulating concentrations of testosterone as in the presence of testosterone levels equivalent to those found in sexually mature stallions. However, longterm persistent EAV infection did not occur in these colts (Holyoak et al., 1993a). The mechanism by which testosterone contributes to the establishment and maintenance of the persistent EAV infection in stallions remains undetermined. It is speculated that testosterone may be involved by stimulating the development of a mature reproductive tract and in the production of androgen-dependent cells in the reproductive tract of stallions. It thus remains to be determined which host or viral factor(s) contribute to the establishment and maintenance of persistent EAV infection in stallions. Analysis of two carrier stallions and a number of their male offspring did not demonstrate a significant association between inherited MHC haplotype and the carrier state (Albright-Fraser, 1998). Moreover, the mechanism accounting for the spontaneous clearance of EAV in some of the carrier stallions is not clear.

3.3. Clinical signs Although the confirmed cases of clinical EVA have increased in recent years, the majority of infections are subclinical. The occurrence of clinical signs depends on the age of the animal, the route of exposure, the virus strain and the virus dose. The clinical signs are generally most severe in old and very young animals and in horses that are immune-compromised or in poor condition. Clinical signs typically develop between 1 and 10dpi (Cole et al., 1986) and may include any combination or all of the following clinical signs: pyrexia (2-12 days), depression, anorexia, conjunctivitis, with lacrimal discharge, rhinitis with serous to mucoid nasal discharge, rhinorrhea, epiphora, lower limb edema, and stiffness of gait. Edema of the periorbital and supraorbital areas, mid-ventral regions, scrotum, prepuce, mammary gland, and urticarial rash may also occur. Less frequently observed are: severe respiratory distress, ataxia, mucosal eruptions, photophobia, diarrhea, icterus, submaxillary lymphadenopathy, and intermandibular and shoulder edema may be present. In general, animals recover completely (Timoney and McCollum, 1993a and 1996). A schematic representation of the main and most common clinical signs, laboratory findings and localization of EVA following respiratory infection is given in Figure 6. The abortion rate varies from 10% to 50-60% and an outbreak may be characterized as “epidemic abortion”. Susceptible mares infected by EAV between 2 and 11 months of gestation can experience abortion and the

21

Introduction

ages of the aborted fetuses may range from 90 to 337 days. At the time of abortion, mares usually exhibit no clear signs but it is possible that they have manifested anorexia, lameness, fever (41°C), conjunctivitis, and nasal discharge prior to abortion (Timoney and McCollum, 1993a). Normally, abortion can occur during either the acute stage of the infection or soon thereafter (indicatively between 1 to 3 weeks following exposure to the virus). There is no evidence that mares can abort more than once due to EAV infection (Timoney and McCollum, 1987). Experimentally, mares aborted fetuses enveloped within their fetal membranes between 10 and 12dpi (Wada et al., 1996) while non-inoculated in contact mares aborted 23 to 57 days after the infection had been started in the inoculated mares (Cole et al., 1986). While mortality is very rare in healthy adult horses, it is nearly 100% in newborn foals. Thus, EAV infected neonates, not protected by maternal immunity, may die suddenly or shortly after showing severe respiratory distress (Timoney and McCollum, 1993a and 1996). Foals infected within a few months of age may develop a life-threatening pneumonia or pneumoenteritis. After experimental EAV infection and particularly during the acute stage, stallions may undergo a period of temporary subfertility, associated with reduced libido, increase of scrotal temperature and change in sperm quality as manifested by decrease in motility, concentration, and percentage of morphologically normal spermatozoa. These abnormalities may persist for up to 16 weeks before returning to pre-exposure levels (Neu et al., 1992). Semen quality is normal in persistently infected stallions, despite presence of the virus. Venereal infection of mares by persistently infected stallions may result in decreased fertility at the initial cycle, but it does not appear to result in subsequent fertility problems (Timoney and McCollum, 1993a).

22

Figure 6: Schematic representation of clinical and laboratory findings and localization of EVA following respiratory infection. Vertical bars represent the chronological occurrence of the respective clinical or laboratory findings and the distribution of virus in body tissues and secretions (adapted from Balasuriya and MacLachlan, 2004).

Introduction _

23

Introduction

3.4. Pathology The North American EAV isolates appear to differ in virulence and, consequently, induce lesions that differ in severity (McCollum, 1981). Data on gross and histological lesions result from studies of natural and experimental infections with North American strains of EAV.

3.4.1. Gross lesions The gross and microscopic lesions reflect the extensive and considerable vascular damage at the level of lymphatic vessels, large and small arteries, veins and capillaries. Edema, congestion, and hemorrhages in the subcutaneous tissues, lymph nodes, and viscera are the most frequently observed gross lesions. The body cavities may contain moderate to abundant amounts of peritoneal, pleural, and pericardial clear to yellowish exudate. Congestion and lymphadenomegaly, edema, and hemorrhages can be observed along the course of the colonic and cecal vessels and are also evident in systemic organs. In lymph nodes, there may be a prominent subcapsular sinus and dilated medullary sinuses. Lungs are wet and increased in weight, with edema, emphysema, interstitial pneumonia and show a prominent lobular pattern. These findings are more severe in infected neonates. When congestion and hemorrhages are present, lungs can be multifocally or diffusely reddish. Enteritis and infarcts in the spleen have been described in fatal cases of the disease in foals. The uterine endometrial surface of aborting mares can be swollen and diffusely congested, sometimes with hemorrhages (Del Piero, 2006 and Prickett et al., 1972).

3.4.2. Histopathology In general, the histopathologic lesions are observed in many organs but the blood vessels are the principal target. Mild vascular lesions include lymphocytic infiltration and endothelial cell hypertrophy. Severe vascular changes include (i) vasculitis with fibrinoid necrosis of the tunica media, (ii) abundant vascular and perivascular lymphocytic and lesser granulocytic infiltration, (iii) loss of endothelium, and (iv) formation of large fibrinocellular stratified thrombi with associated tissue infarction. Capillary vessels are often obliterated by swollen endothelial cells, platelet thrombi, or neutrophils (Estes and Cheville, 1970). Detailed descriptions of the histopathological changes in different organs were provided following experimental infections with EAV North American isolates (Del Piero, 2006; Jones et al., 1957 and Prickett et al., 1972). The type and severity of lesions described and the organs affected vary from one animal to another and from one virus strain to another. In lungs, mild to severe interstitial pneumonia characterized by alveolar infiltration with macrophages and neutrophils, hyaline membrane formation, and fluid-filled alveoli are common lesions. In addition, pulmonary arteritis and phlebitis are frequently observed. Within lymphoid organs, lymphoid follicle necrosis, edema and slight hemorrhage with histiocytic erythrophagocytosis are common findings. In lymph node sinuses, prominent and sometimes highly pleomorphic histiocytic cells and lymphocytes are often detected. Cardiac vasculitis with myocyte necrosis and associated EAV-antigens is rarely observed. In the liver, a portal vasculitis consisting of a severe inflammatory cell infiltrate that erodes

