GENERAL INTRODUCTION: ARTERIVIRIDAE

Introduction

Equine viral arteritis (EVA) is a reproductive and respiratory disease of equids (horses, donkeys, mules, and zebras) caused by equine arteritis virus (EAV).⁶⁵ EVA is an acute contagious disease principally characterized by fever, dependent oedema, respiratory signs, neonatal mortality and abortion in pregnant mares.¹⁸¹ Most importantly, EAV establishes persistent infection of variable duration in the reproductive tract in the majority of stallions infected with the virus (carrier state).^{181, 187, 188}

The older veterinary literature dating back to the late nineteenth century carries several reports of a disease clinically indistinguishable from EVA, which was referred to by various terms, such as infectious cellulitis-pinkeye, fièvre typhoide du cheval and Pferdestaupe.^{26, 49, 156, 159} It was not until 1953, however, following an outbreak of respiratory illness and abortion on a Standardbred breeding farm near Bucyrus, Ohio, USA, that EVA was defined as a separate equine viral disease and the aetiological agent (i.e., EAV) was isolated for the first time.⁶⁵ Little international significance was attached to EVA for many years until 1984, when it occurred on a widespread scale in Kentucky, involving an estimated 41 Thoroughbred breeding farms.⁹⁹ Since then, periodic outbreaks of EVA have been reported in the United States and a number of countries around the world. The major concern over the potential risk of the virus spreading to susceptible Thoroughbred populations in the USA and abroad resulted in the imposition of stringent measures governing the international movement of horses between most countries.¹⁸² Many of these restrictions continue to impact on the international trade of horses and equine semen. This disease may result in significant economic loss to the equine breeding industry due to the occurrence of abortion in pregnant mares, neonatal mortality, and the establishment of the carrier state in stallions.

Of the changing trends that have taken place in the horse industry worldwide over the past 40 to 50 years, two have contributed significantly to the  global dissemination  of EAV.¹⁷⁶ First, the continued growth in the volume of international movement of horses for performance and breeding purposes has dramatically enhanced the risk of the spread of the virus. The second factor has been acceptance of the use of frozen semen for breeding by most major horse breed registries worldwide. As a result, the international trade in equine semen has expanded and outbreaks of EVA have been linked directly to the use of imported infective  semen from particular carrier stallions.^{20, 175}

EVA can have economic consequences for both the breeding and performance sectors of the horse industry.¹⁸⁰ A range of financial losses can result directly from outbreaks of the disease on breeding farms. These include losses due to abortion and/or disease and death in young foals; decreased commercial value of stallions that become carriers of the virus; reduced demand to breed to carrier stallions because of the associated inconvenience and added expense; denied export markets for carrier stallions or EAV-infective semen; and reduced export markets for fillies, mares, colts, geldings and non-carrier stallions which are EAV seropositive. An outbreak of EVA at a racecourse, equestrian event or sale can have considerable impact. Such occurrences can result in direct financial losses through disruption of training schedules, reduced entries and even fixture cancellations. At the international level, EVA continues to have a significant effect on trade in horses and semen, with all the major horse breeding countries, except the USA, denying entry to EAV carrier stallions and EAV-infective semen.

During the past several decades, there have been significant advances in characterization of the molecular biology of EAV including identification of neutralization and virulence determinants of the virus, the molecular epidemiology of EAV infection, and host-pathogen interactions including the genetic basis of persistent infection in stallions and the cellular and molecular basis of EAV persistent infection in the stallion reproductive tract. The authors of this chapter have authored or co-authored a significant number of recent research and review articles on EAV and EVA^{2, 5, 17-19, 21, 36-41, 161, 162} and, thus, some of the published material is reused in this book chapter, and the relevant references are cited in the text. The readership is encouraged to read the original publications and citations therein for more information.