24

Introduction

_

the hepatocellular periportal limiting plate, is occasionally observed. Distention of the submucosal lymphatics of the large intestine, with mild crypt and lamina propria necrosis, is sporadically observed. The adrenal gland occasionally show multifocal vasculitis, hemorrhages and infarcts. Renal lesions occur when the infection is at an advanced stage and consists of tubular necrosis, lymphocytic interstitial nephritis, glomerular tuft disorganization, and hypercellularity. The dermis is occasionally involved with vasculitis with or without association of thrombosis and ulcerative dermatitis. The nervous system is generally not affected, although cerebral vascular necrosis has been reported in fetuses. The uterine propria and submucosa are sometimes edematous with infiltration of neutrophils and macrophages and endothelial cell swelling, while necrotic myocytes, and macrophages are commonly found (Jones et al., 1957 and Coignoul and Cheville, 1984). Experimentally infected prepuberal and peripuberal colts, euthanized between the 7th and the 14th day following viral inoculation, show necrotizing vasculitis in the testes, epididymides, vasa deferentia, ampullae, prostate glands, and vesicular and bulbourethral glands. This vasculitis is characterized by severe fibrinoid necrosis of the small muscular arteries with edema and hemorrhage. In colts examined between the 28th and 180th dpi, lymphocytic and plasmacytic infiltrates were found in the lamina propria and tunica muscularis of the epididymides and accessory genital glands. One of the prepuberal foals, infected for 15 months, had marked lymphoplasmacytic infiltration of ampullae (Holyoak et al., 1993b). Fetuses and fetal membranes are often expelled without premonitory signs of abortion, either autolyzed or well preserved. Lesions in the fetus, when present, consist of mild perivascular lymphocytic infiltrate and mild interstitial pneumonia. Mild vasculitis involving the allantochorion, brain, liver, spleen, and lung is sporadically found. In foals, where the pulmonary lesions generally prevail, a pneumoenteric syndrome with pathologic changes involving intestinal crypts, intestinal mucosal blood vessels and gastrointestinal tractassociated lymphoid tissues is sometimes observed; infarcts of caecum and colon are rare (Golnik et al., 1981 and Johnson et al., 1991).

3.5. Clinical signs and pathology of infection with European EAV strains A natural outbreak of EAV caused by European strains may be characterized by fever, anorexia, depression, edema of the limbs, conjunctivitis with lacrimation, rhinitis and nasal discharge, urticaria of the head, abortion and death in foals (Hans et al., 2008). In a Bulgarian outbreak for example (Chenchev, 2008) the first clinical signs were depression, anorexia and conjunctivitis with tearing from the medial canthus. The body temperature increased to approximately 40-40.2°C. In some animals dermatitis was noted. Sick animals were retarded in growth and frequently experienced secondary bacterial infections, such as salmonellosis and pneumonia. The percentage of cases of abortion was 18%. Abortions were mainly observed during the first part of gestation and the mares recovered without treatment. Pathological changes included: cyanosis of skin; typical lesions of interstitial pneumonia with multiple nodular masses in the lungs which were pink to bloody-red in color. The trachea and bronchi contained frothy fluid.

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Introduction

3.6. Immunity Since natural EAV infection occurs via respiratory or reproductive tract, the innate response of the mucosa lining the respiratory and genital tracts provides the first line of defense. Although little is known on the innate immune response evoked by EAV, it is assumed not to differ from that induced by other viruses (Balasuriya and MacLachlan, 2004). The adaptive immune response is divided in humoral and cell-mediated. Antibodies have been detected after infection using the complement fixing (CF) and serum neutralization (SN) tests. The CF antibodies peak 2–3 weeks post infection and persist for 8 months. EAV-neutralizing antibodies, induced after natural or experimental infection or by vaccination, peak within 1-2 months (Chirnside, 1992) and last for several years (possibly life-long). With the exception of persistently infected carrier stallions, EAV is generally eliminated from the tissues of infected horses by 28 days after infection and virus clearance correlates with the appearance of antibodies in serum (Timoney and McCollum, 1993a). Carrier stallions generally have particularly high titers of neutralizing antibodies (Timoney and McCollum, 1993a and 2000 and Balasuriya and MacLachlan, 2004). SN antibodies protect horses against re-infection upon subsequent challenge (McCollum, 1970; 1976 and 1986). Maternally derived SN antibodies appear a few hours after colostrum uptake and are detectable until 2 to 6 months of age. The mean biological half-life of the maternally derived antibodies in foals is estimated to be 32 days (Hullinger et al., 1998). The immunoblotting assay showed that sera of non-carrier animals most consistently recognize the conserved M envelope protein, that the serological response of horses to the GP5 and N proteins is variable and that the GP2b protein is rarely recognized. Persistently infected stallions and horses immunized repeatedly with vaccines develop antibodies specific for GP3 (100 and 81%, respectively), while such antibodies were found only in 16–22% of animals that were in contact with the virus only once. These findings clearly suggest that GP3 is immunogenic in horses but only after repeated exposure (Balasuriya and MacLachlan, 2004). Several laboratories have developed neutralizing monoclonal antibodies to EAV and all of them recognize the GP5 protein using Western blot and/or immunoprecipitation assays (Glaser et al., 1995). Several studies have demonstrated that the ectodomain (aa 19-116) of the GP5 protein is immunedominant and that, more specifically, the amino acid residues (i) 49 and 61; (ii) from 67 to 90 and (iii) from 99 to 106 are the sites for induction of virus neutralization (Balasuriya et al., 1993; 1995b and 1997). Taken together, these studies demonstrated that the ectodomain of the GP5 protein contains EAV neutralization determinants (Balasuriya et al., 1997 and 2004b). The mechanism by which antibodies act to neutralize viral infectivity are not clear but it is shown that neutralization of EAV is complement-dependent and that the addition of 10% guinea pig complement into the SN assay increases its sensitivity (Fukunaga et al., 1994). Cell-mediated immunity to EAV is poorly studied. The detection of CD8+ CTL precursors from EAVconvalescent animals indicates that cell-mediated immunity may play an important role in the ultimate

26

Introduction

_

clearance of the viraemia. It was noticed that upon induction with EAV, an activation occurred of both CD8+ and CD4+ cells and an increase in the CD8+/CD4+ ratio which may indicate that CD8+ cells can be responsible for the lysis of EAV-infected target cells (Castillo-Olivares et al., 2003). Taking into consideration that (i) EAV replication in chronically infected stallions persists for several months to years despite high levels of circulating SN Abs; and (ii) experimental infections with EAV can cause a cell-associated viraemia that lasts several weeks after serum SN antibodies become detectable, it can be deduced that EAV, as well as the other members of its family, is able to escape to some extend from the host immunity.

3.7. Diagnosis 3.7.1. Clinical diagnosis EVA cannot be diagnosed based solely on clinical or clinical-pathological findings. Therefore, virological or serological laboratory confirmation is required. EAV should be considered when the clinical signs include fever, depression, edema, conjunctivitis, nasal discharge and abortion. Differential diagnoses are numerous and include other viral infections such as EHV-1 and 4, equine influenza viruses, equine rhinoviruses, equine infectious anemia, Hendra disease, African horse sickness, and Getah virus. Also diseases of non-infectious nature such as urticaria, shock, purpura hemorrhagica, Hoary alyssum toxicity, hemolytic uremic syndrome may resemble EAV infection (Del Piero, 2000).

3.7.2. Detection of EAV virus, viral components or antibodies When EAV is suspected, virus isolation should be performed on nasopharyngeal and conjunctival swabs, bronchoalveolar lavage fluid, unclotted citrated or EDTA blood samples from live animals (Timoney and McCollum, 1993a). To optimize the chances of virus isolation, the relevant specimens should be obtained as soon as possible after the onset of fever in affected horses. In cases of mortality in young foals or older animals, virus isolation can be attempted from a variety of tissues, especially the alimentary tract with its associated lymph nodes, the lungs, liver and spleen (McCollum et al., 1971). In outbreaks of EVA-related abortion and/or cases of stillborn foals, placenta, fetal fluids and therefore, tissues (especially lungs) are sources of virus (Timoney and McCollum, 1993a). Often, an aborted fetus does not contain detectable levels of EAV-antigens and virus isolation and PCR on endometrial tissue should be performed. The presence of EAV in body fluid, tissue samples, blood, semen and placenta can usually be detected by virus isolation in cell culture. The most appropriate specimens for virus isolation from live animals are nasopharyngeal swabs, conjunctival swabs and citrated or EDTA blood samples for separation of buffy coat cells. Isolation of EAV is currently performed using RK-13 cell line. Virus isolation (VI) is the current gold standard test, approved by the World Organization for Animal Health (OIE), for the detection of EAV in semen from stallions and is the prescribed test for international trade. The cytopathic effect (CPE) and virus

27

Introduction

titer can vary significantly among cell lines. The viral cytopathic effects (CPE) in EAV infected cells appear within 2–6 days and are usually characterized by rounding, vacuolation, increased optical density, refraction and detachment from culture vessels. It should be considered that while the vast majority of isolations of EAV are made on the first passage in cell culture, a small minority will only become evident during the second or subsequent passages. Immunocytochemical methods (Little et al., 1995), such as indirect immunofluorescence (Crawford and Henson, 1972) or the avidin–biotin–peroxidase staining (Del Piero et al., 1997) have also been used to detect viral antigens in fetal membranes, placenta, fetal tissues (especially lungs, lymph nodes, liver, and intestine) and tissues (such as lungs, lymph nodes, heart, liver, spleen, intestine and testis or uterus) collected from experimentally infected animals, as well as in skin biopsies from acutely infected horses. RT-PCR can detect viral nucleic acids in clinical samples such as body fluids and semen from live horses or tissues (fetal membranes, placenta, fetal lungs, lymph nodes, heart, liver, spleen, intestine, testis or uterus) collected at necropsy. RT-PCR is a powerful and sensitive diagnostic technique even if genetic variability of EAV quasi-species has to be taken into consideration. Multiple PCRs using different couples of primers are advised to reduce false negatives (Balasuriya et al., 1998). Finally, seropositive stallions without vaccination history or with a certificate that the animal was seronegative before the first vaccination, should be screened for carrier status. The carrier status of a stallion can be determined by RT- PCR on the sperm-rich fraction or by breeding two seronegative mares and to test the mares for development of antibodies. If the mares seroconvert four weeks after breeding, then the virus was present in the semen and the stallion is diagnosed as a carrier. A variety of serological tests, including virus neutralization (VN), complement fixation (CF), agar gel immuno-diffusion,

enzyme-linked

immunosorbent

assay

(ELISA),

and

fluorescent

microsphere

immunoassay (MIA) have been used to detect antibodies to EAV. In acute cases, a four-fold increase in titers in paired serum samples should be observed. The VN test is considered the “gold standard” for detection and titration of antibodies to EAV and, currently, is the only validated test accepted for international trade. Although, the VN test is highly sensitive and accurate, it has several disadvantages: it is expensive, laborintensive and time-consuming to perform (Westcott et al., 1995). In addition, results can vary among laboratories when adequate attention is not paid to standardization of both test reagents and procedure. Moreover, some sera when used undiluted or at low dilutions, may induce cytotoxicity in the cell cultures possibly leading to misinterpretation. Further, the VN test cannot differentiate the antibody response of vaccinated from naturally infected horses. To overcome these disadvantages, several ELISAs have been developed. Even though they have not been as extensively validated as the VN test, some appear to offer comparable specificity and close to equivalent sensitivity (Cho et al., 2000). The CF test is less sensitive than the aforementioned assays, but can be used for diagnosing recent infections.

28

Introduction

_

4. EQUINE ARTERITIS VIRUS - UPPER RESPIRATORY MUCOSA INTERACTION: EX VIVO MODEL 4.1. Mucosal explants in research Experiments in animals should be limited as much as possible for ethical reasons. In vivo work involving large animals is difficult, as purchase and maintenance costs are high and suitable experimental animals are often difficult to obtain. Further, for the horse, specific-pathogen-free animals are not available. Thus, there is a constant search for in vitro models that minimize the number of in vivo experiments and the use of experimental animals, as required by the principles of the 3 R’s (Russel and Burch, 1959). These include reduction of the number of experimental animals, refinement of the experiments to minimize pain and distress and replacement of experimental animals by using in vitro cell or tissue models. In some models such as in one- or two-dimensional monolayer cultures, the possibility exists that the normal behavior of cells is compromised if they are removed from their surrounding micro-environment and lose their normal threedimensional association. Explant models may fulfill many of the above mentioned requirements. In fact, the three-dimensional structure and normal cell-cell contacts are maintained in these models, hereby providing accessible means to mimic the in vivo situation. Explant models are powerful ex vivo tools permitting controlled experimental manipulation while maintaining micro-environmental architecture (Anderson and Jenkinson, 1998). Also, the use of explant models minimizes inter-animal and inter-experiment variations as tissues can be obtained from the same animal and samples can be collected at different time points. In general, the organ culture method can provide a valuable research tool for physiological and pathological studies of the respiratory mucosa, for studying the effect of numerous non-infectious and infectious respiratory agents upon interaction with the respiratory mucosa. Explant models of nasal mucosa have already been described for a wide variety of species including pigs, horses, cows, chickens, rats, and humans (Ali et al., 1996; Butler and Ellaway, 1972; Fanucchi et al., 1999; Glorieux et al., 2007; Steukers et al., 2011 and Vandekerckhove et al., 2009). All these models have two important features in common: (i) the cultivation of the mucosa at an air-liquid interface which implies that the tissue is in contact with air on one side, while it is in contact with culture medium on the other side, hence creating a physiologically relevant environment (Middleton et al., 2003) and (ii) serum-free conditions used for all cultivation medium since serum-supplemented medium causes an enlargement of the epithelial cells with loss of cells-cells contacts and, consequently, reduced integrity of the epithelium and a decreased cultivation period of epithelial cells (Glorieux et al., 2007).

4.2. General characteristics of the respiratory mucosa The respiratory mucosa lines the respiratory tract, including nasal cavity, nasopharynx, larynx, trachea, and bronchial tree and represents the first line of defense against pathogens such as EAV. The respiratory mucosa consists of the luminal surface epithelium and the underlying connective tissue or lamina propria separated

29

Introduction

from each other by a firm barrier, the basement membrane (BM). Epithelium, BM and lamina propria are firm barriers that a virus needs to cross prior to cause a generalized infection.

4.3. Histology of the upper respiratory tract mucosa 4.3.1. Respiratory epithelial components and their functions The upper respiratory tract (URT) and deep respiratory tract (DRT) are covered by an epithelium designated as respiratory epithelium, which varies in composition depending on the site in the respiratory tract. Since the ex vivo research part of this thesis was mainly conducted on respiratory epithelium of the nasal cavities and nasopharynx, the respiratory epithelium of these two regions will be described here in detail. The left and right nasal cavities are completely separated by a cartilaginous nasal septum and extend from the external nares to the nasopharynx with which they communicate caudally (Kumar et al., 2000). The main portion of the nasal cavities is formed by the pars respiratoria, which is characterized by ciliated pseudostratified columnar respiratory epithelium. The nasopharynx has also a ciliated pseudostratified columnar respiratory epithelium. An epithelium is defined as pseudostratified when, although all cells make contact with the BM and represent a single layer of cells, the nuclei are not aligned in the same plane (Eurell and Frappier, 2006). The nasal and nasopharyngeal ciliated pseudostratified epithelium contains several morphologically different cell types, including basal cells, ciliated cells, brush cells and goblet cells, which are unevenly distributed along the mucosal surface (Figure 7) (Plopper and Adams, 1993).