Aetiology

Equine arteritis virus is an enveloped, positive-sense, single-stranded RNA (ssRNA) virus that belongs to the family Arteriviridae (genus: Equartevirus, order: Nidovirales), which includes lactate dehydrogenase-elevating virus (LDV), simian hemorrhagic fever virus (SHFV), porcine reproductive and respiratory syndrome virus (PRRSV), wobbly possum disease virus (WPDV) and African pouched rat arterivirus (APRAV-1).^{43, 62, 105, 155, 167} The ssRNA genome of EAV strains varies between 12,704 to 12,731 bp, and includes a 5¢ leader sequence (224 nucleotides), at least ten open reading frames (ORFs) and a 3¢- untranslated region followed by a poly (A) tail.^{9, 15, 62, 130, 165, 166, 199, 213} The two most 5¢-proximal ORFs (1a and 1b) occupy approximately 75%  of the genome. The replicase ORFs 1a and 1b are translated to produce polyproteins pp1a (1725 amino acids) and pp1ab (3176 amino acids; based on the published sequence of the EAV VBS strain [ATCC VR-796], GenBank accession number DQ846750), with the latter being a C-terminally extended version of the former.⁶² The translation of ORF 1b depends on a -1 ribosomal frameshift just before termination of ORF 1a translation.⁶² These two precursor polyproteins are extensively processed after translation into at least 13 nsps (nsp1-12, including nsp7 α/β) by three viral proteases (nsp1, nsp2, and nsp4).^{24, 60, 165, 168-172, 194-197, 200, 201, 203, 214} The remaining eight ORFs (2-7) are located in the 3’ quarter of the genome and encode seven envelope proteins (E, GP2, GP3, GP4, ORF5a protein, GP5, and M) and a nucleocapsid (N) protein, respectively encoded by ORFs 2a, 2b, 3-4, 5a, 5b, and 6-7.^{57, 69, 166, 167, 207} These structural proteins are expressed from six subgenomic viral messenger RNAs (sg mRNAs) that form a 3¢-co-terminal nested set and contain a common leader sequence encoded by the 5¢-end of the genome.⁶¹

EAV particles have a spherical diameter of 40 to 60 nm and consist of a genome-containing nucleocapsid core (25 to 35 nm in diameter) composed of the N protein that is surrounded by a relatively smooth lipid envelope containing the envelope proteins.^{57, 62, 64, 69, 93, 94, 111, 165-167, 207} The non-glycosylated membrane protein M and glycosylated GP5 protein are present as disulfide-linked heterodimers in the mature virus particles.

Heterodimerization of the GP5 and M proteins is critical for the authentic post-translational modification (glycosylation) and conformational maturation of the neutralization determinants in GP5.^{6, 14} Four distinct neutralization determinants of the virus have been identified in GP5: aa 49 (site A), 61 (site B), 67 to 90 (site C), and 98 to 106 (site D).^{12-14, 44, 63, 76, 145, 211} Site D expresses several overlapping linear epitopes in the protein that may possibly interact with the three other sites to form conformational epitopes.^{6, 12, 13, 76} The M protein may act as an essential scaffold on which the GP5 protein folds to form the conformational epitopes that induce neutralizing antibodies in infected animals. The GP2, GP3, and GP4 minor envelope glycoproteins are covalently associated and form a heterotrimeric complex on the surface of the virion.^{57, 204-207} The major envelope proteins (GP5 and M) and the N protein are essential for EAV particle formation, whereas neither E protein nor the minor envelope proteins are required for the production of viral particles since the absence of any of these proteins does not inhibit incorporation of viral genomic RNA.¹³² In contrast, with the exception of the ORF5a protein, all major (N, GP5, and M) and minor (E, GP2, GP3, and GP4) structural proteins are required for the production of infectious progeny virus.²⁰⁷ Furthermore, it has been shown that elimination of ORF5a protein expression cripples EAV, leading to production of progeny virus with a small plaque phenotype and a significantly reduced virus titer.⁶⁹ Equine arteritis virus possesses a complement-fixing antigen but no haemagglutinin.¹⁸¹

There is only one serotype of EAV and all strains evaluated thus far are neutralized by polyclonal antiserum raised against the prototype virulent Bucyrus strain (VBS).^{3, 6, 13, 14, 23, 44, 63, 70, 76, 181, 213} However, field strains of EAV are frequently distinguished on the basis of differences in their neutralization phenotype with polyclonal antisera and monoclonal antibodies and, similarly, geographically and temporally distinct strains of EAV can differ in the severity of the clinical disease they induce and in their abortigenic potential.^{13, 20, 22, 124, 125, 136, 137, 148, 181} Experimental infectivity studies have identified lentogenic, mesogenic and velogenic strains of EAV.¹²⁴ Although many carrier stallions shed virus of low inherent pathogenicity, there is evidence that specific viral variants present in the semen of some persistently infected stallions have been responsible for initiating outbreaks of EVA.²² In contrast to the heterogenic viral population that can exist in the semen of carrier stallions, the viruses that circulate during an outbreak of EAV infection are genomically relatively homogenous.²²