A

B

Figure 7. Schematic representation (A) and haematoxylin-eosin photogram (B) of the different cell types present in the equine respiratory epithelium. (a): ciliated cell, (b): goblet cell, (c): brush cell, (d): basal cell, (e): basement membrane, (f): mucus blanket. (A: adapted from Vandekerckhove PhD dissertation, 2011).

30

Introduction

_

Basal cells may function as progenitor cells and, through division and differentiation, replace epithelial cells. By flattening out and covering the BM, basal cells can act as a defense mechanism when neighboring columnar cells are lost. Furthermore, they are firmly attached at the base of the epithelium to the basal lamina by hemidesmosomes and to adjacent columnar cells by desmosomes (Evans et al., 2001). Ciliated cells are the main cell type in the ciliated pseudostratified columnar epithelium. They are supporting, columnar epithelial cells with 200 to 300 motile cilia and numerous microvilli projecting into the nasal lumen (Plopper and Adams, 1993). Their rod-shaped nuclei are distributed irregularly, occupying the entire thickness of the epithelium (Kumar et al., 2000). They have a wide apical side and a narrow basal side directed towards the BM. Brush cells, also known as tuft cells, represent a population of epithelial cells scattered throughout the epithelial lining of the respiratory apparatus. Their most characteristic morphologic features are the brush of relatively long, rigid and thick microvilli on their apical cell surface and a cytoplasm containing many filaments. Despite numerous morphological studies, the function of brush cells remains obscure. The two currently proposed functions of tuft cells are secretion and absorption (Sato, 2007). Goblet cells, so called because they are shaped like a wine goblet, are widely distributed throughout the mammalian airway tract. They are located towards the supranuclear zone of the epithelium and present a strong periodic acid Schiff’s (PAS) reaction indicating the presence of neutral mucopolysaccharides (Kumar et al., 2000). They are columnar epithelial cells containing membrane-bound mucous granules and secreting mucin, which dissolves in water to form mucus. Mucus in the upper airway is important for defense, maintenance of epithelial moisture and filtering the inhaled air by trapping inhaled particles and pathogens (Davis and Dickey, 2008). Synchronized beating of surface cilia propels the mucus with the entrapped materials to the naso- and oropharynx. It is then swallowed into the esophagus and hence, cleared from the respiratory tract through the digestive apparatus (Harkema et al., 2006).

4.3.2. Cell-cell and cell-matrix adhesions Specialized cell junctions are abundantly present at the level of cell-cell and cell-matrix contact. These contacts can be partially disassembled and reassembled to facilitate physiological processes such as tissue turnover, leukocyte extravasation, wound healing and tissue repair (Ebnet, 2008). Three functional groups of cell junctions have been described: tight junctions, anchoring junctions and communicating junctions. Tight junctions (or zonula occludens) are the sites where the membranes of two cells come very close together. They function as a gate, limiting movement between adjacent epithelial cells and as a barrier preventing diffusion of proteins from the apical to the basolateral surface of the cell (Van Itallie and Anderson, 2006). Epithelial cells can alter their tight junctions to permit an increased flow of solutes and water through breaches in the junctional barriers.

31

Introduction

Anchoring junctions are responsible for the mechanical attachment of the cytoskeleton of a cell either to the cytoskeleton of a neighboring cell or to the extracellular matrix. Functionally, anchoring junctions can be classified in adherens junctions (or zonula adherens) and desmosomes (or macula adherens). Adherens junctions are responsible for cell-cell adhesion forming a continuous adhesion belt just below the tight junctions (Gumbiner, 1996). Desmosomes are button-like points of intercellular contact and connect intermediate filaments of adjacent cells forming a structural framework of great strength and resistance against mechanical stress (Alberts et al., 2002). Communicating junctions (or gap junctions) put cells in communication with their neighboring cells by creating a channel between the membranes of contacting cells, hence connecting the cytoplasm of two neighboring cells. Gap junctions enable small molecules to pass directly from cell to cell and are indispensable for cell synchronization, growth, differentiation and migration.

4.3.3. Extracellular matrix The extracellular matrix (ECM) provides the scaffolding, support and strength to tissue and organs. The ECM contains collagens, proteoglycans and glycoproteins and provides a framework for cell adhesion and tissue development (Tanzer, 2006). The ECM can be subdivided in the BM and the ECM of the lamina propria.

4.3.3.1. The basement membrane The basement membrane (BM) is a thin, sheet-like structure of fibers underlying the epithelium which individual components are regulators of biological activities such as cell growth, differentiation and migration, influencing tissue development and repair (Aumailley and Gayraud, 1998). With a pore size of approximately 50 nm, only small molecules are able to passively diffuse across this thin, rugged barrier (Kalluri, 2003). Nonetheless, there are specific mechanisms that permit normal cells to traffic freely and rapidly across the BM during morphogenesis and immune surveillance (Rowe and Weiss, 2008). Thus, leukocytes can breach the BM barrier when they are recruited from capillaries for body defense tasks. The BM is the fusion of two laminae: the lamina densa (or lamina basalis) and the lamina reticularis (Ham and Cormack, 1979). Between the epithelium and the lamina densa, a clear area is visible: the lamina lucida, which functions as the region of attachment between the epithelium and the lamina densa. The lamina densa is a sheet of connective tissue predominantly made up of type IV collagen, adhesive glycoproteins (laminin and fibronectin), proteoglycan (perlecan) and entactin. Type IV collagen, a non-fibrous polymer, is the major constituent of the lamina densa and thus represents the backbone of the BM, which provides mechanical strength and stability. The lamina reticularis is considered to be a specialized extension of the extracellular matrix (Alberts et al., 2002). With transmission electron microscopy, it was seen that the lamina reticularis is made up of numerous collagen fibers. The predominant collagen is type III, followed by collagens I, V, VI and VII. In particular, collagen VII is the main structural component of anchoring fibrils, linking the lamina densa to the underlying lamina propria (Evans et al., 2000).

32

Introduction

_

4.3.3.2. The lamina propria The lamina propria of the URT consists of proteoglycans and fibrous proteins (collagen fibers, elastin and fibronectin), which are forming a three-dimensional network wherein cells, mostly fibroblasts, and matrix are situated. Proteoglycans are heavily glycosylated glycoproteins. They can resist compressive forces by forming a highly hydrated gel. This gel forms a physicochemical environment for free diffusion of nutrients and chemical messengers (LeBleu et al., 2007). Furthermore, proteoglycans have a major role in chemical signaling between cells since they can bind various secreted signal molecules, such as protein growth factors, enhancing or inhibiting their signaling activity. Proteoglycans can also bind and, as such, regulate the activities of other types of secreted proteins, including proteolytic enzymes and protease inhibitors (Alberts et al., 2002). Collagens are the structural macromolecules and the most abundant proteins of the ECM. Collagens, mainly type I, II, III, V and VI, form fibers made by alloys of fibrillar collagens. Their most important task is to give structural support to resident cells (van der Rest and Garrone, 1991). Fibronectins are proteins connected with collagen fibers. They facilitate cell movement by binding collagen and cell surface integrins, hence reorganizing the cytoskeleton of the cell allowing them to migrate through the ECM. Furthermore, they have a function in wound healing by binding to platelets during blood clotting and facilitating cell movement to the affected area (Plopper, 2007). Fibroblasts secrete the precursors of all the components of the extracellular matrix and a variety of fibers. The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuously secreting precursors of the extracellular matrix.

33

Introduction

Figure 8: Schematic representation of the different immune cell types present in the equine respiratory epithelium and in the respiratory mucosa (adapted from Vandekerckhove Ph.D. dissertation, 2011).

4.4. Mucosal immune cells The mucosal surface of the respiratory tract is a cellular barrier that faces environments rich in pathogens. Several of these pathogens have developed effective mechanisms for colonization of epithelial surfaces and invasion of mucosal tissues. In defense, the respiratory mucosa is heavily populated with cells of the immune system (Figure 8) (Neutra et al., 1996). The mucosal immune system consists of specialized local inductive sites, the organized mucosa-associated lymphoid tissue or O-MALT and a widespread network of effector sites, the diffuse mucosa-associated lymphoid tissue or D-MALT (Kraenenbuhl and Neutra, 1992). In the respiratory epithelium, the D-MALT consists of lymphocytes in diapedesis between epithelial cells. It lies in an intercellular position and at all depths of the epithelium with greater concentration towards the base. A diffuse distribution of lymphocytes can also be found throughout the lamina propria and submucosa with greater concentration just beneath the epithelium (Figure 8) (Mair et al., 1987a). D-MALT includes CD3+ T lymphocytes, CD172a+ macrophages, dendritic cells and IgM+ B lymphocytes. Specifically, the CD3+ lymphocytes located between epithelial cells are mainly CD8+ T cells, whereas the CD4+ subset of T cells mainly localize in the stroma (Brandtzaeg, 1989 and 1996). CD8+ T cells (or

34

Introduction

_

cytotoxic T cells, killer T cells) are a subgroup of CD3+ T lymphocytes, capable of recognizing antigens presented in association with major histocompatibility (MHC) class I molecules and thus inducing dysfunction or damage or death of infected cells. CD4+ T cells (or helper T cells) are a subgroup of CD3+ T lymphocytes that generally do not display cytotoxic effects against pathogens. They assist other leukocytes in processes such as maturation of B cells and activation of CD8+ T lymphocytes and macrophages (Harrington et al., 2005). CD172a+ macrophages are produced by differentiation of monocytes and their main function is phagocytosis of cellular debris and pathogens. In addition, they stimulate lymphocytes and other immune cells. They are present between the epithelium and in the lamina propria of the URT mucosa. Macrophages in the airways are capable of effectively eliminating invading antigens or allergens by phagocytosis (Holt, 1993). Numerous MHC class II and CD172a-positive DCs are located within the airway epithelium. DCs form a network of antigen-presenting cells (APC) in the respiratory mucosa (Holt et al., 1990). DCs of mucosal surfaces can serve as APCs after migration out of mucosal tissues to draining lymph nodes (Hamilton-Easton and Eichelberger, 1995). The DC most extensively studied in the nasal mucosa is the Langerhans cell (LC), found in both the epithelial layer and in the lamina propria (Fokkens et al., 1989 and 1991). Immature LCs are well equipped for antigen binding and processing, but weak in stimulating resting T cells (Romani and Schuler, 1992). After maturation, LCs lose their typical characteristics changing their structure, phenotype and functional capacities into those of DCs. Mature DCs are weak in binding and processing antigens, but extremely powerful in stimulating resting T cells (de la Salle et al., 1997). The DCs act as ‘sensory cells’ of the immune system recognizing danger signals. In addition to their central role in the sensitization against environmental antigens, their ability to activate T cells in the airway mucosa enables them to maintain a prolonged inflammation (Huh et al., 2003). Because of their ability to take up antigens, their migratory activities and their ability to establish close associations with T cells, DCs run the risk of becoming infected by and serving as transport vehicles for viruses that infect mucosal surfaces (e.g. human immunodeficiency virus) (Pope et al., 1994). IgM+ B lymphocytes that produce immunoglobulins (Ig) have been identified in the respiratory mucosa, lamina propria and submucosa of many domestic animals, revealing a predominance of IgA-secreting plasma cells, over IgG-secreting cells and low numbers of IgM-secreting cells. In the lamina propria adjacent to mucosal surfaces, plasma cells act as a first line of defense by producing IgA in a dimeric form (Mair et al., 1987b and 1988). IgA then binds to the immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up via endocytosis. The receptor-IgA complex then passes through the cells in a process called transcytosis and is secreted at the luminal surface of epithelial cells. After proteolysis of the receptor, the dimeric IgA can diffuse throughout the lumen along with the secretory component of the receptor (Snoeck et al., 2006). Natural killer (NK) cells are an important part of the innate immune system and they contribute to host defenses against tumors and infections, particularly with intracellular pathogens (Lodoen and Lanier, 2006).

35

Introduction

NK cells are lymphocytes which do not express a T or B cell receptor. A wide array of cell surface receptors allows them to recognize different self-proteins and non-self-proteins on other cells. Some of these receptors induce NK cells activation, while other receptors inhibit NK cells function (Lanier, 2005). The balance between these activating and inhibitory signals determines whether or not an NK cell becomes activated by a particular cell. Although cell cytotoxicity is the hallmark of NK cells and the basis for their name as ‘natural killer’ cells, NK cells are able to produce several different cytokines, many of which are proinflammatory (interferon-γ, tumor necrosis factor-α, granulocyte macrophage-colony stimulating factor and interleukins) (Cooper et al., 2001). There are significant clinical data showing that human individuals with defects in NK cell activity are more susceptible to infections, especially with herpes-type viruses (Vossen et al., 2005). Mast cells are resident cells, present at all levels of the respiratory tract, with the greatest density in the nasopharynx. Up to 94% of this cell population is located within the connective tissue of the lamina propria, while only small numbers are present in the surface epithelium. They contain granules rich in histamine and heparin and mast cells armed with IgE are common in allergic patients, both in the connective tissue and in the epithelium. They are also involved in defense against (mainly parasitic) pathogens and wound healing (Mair et al., 1988). Neutrophils form an essential part of the innate immune response and are the hallmark of acute inflammation, being the first-responders to migrate towards the site of inflammation, attracted by chemokines (Nathan, 2006).