EAV readily infects cells from equine origin such as endothelial cells, fibroblasts, monocytes, macrophages and a subpopulation of CD3⁺ T lymphocytes. Although EAV is highly species-specific, it can infect a variety of mammalian cells in vitro, which include rabbit kidney 13 (RK-13), baby hamster kidney (BHK-21), African green monkey kidney (Vero) and HeLa cells. In contrast, mouse connective tissue cells (L-M [ATC CCL-1.2], human epidermoid larynx cells (Hep-2 cells [ATCC CCL-23]), and human embryonic kidney cells (HEK-293T [ATCC CRL-3216]) are resistant to EAV infection.²¹² EAV is readily inactivated by lipid solvents (e.g., ether and chloroform) and by common disinfectants and detergents.  The virus is highly heat labile, and its half-life progressively decreases with increasing temperatures.  EAV remains infectious for 75 days at 4 °C, between 2 to 3 days at 37 °C, and 20 to 30 minutes at 56 °C.  EAV-infected tissue samples, tissue culture fluid, semen samples, and embryos can be stored frozen (-70 °C to -80 °C) for more than six decades without any significant loss of virus infectivity (Balasuriya and Timoney, unpublished data). The virus can also remain viable in organ samples stored at -20 °C for more than five years.¹¹⁹ EAV is highly susceptible to inactivation by UV light.

Epidemiology

A range of viral, host and environmental factors are involved in the epidemiology of EVA.^{178, 181} The most significant of these are: variation in pathogenicity and other phenotypic characteristics among naturally occurring strains of EAV, routes of virus transmission during acute and chronic phases of the infection, host genetics associated with the establishment and maintenance of the carrier state in the stallion, nature and duration of acquired immunity to infection, and changing trends in the horse industry.

Consideration has been given to the potential for equine species other than horses to serve as a reservoir of EAV. Specifically, evidence of EAV infection has been found in the donkey populations of many countries including South Africa, Chile, Argentina and Brazil.^{149, 151}  Studies in South Africa confirmed a high seroprevalence of EAV infection in donkeys in some areas of the country and close genomic homology between asinine and horse isolates of the virus.^{151, 153} However, experimental studies with a donkey strain of EAV showed that the virus was of very low transmissibility and pathogenicity for horses.¹⁵⁰ This finding, and the fact that attempts to achieve lateral transmission from experimentally infected donkeys to horses were unsuccessful, indicates that donkeys are unlikely to play a significant epidemiological role as reservoirs of EAV for horses.

The results of various serological surveys conducted over the years have confirmed that EAV has a worldwide distribution primarily in domestic equine populations in North and South America,^{118, 122, 143} Europe,^{56, 98, 135} Australia,⁹⁶ Africa,^{135, 152}   and various countries in Asia.^{104, 112, 181}  Other countries, such as Iceland, Japan, and New Zealand, are currently and/or historically free of the disease.^{96, 104, 128} Outbreaks of the EVA have also been reported from multiple countries including Switzerland, Austria, Poland, Italy, the United Kingdom, Ireland, Spain, the Netherlands, Canada, the United States, and Argentina.^{1, 20, 30, 32, 34, 50, 53, 68, 73, 86, 90, 91, 102, 118, 122, 124, 133, 144, 147, 173, 181, 202, 209} Interestingly, EAV seroprevalence varies between countries and between horses of different breeds and ages within the same country.^{139, 181, 183} In the United States, a very high percentage of adult Standardbred and Saddlebred horses are seropositive for EAV (70%-90% and 8%-25%, respectively), whereas the seroprevalence in the Thoroughbred population is very low (<5.4%).^{118, 127, 129, 179, 181} The 1998 National Animal Health Monitoring System (NAHMS) equine survey showed that only 0.6% of the US American Quarter Horse (AQH) population was seropositive to EAV,^{139, 140} but the extensive multistate EVA occurrence in the  United States during 2006-2007 has probably increased the seroprevalence within this breed.²¹³ The seroprevalence in Warmblood stallions is also very high in several European countries, with some 55% to 93% of Austrian Warmblood stallions being seropositive to EAV.^{31, 135} Similarly, there is a high seroprevalence among mares and stallions of Hucul horses in Poland (53.2% and 68.2%, respectively),¹⁶⁰ and a recent study performed in a population of Spanish purebred horses demonstrated a seroprevalence of 17.3%.⁵⁵ Seroprevalence of EAV increases with age, indicating that horses may be repeatedly exposed to the virus during their lifespan. Differences in the breed-specific seroprevalence of EAV infection may reflect genetic differences that confer susceptibility of stallions to becoming persistently infected carriers which favours the circulation of the virus in the farms.⁷⁸ In addition, several studies have identified a correlation between the number of breeding mares and EAV seropositivity suggesting virus circulating in the farm.^{55, 181}