36

Introduction

_

REFERENCES Albright-Fraser D.G. (1998). Studies on the organization and polymorphism of equine MHC Class II genes. Ph.D. dissertation, University of Kentucky. Ali M., Maniscalco J. and Baraniuk J.N. (1996). Spontaneous release of submucosal gland serous and mucous cell macromolecules from human nasal explants in vitro. Am J Physiol, 270, 595-600. Anderson G. and Jenkinson E.J. (1998). Use of explants technology in this study of in vitro immune responses. J Immunol Methods, 216, 155-163. Asagoe T., Inaba Y., Jusa E.R., Kouno M., Uwatoko K. and Fukunaga Y. (1997). Effect of heparin on infection of cells by equine arteritis virus. J Vet Med Sci, 59, 727-728. Aumailley M. and Gayraud B. (1998). Structure and biological activity of the extracellular matrix. J Mol Med, 76, 253-265. Balasuriya U.B. and MacLachlan N.J. (2004). The immune response to equine arteritis virus: potential lessons for other arteriviruses. Vet Immunol Immunop, 102, 107-129. Balasuriya U.B., Rossitto P.V., DeMaula C.D. and MacLachlan N.J. (1993). A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies. J Gen Virol, 74, 2525-2529. Balasuriya U.B., Timoney P.J., McCollum W.H. and MacLachlan N.J. (1995a). Phylogenetic analysis of open reading frame 5 of field isolates of equine arteritis virus and identification of conserved and nonconserved regions in the GL envelope glycoprotein. Virology, 214, 690-697. Balasuriya U.B., Maclachlan N.J., De Vries A.A., Rossitto P.V. and Rottier P.J. (1995b). Identification of a neutralization site in the major envelope glycoprotein (GL) of equine arteritis virus. Virology, 207, 518-527. Balasuriya U.B., Patton J.F., Rossitto P.V., Timoney P.J., McCollum W.H. and MacLachlan N.J. (1997). Neutralization determinants of laboratory strains and field isolates of equine arteritis virus: identification of four neutralization sites in the amino-terminal ectodomain of the G(L) envelope glycoprotein. Virology, 232, 114-128. Balasuriya U.B., Evermann J.F., Hedges J.F., McKeirnan A.J., Mitten J.Q., Beyer J.C., McCollum W.H., Timoney P.J. and MacLachlan N.J. (1998). Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus. J Am Vet Med Assoc, 213, 1586-1589. Balasuriya U.B., Hedges J.F., Nadler S.A., McCollum W.H., Timoney P.J. and MacLachlan N.J. (1999). Genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis. J Gen Virol, 80, 1949-1958. Balasuriya U.B., Hedges J.F., Smalley V.L., Navarrette A., McCollum W.H., Timoney P.J., Snijder E.J. and MacLachlan N.J. (2004a). Genetic characterization of equine arteritis virus during persistent infection of stallions. J Gen Virol, 85, 379-390. Balasuriya U.B., Dobbe J.C., Heidner H.W., Smalley V.L., Navarrette A., Snijder E.J. and MacLachlan N.J. (2004b). Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone. Virology, 321, 235-246. Bergman A.M. (1913). Beiträge zur Kenntnis der Virustrager bei Rotlaufseuche, Influenza erysipelatosa, des Pferdes. Z Infektionskrankh, 13, 161-174. Borchers K., Wiik H., Frolich K., Ludwig H. and East M.L. (2005). Antibodies against equine herpesviruses and equine arteritis virus in Burchell's zebras (Equus burchelli ) from the Serengeti ecosystem. J Wild Dis, 41, 80-86. Brandtzaeg P. (1989). Overview of the mucosal immune system. Curr Top Microbiol Immunol, 146, 13-25. Brandtzaeg P., Jahnsen F.L. and Farstad I.N. (1996). Immune functions and immunopathology of the mucosa of the upper respiratory airways. Acta otolaryngol, 116, 149-159. Broaddus C.C., Balasuriya U.B., Timoney P.J., White J.L., Makloski C., Torrisi K., Payton M. and Holyoak G.R. (2011). Infection of embryos following insemination of donor mares with equine arteritis virus infective semen. Theriogenology, 76, 47-60.

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AIMS OF THE THESIS

Aims of the study

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Although equine arteritis virus (EAV) is not often included as etiologic agent in the differential diagnosis of abortion and neonatal mortality, equine viral arteritis (EVA) is an important disease that can result in severe economic losses for the equine industry. Breeders, racehorse and show horse owners have strong economic reasons to prevent and control this disease. While it does not kill mature horses, EVA can markedly disturb the breeding season by causing abortion in numerous mares. In addition, stallions may become persistent carriers of the virus and transmit it during breeding, with a consequent decrease in their desirability as breeding animals. EVA has recently increased in prevalence worldwide, possibly due to an increased transportation of infected horses and infectious semen. Outbreaks of EVA were first and most frequently described in the USA and, therefore, the main body of EAV literature and studies on the virus-animal interactions originates from that continent. Virus strains show a wide variety of virulence characteristics as manifested by the type and severity of clinical signs and lesions upon experimental inoculation of horses. The appearance of EVA in Europe is more recent. The phylogenetic analysis of ORF5 of different EAV isolates has revealed that they can be clustered in North American and European clades. However, it is not clear if such genetic differences have an impact on the virulence and the pathogenetic features of the European strains compared to the North American ones. At the occasion of the isolation of an EAV strain (designated 08P178) from an infected foal that died shortly after birth in Belgium in 2008, the question rose how this European strain would behave with regard to organ tropism, clinical signs, virological aspects of pathogenesis and development of lesions when inoculated in experimental horses. These points were particularly interesting since no clinical signs were observed in young or adult horses on the stud where the Belgian isolate originated from. By performing such experimental infections, an answer on the different aspects of the course of infection with this European strain was sought. All the experimental studies in horses performed using North American EAV isolates showed that the virus first caused an infection of the respiratory tract followed by spread to internal organs via a cell-associated viraemia. Pathogenetic events of viral infections may be a result of virulence genes expression. It is unknown whether or not a European strain, such as the Belgian isolate, show a profile of infection with regard to tropism for cell types and organs that could be similar or different from that previously published for North American strains. In general, the type of cells that become infected with EAV and that may be responsible for further virus invasion at the respiratory tract as portal of entry is largely unknown. More detailed information in these initial phases of EAV pathogenesis are essential and could give some insights not only on which anatomical structure(s) of the mucosal layer is (are) involved but also on the local immunological processes allowing to adapt approaches for prevention at the primary site of replication.

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Aims of the study

Also, as the name “Equine Viral Arteritis “ implies, it has almost automatically been accepted that blood vessels and possibly endothelial cells can be heavily involved in the infection and the genesis of lesions. It is intriguing to find out if such generally accepted concept is justified. Also, it could be that the degree of the endothelial cell involvement and the lesions in internal organs are directly related to the virulence characteristics of the virus strain. It was thus interesting to know if, and possibly to which extent, endothelial cells in blood vessels of infected horses serve as target cells for this European EAV strain. Thus, the general aim of the research carried out for this doctoral thesis was to obtain a better understanding of the EAV pathogenesis with a European strain starting from virus replication at the portal of entry, over the viraemia and including replication in the internal organs. The more specific aims were (i) to inoculate horses with the Belgian field EAV strain (08P178) and follow the animals, at different time intervals after inoculation, for virus quantities in different organ tissues and secretions, for humoral immune response and for clinical signs and gross pathology. (ii) to define the cell type(s) in the EAV inoculated horses (in vivo) in which the virus replicates at the portal of entry and in internal organs, in the latter with special reference to endothelial cells. (iii) to evaluate a polarized explant system of equine nasal and nasopharyngeal mucosae (ex vivo) for determining and characterizing the cellular population in which EAV replicates at the level of the upper respiratory mucosal tissues and, thus, to learn about the usefulness of this system for studying viral pathogenesis at the respiratory portal of entry.

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Sabrina Vairo, Annelies Vandekerckhove, Lennert Steukers, Sarah Glorieux, Wim Van den Broeck, Hans Nauwynck

Clinical and virological outcome of an infection with the Belgian equine arteritis virus strain 08P178

Published in VET MIC 2012; 333-344.

Clinical and virological outcome of an infection with the Belgian EAV strain 08P178

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Abstract Equine viral arteritis (EVA) is an infectious disease with variable clinical outcome. Outbreaks, causing important economic losses, are becoming more frequent. Currently, there is a shortage of pathogenesis studies performed with European strains. In the present study, eight seronegative ponies were experimentally inoculated with the Belgian strain of equine arteritis virus (EAV) 08P178 (EU-1 clade) and monitored daily for clinical signs of EVA. Nasopharyngeal swabs, ocular swabs, bronchoalveolar cells and blood were collected for virological and serological testing. At 3, 7, 14, and 28 days post inoculation (dpi) two ponies were euthanized After necropsy, specimens were collected for virus titration and immunofluorescence. EVA clinical signs such as fever and lymphadenomegaly were evident from 3 to 10dpi. Virus was isolated in nasal secretions from 2 to 9dpi and in bronchoalveolar cells from 3 to 7dpi. A cell-associated viraemia was detected from 3 to 10dpi. After replication in the respiratory tract and draining lymph nodes, EAV reached secondary target organs (high virus titers in internal organs sampled at 7dpi). At 14dpi, virus titers dropped drastically and, at 28dpi, only tonsils were positive. Immunofluorescence revealed both individual and clustered EAV-infected cells. Antibodies were detected starting from 7dpi. It can be concluded that the Belgian strain 08P178 is a European mildly virulent subtype. At present, most European EAV strain infections were thought to run a subclinical course. This study is a proof that mildly virulent European EAV strains do exist in the field.