The most important means of transmission of EAV are via the respiratory route from  acutely infected animals^{65, 121, 122} and via the venereal route by acutely or chronically infected stallions.^{173, 185} Horizontal transmission occurs through the respiratory route after aerosolization of viral particles in respiratory secretions originating from acutely infected horses and is facilitated by direct and/or close contact between an infected and a naïve horse. Nasal shedding of EAV frequently lasts 7 to 14 days (or up to 16 days) during the acute phase of the disease, with viral titers ranging from 10 - >2x10³ plaque-forming units per ml (PFU/ml).^{16, 121, 126} EAV can also be aerosolized from urine and other body secretions of acutely infected horses, aborted fetuses, and fetal membranes. Aerosolization is the principal means of virus dissemination during outbreaks at racecourses, sales, shows and veterinary hospitals where horses are closely congregated together. It has been demonstrated that EAV is shed in feces of experimentally infected stallions up to 8 days postinfection,¹⁴² and the virus is also present in the female reproductive tract for a brief period after infection.¹²⁶

The venereal mode of transmission occurs exclusively via semen from acutely or chronically infected stallions (carrier stallions) during natural or artificial breeding.^{173, 185} Following infection, 10 to 70% of EAV infected stallions become persistently infected and continuously shed virus in their semen for a variable and frequently extended period of time, ranging from several weeks to years’ post-infection or even life-long, despite the presence of high levels of neutralizing antibodies in serum.^{181, 185, 187} Thus, carrier stallions play a major epidemiological role since they constitute the natural reservoir for EAV and, thus, are responsible for the maintenance, perpetuation, and evolution of EAV in equine populations between breeding seasons.^{8, 9, 22, 89, 130, 179, 181, 213} Furthermore, the carrier stallion plays a major role in the venereal transmission of the virus whether mares are bred by live cover or artificial insemination.^{181, 188} Dissemination of EAV can also occur through the use of infective fresh-cooled or frozen semen.^{20, 87, 175} The increasing prevalence of EVA¹⁷⁴  is related in most instances to the movement of infected horses or use of virus-infective semen.^{87, 88, 189}

Persistent EAV infection (carrier state) can occur in the stallion or pubertal colt but not in the mare, gelding, filly or sexually immature colt.^{92, 120, 181, 187} Carrier stallions are the primary reservoirs of EAV and are of major epidemiological importance in the dissemination and persistence of the virus in equine populations around the world.¹⁸⁸ Experimental studies have demonstrated that establishment and maintenance of the carrier state is testosterone-dependent.¹⁰⁶ It has been shown that persistently infected stallions that are castrated cease shedding virus in semen, whereas those supplemented with testosterone after castration continue to shed virus.¹⁰⁶ Carrier stallions show no clinical signs of disease nor is their fertility adversely affected.^{33, 39} Stallions may become short-term (duration of viral shedding in semen £ < 1 year) or long-term (duration of viral shedding in semen > 1 year) carriers of EAV.¹⁸¹ Long-term persistence of EAV is common among carrier stallions, in some instances, which may last for many years or life-long. A variable number of carrier stallions spontaneously eliminate the virus from their reproductive tract and are no longer a source of infection. EAV is constantly shed in the semen (sperm-rich fraction) of carrier stallions.¹⁸⁵ While the virus infectivity titre in semen can vary among stallions, the results of one study have shown that more than 83 percent of samples tested had infectivity titres of 3 - >7 log of EAV per 1.0 ml of seminal plasma.¹⁸⁴