1. Introduction Equine viral arteritis (EVA) is a horse infectious disease caused by equine arteritis virus (EAV). EAV is a single-stranded, positive-sense RNA virus with a genome of approximately 12.7kb (Snijder and Meulenberg, 1998 and Cavanagh, 1997). Within the order of the Nidovirales, EAV belongs to the family Arteriviridae. EAV was first isolated from lungs of an aborted fetus following an extensive abortion outbreak in Bucyrus, Ohio (USA, 1953) (Doll et al., 1957). Since then, several different strains have been identified. Serological and virological studies have indicated that EAV is widely distributed in equine populations around the world (Huntington et al., 1990 and Moraillon and Moraillon, 1978). Based on phylogenetic analysis of EAV-ORF5 sequences, isolates of EAV are clustered into two distinct clades: a North American and a European group. The latter can be further divided into two subgroups: EU-1 and EU-2 (Zhang et al., 2007). It has been previously reported that North American and European EAV isolates have 85% nucleotide identity. This genetic diversity among different field isolates may lead to a clinical outcome with varying severity (McCollum and Timoney, 2003; Timoney, 2000a and Patton et al., 1999). The vast majority of EAV strains causes subclinical infections. Nevertheless, in the past, extensive clinical outbreaks caused by certain EAV strains have been described in various countries (Timoney et al., 2006; Van der Meulen et al., 2001; Eichhorn et al., 1995; Wood et al., 1995 and Boer et al., 1979). A clinical infection with EAV is characterized by an influenza-like illness in adult horses, abortion in 10 to 50% of susceptible mares and interstitial pneumonia in newborn animals (Timoney, 2000a). Mortality is rare, except in foals and in old and debilitated horses. The direct consequences of EVA outbreaks are financial losses mainly caused by the

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Clinical and virological outcome of an infection with the Belgian EAV strain 08P178_

previously mentioned clinical signs in mares and newborn animals. Up to 10-70% of the stallions infected with EAV become carrier (Holyoak et al., 1993a and Holyoak et al., 1993b), constantly shedding virus into the semen (Timoney, 2000a). The commercial value of persistently infected stallions decreases as the export of these animals and their infected semen is prohibited in many countries (Timoney, 2000b). EAV can be transmitted via the respiratory and venereal route. Aerosol transmission predominates, especially when horses are gathered at racetracks, sales, shows and other events. Venereal transmission from carrier stallions is particularly significant on breeding farms (Timoney, 2000b). The virus can be found in nasal secretions of acutely infected animals for up to 16 days (Del Piero, 2000) and in infected semen for several years. Although most EAV infections are subclinical, one outbreak in 2000 (Van der Meulen et al., 2001) and one in 2008 (Gryspeerdt et al., 2009) entailed important economic losses in Belgium. In addition, a serological survey, performed on samples submitted over the last ten years to the Faculty of Veterinary Medicine of Ghent University (Belgium), reported an increase from 10 to 30% in seropositivity for EAV in horses. In this decade, the authors obtained 3 EAV isolates from horses with different clinical signs. The first was isolated from newborn animals which have died during the EAV outbreak in 2008 (Gryspeerdt et al., 2009). A second strain was isolated from an aborted fetus during an abortion outbreak on a stud farm in 2000 (Van der Meulen et al., 2001). The third strain was isolated from a healthy but persistently EAV shedding stallion. The worldwide spread of EAV and its genetic variability increase the risk of new clinical outbreaks of EVA. Currently, there is a lack of data on the pathogenic mechanisms of European EAV strains. The aim of this research was to elucidate the pathogenesis of an infection with the Belgian EAV strain 08P178. Full knowledge of this pathogenesis (i) will improve epidemiological insights in EVA, (ii) allow the identification of clinical signs that can be attributed to EAV, (iii) ameliorate approaches to diagnose EVA and (iv) enable the setting up of improved control strategies (blocking of invasion, modeling for the testing of vaccines and antivirals).

2. Materials and methods 2.1. Animals Ten Shetland ponies, 4 to 8 months old, were used in this study. Prior to the start of the experiment, an acclimatization period of two weeks was respected. During this period, the animals, 6 males (M) and 4 females (F), were tested weekly for their seronegativity to EAV by means of a complement-dependent seroneutralisation (SN)-test and an immunoperoxidase monolayer assay (IPMA). The ponies were housed in isolation units and fed daily with a commercial, complete food. Drinking water and hay were supplied ad libitum. EAV-infected animals were euthanized at different time points post inoculation: M3 and F3 at 3 days post inoculation (dpi); M7 and F7 at 7dpi; M14 and F14 at 14dpi and M28a and M28b at 28dpi. Two mock inoculated, control animals M28ac and M28bc were euthanized at the end of the experiment. The experiment was approved by the ethical committee of the veterinary faculty, Ghent university (EC 2009/008).

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Clinical and virological outcome of an infection with the Belgian EAV strain 08P178

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2.2. Virus and virus inoculation 2.2.1. Virus The strain used in this study was isolated from neonatal foals that died after EAV infection in Belgium in 2008. The outbreak started with one foal suffering from acute dyspnea four days after birth. Despite intensive treatment, this foal died within a few hours. Three weeks later, on the same farm, another foal was born in healthy condition, although placental edema was found. Ten days later, the foal showed acute respiratory distress with severe dyspnea and died shortly after (Gryspeerdt et al., 2009). Upon inoculation of rabbit kidney (RK13) cells with a 20% suspension of lung tissue of these foals, a cytopathic effect (CPE) was observed. The agent was identified as EAV by means of immunofluorescence staining using a mixture of three mouse monoclonal antibodies (mAbs): two mAbs against the GP5 protein and one mAb against the M protein of EAV (Van der Meulen et al., 2001). In addition, the virus was confirmed to belong to the subgroup EU-1 by partially sequencing (916 nucleotides) the ORF5 (GenBank accession number: JN25761) (Department of Health Care and Biotechnology, KATHO, Catholic University College of South-West Flanders, Belgium).

2.2.2. Virus inoculation Eight animals were inoculated both intranasally and orally with 20 ml of phosphate buffered saline (PBS) containing 107.6 tissue culture infectious dose 50% endpoint (TCID50) of EAV [08P178 respiratory strain, 4° passage on RK13 cells] using a fenestrated polypropylene 6.8 French, 400 mm length catheter. A dose of 5 ml of the viral suspension was administered into each nostril and 10 ml into the mouth. The exact virus titer was controlled by titration of the inoculum. Two control animals were mock inoculated with 20 ml of PBS.

2.3. Scoring of clinical signs On a daily basis, body temperature, heart and respiratory rates were determined. The animals were also scored for appetite, behavior, swelling of the lymph nodes, congestion and/or presence of petechiae at the level of mucosal membranes, respiratory distress, ocular and nasal discharge, conjunctivitis, edema, skin rash and ataxia. The scoring system used ranged from 0 (no clinical signs or normal) to 3 (severe clinical signs) (Table 1).