Transmission of the virus by carrier stallions occurs only by the venereal route, with infection rates in mares as high as 85 to 100%. Most of the seronegative mares naturally or artificially bred to carrier stallions become infected and seroconvert within 28 days.^{20, 126, 186} Infected mares may develop clinical signs of EVA; irrespective of clinical status, acutely infected mares can readily transmit the virus by the respiratory route to susceptible cohorts in close proximity and can rapidly initiate an EVA outbreak.  The virus can also be transmitted from donor mares inseminated with EAV-infective semen to naïve recipient mares via embryo transfer²⁷ and  across the placenta from an infected mare to her unborn foal.¹⁹⁰ In such instances, the foetus, foetal fluids, placenta and placental fluids constitute important sources of virus. Furthermore, congenital infection of foals following transplacental transmission of the virus in mares infected in late gestation can occur, and congenitally infected foals develop a rapidly progressive, fulminating interstitial pneumonia and fibrinonecrotic enteritis.^{35, 59, 85, 190, 208} Lateral transmission of EAV can also occur through contaminated fomites (e.g., personnel, clothing, vehicles, tack, and equipment such as artificial vaginas and phantoms).^{88, 173, 179, 181}

Several studies confirm  that EAV carrier stallions are a source of genetic and phenotypic divergence of the virus, i.e., EAV evolves in the reproductive tract of carrier stallions causing the emergence of novel viral variants with neutralization phenotypes that allow immune escape and which can lead to outbreaks of EVA.^{8, 9, 22, 89} Recent studies on sequential semen isolates from long-term carrier stallions have shown non-stochastic EAV evolution during persistent infection was driven by active intra-host selective pressure.¹³⁸ Specifically, open reading frame (ORF)3, ORF5 and the nsp2 encoding region of ORF1a were preferentially targeted, while ORF3 and ORF5 had the highest intra-host evolutionary rates. Moreover, amino acid substitutions at multiple codon positions of ORF3 demonstrated phenotypic convergence of variants during persistent infection in stallions. During persistent infection in the stallion reproductive tract, the EAV tends to evolve at a rate of 4 x 10^-4 nucleotide substitutions/site/year.

Pathogenesis

The majority of the field strains of EAV are avirulent or cause mild to moderate respiratory disease in horses.^{146, 158, 181} Thus, the clinical signs and lesions caused by the horse-adapted highly virulent Bucyrus strain (VBS) of EAV are not representative of the disease caused by field strains of the virus in circulation during recent  decades. The pathogenesis of EVA has been studied in both natural outbreaks and horses experimentally infected with different viral strains.^{7, 10, 52, 54, 58, 100, 109, 116, 117, 121, 124, 125} Original studies suggested initial virus replication within 24 hours in the respiratory epithelium and bronchial and alveolar macrophages. However, recent ex vivo (i.e., mucosal explants) and in vitro studies to elucidate the early events in the pathogenesis of EAV infection demonstrated that the virus localizes in CD3⁺ T lymphocytes, CD172a⁺ myeloid cells, and a small population of IgM⁺ B lymphocytes present  in  connective tissue.¹⁹² Interestingly, the virus was not detected in epithelial cells from the upper respiratory tract during the early phase of infection.^{191, 192} These studies have shown that the nasopharynx and tubal-nasopharyngeal tonsils play an important role as primary sites of EAV replication during early infection. By 48 hours post-infection, the virus can be detected in the satellite lymph nodes, especially the bronchial lymph nodes. Within three days of infection, the virus spreads from the bronchial lymph nodes to induce the development of a leukocyte-associated viraemia,¹⁹³ by which the virus becomes distributed in various body fluids and tissues.⁵⁸ At approximately six to eight days post-infection, the virus has localized in the endothelium and medial myocytes of blood vessels. Development of the characteristic vascular lesions associated with EAV infection is evident initially in the blood vessels of the lungs in animals exposed by the respiratory route, and then in the small arteries and veins throughout the body. Endothelial damage and lymphocytic infiltration of vessel walls result in damage to the internal elastic lamina of arterioles. The vascular lesions give rise to oedema and haemorrhage in many tissues and organs. Most severe damage to the vasculature is evident about 10 days after infection, following which the lesions begin to resolve. Viral replication also occurs in various organs, especially the adrenal and thyroid glands, liver, and testes. With the exception of the reproductive tract in colts and stallions that become carriers,¹⁴²  infectious virus is no longer detectable in most body fluids and tissues after 28 days post-infection.