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Clinical and virological outcome of an infection with the Belgian EAV strain 08P178_

Table I: Scoring system used to evaluate the clinical manifestations of the Belgian EAV strain 08P178. Description of clinical signs that get a score … Symptom 0 1 2 3 Appetite Normal Diminished Anorexia Cachexia Mild Moderate Severe Behaviour Alert depression depression depression Swelling lymph nodes None Mild Moderate Severe Congestion of mucosal None Mild Moderate Severe membranes Petechiation of mucosal None Local Moderate Diffuse membranes Moderate Severe Respiratory distress None Dispnea tachypnea tachypnea Nasal discharge None Serous Mucous Purulent Ocular discharge None Serous Mucous Purulent Conjunctivitis / lacrimation None Mild Moderate Severe Periorbital / supraorbital edema None Mild Moderate Severe None Edema of ventral abdomen / legs Mild Moderate Severe Edema of mammary glands or None Mild Moderate Severe preputium/ scrotum Diarrhoea None Mild Moderate Severe Urticarial skin rash None Mild Moderate Severe Ataxia None Slight Mild Moderate Death None Yes

2.4. Collection of nasopharyngeal and ocular swabs, blood and tissues Nasopharyngeal swabs, ocular swabs and 30 ml of blood on EDTA (VWR, Bridgeport, CT, USA) were collected daily during the first 14dpi and then at 16, 18, 20, 22, 24, 26, and 28dpi. Bronchoalveolar cells were harvested by bronchoalveolar lavage (BAL) as previously described (Clark et al., 1995) at -1, 3, 7, 10, 14, 21, and 28dpi. At euthanasia, forty-four different samples (Table 2) were collected for histopathology, virus titration and immunofluorescence staining. Abdominal and pleural fluids, when present, were submitted for virus titration and antibody detection (IPMA). Lungs, liver, spleen and kidneys were collected in duplicate with one superficial and one deep portion.

2.5. Virus titration Nasal and ocular swabs, plasma and tissues were processed for virus titration. Briefly, RK13 cells were inoculated for 1h with serial 10-fold dilutions (100-10-7 in quadruplicate) of nasal and ocular secretions, plasma and 20% tissue suspensions, overlaid with medium and observed daily for CPE for 5-7 days. Presence of EAV-infected cells was confirmed by indirect immunohistochemistry (IHC) staining using polyclonal antibodies against EAV 08P178 strain (1:160) as primary antibodies and peroxidase-labeled goat anti-horse immunoglobulins (Molecular Probes, Oregon, USA) (1:500) as secondary antibodies. Viral antigen-positive cells were detected with a light microscope (Olympus Optical Co., Hamburg, Germany) and TCID50 was determined with the method of Reed and Muench (Reed and Muench, 1938).

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Clinical and virological outcome of an infection with the Belgian EAV strain 08P178

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2.6. Co-culture of equine bronchoalveolar and peripheral blood mononuclear cells Cells in the aspirated BAL fluid were pelleted by centrifugation at 400g for 20 min, washed twice and serial five-fold dilutions of each sample were co-cultured with a monolayer of RK13 cells at 37°C and 5% CO2 for 5 days. Peripheral blood mononuclear cells (PBMC) were isolated from 30 ml of EDTA anticoagulated blood by density gradient centrifugation on 95% Ficoll-Paque (Sigma-Aldrich, Bornem, Belgium). Five-fold dilutions of the PBMC were co-cultured with a monolayer of RK13 cells at 37°C and 5% CO2 for 5 days. Afterward, the plates were air-dried and submitted for IHC staining. Briefly, the plates were incubated with 4% paraformaldehyde for 10min at room temperature, washed twice with PBS and further incubated with methanol supplemented with 0.1% H2O2. After 1h of incubation with polyclonal antibodies against EAV strain 08P178 (1:160), the plates were further incubated for 1h with peroxidase-labeled goat anti-horse immunoglobulin (Molecular Probes) (1:500). Primary and secondary antibodies were diluted in PBS supplemented with 0.8% Tween and 10% complement-inactivated horse serum. Before each incubation step, the wells were extensively washed with PBS supplemented with 0.8% Tween. Infected cells were visualized with a substrate solution of 3-amino-9-ethylcarbazole in 0.05 M acetate buffer (pH 5) with 0.05% H2O2. Viral antigen-positive cells were detected with a light microscope (Olympus Optical Co., Hamburg, Germany) and TCID50 was determined with the method of Reed and Muench (Reed and Muench, 1938). 2.7. Immunofluorescence staining Only tissues with titers equal or higher than 105.0 TCID50/g were investigated by means of immunofluorescence staining. After fixation with 100% methanol for 20min at -20 °C, 8µm tissue cryosections were incubated for 1h at 37°C with monoclonal antibodies (17D3), specific for the nucleocapsid (N) protein of EAV (VMRD, Pullman, USA), diluted 1:100 in PBS containing 10% negative goat serum. Afterwards, the samples were incubated for 1h at 37°C with goat anti mouse-fluorescein isothiocyanate (FITC) antibodies (Molecular Probes) (dilution 1:100 in PBS containing 10% negative goat serum). After fixation with 100% methanol for 20min at -20°C, PBMC cytospins were incubated with biotinylated polyclonal antibodies against EAV (dilution 1:5 in PBS containing 10% negative horse serum) for 1h at 37°C and, afterwards, with streptavidin-FITC (dilution 1:100 in PBS containing 10% negative horse serum) for another hour at 37°C. In order to detect the presence and the localization of viral antigen-positive cells, the samples were analyzed with a Leica DMI 4000 B inverted fluorescence microscope (Leica microsystems, Bannockburn, USA). 2.8. Quantification of the humoral immune response 2.8.1. Serology Antibody titers were determined at -14, -7, 0, 7, 14, 21, and 28dpi by means of a complement-dependent SNtest and IPMA. The standard OIE SN test was used for the detection of EAV specific neutralizing antibodies. The SN titer was calculated following the OIE instructions (cut off 1:2).

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Clinical and virological outcome of an infection with the Belgian EAV strain 08P178_

For the IPMA, RK13 cells were seeded in 96-well microtiter plates, grown to confluency and inoculated with 103 TCID50 of 08P178 isolate. After 28h, cells were washed, dried for 1h at 37°C and stored at -20°C until use. Plates were then thawed, fixed with 4% paraformaldehyde, incubated with 0.1% hydrogen peroxide in methanol, washed extensively and serial 2-fold dilutions (starting dilution 1:10) of the sera were incubated for 1h at 37°C. After 1h incubation with peroxidase-labeled goat anti-horse antibodies (1:500) (Jackson ImmunoResearch Laboratories Inc.), a substrate solution of 3-amino-9-ethylcarbazole in 0.05 M acetate buffer with 0.05% hydrogen peroxide was added to each well and, after 20min of incubation at 37°C, replaced with acetate buffer (pH 5). The IPMA titer was calculated as the reciprocal value of the highest serum dilution that gives a visual staining of infected RK13 cells, as determined by light microscopy (cut off 1:10). 2.8.2. Antibodies in pleural and abdominal fluid The described IPMA procedures were used to determine the antibody titers of pleural and abdominal fluid collected at necropsy.

3. Results 3.1. Status of the animals before inoculation During the acclimatization period and prior to the experimental inoculation, none of the ponies showed a

Figure 1: Rectal temperature (°C) at different time poi nts after inoculation with EAV 08P178. Rectal (°C) was daily upantibodies to 28 days inoculation with EAV 08P178. raise intemperature rectal temperature andmeasured no EAV-specific wereafter detected (EAV-specific antibody titers Symbols represent individual animals classified by gender (M and F) and time of euthanasia (3, 7, 14 and DPI), the