It appears that EAV strains differ in their abortigenic potential, as they do in their virulence characteristics. EAV-induced abortions can occur in pregnant mares at any time between 2 and 10 months of gestation without premonitory signs. Abortion in pregnant mares may occur late in the acute phase or early in the convalescent phase of infection and results in the expulsion of fetuses that are usually partially autolyzed. The abortion rates during natural outbreaks of EVA can vary from <10->70%.^{28, 29, 50-52, 123, 181} EAV is vertically transmitted to the fetus as evidenced by the high viral titers and abundance of viral antigen in diverse fetal tissues including placenta; specifically,  trophoblastic, amniotic, and allantoic epithelium, chorioallantoic mesenchyma, pneumocytes, alveolar macrophages, thymic epithelium and lymphocytes, splenic reticular cells and mononuclear cells in the white and red pulp, renal interstitial cells and tubular epithelium, extramedullary hemopoietic cells in the liver and rare hepatocytes, endothelial cells, vascular smooth muscle cells and enterocytes.^{40, 58} It is speculated that abortion occurs as a consequence of vasculitis of myometrial blood vessels and myometrial necrosis, which leads to placental dysfunction and chorionic detachment.^{51, 58, 109, 110} Furthermore, congenital infection of foals following transplacental transmission of the virus in mares infected in late gestation can occur and results in congenitally infected foals that develop a rapidly progressive, fulminating bronchointerstitial pneumonia and fibrinonecrotic enteritis.¹⁹⁰

Recently, in vitro studies using equine peripheral blood mononuclear cells demonstrated that EAV cannot infect CD14⁺ monocytes but, most importantly, it can infect a subset of CD3⁺ T lymphocytes in horses that carry a specific haplotype of the C-X-C chemokine ligand 16 (CXCL16) gene (see below). Also, in vitro studies in equine macrophages and endothelial cells have demonstrated increased transcription of genes encoding proinflammatory mediators (interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor α [TNF α]) following EAV infection, suggesting that these cytokines are critical in determining the severity of the disease.¹³⁴ It has been also demonstrated that experimental inoculation of horses with in vitro CD3⁺ T lymphocyte susceptible or resistant phenotype could express different levels of proinflammatory and immunomodulatory cytokine mRNAs and, furthermore, horses possessing the in vitro CD3⁺ T lymphocytes resistant phenotype tend to develop more severe clinical signs compared to the horses that had CD3⁺ T lymphocyte susceptible phenotype.⁸¹

EAV has the capacity to establish persistent infection in the reproductive tract of the vast majority of infected stallions. Interestingly, persistent EAV infection does not involve immunologically privileged tissues (i.e., testes) and the ampullae are the main tissue reservoir for EAV infection, among other accessory sex glands.^{39, 106, 142} While EAV is present in other accessory glands (vesicular, prostate, and bulbourethral glands), the number of infected cells is considerably lower than in the ampullae and, thus, these other accessory glands are best regarded as minor viral reservoirs. Recently, there has been a significant amount of research focused on understanding the pathogenesis of persistent EAV infection in the stallion reproductive tract. These studies suggested that there is a genetic difference between stallions that become long-term carriers and those that clear the virus from the reproductive tract shortly after infection.⁸¹ Subsequently, this trait was linked to the susceptibility of CD3⁺ T lymphocytes to in vitro EAV infection, and it was demonstrated that those stallions with an in vitro CD3⁺ T lymphocyte susceptible phenotype are at a higher risk of becoming long-term carriers after EAV infection than those that have a resistant one.^{78, 80, 83} Further studies⁷⁹ demonstrated that long-term persistence is strongly associated with a specific allele of the CXCL16 gene (i.e., CXCL16S) and that CXCL16S acts as an entry receptor for EAV.^{161, 162} During persistent infection, EAV specific cellular tropism involves vimentin+ stromal cells (including fibrocytes and possibly tissue macrophages) and CD8⁺ T and CD21⁺ B lymphocytes but not the glandular epithelium in the ampullae or other accessory sex glands. Furthermore, there is a moderate to severe local inflammatory response in the ampullae of long-term EAV carrier stallions.  Extensive characterization of the local inflammatory response determined that persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21⁺) lymphocytes and significant infiltration of T (CD3⁺, CD4⁺, CD8⁺ and CD25⁺) lymphocytes, tissue macrophages and dendritic cells (Iba-1⁺ and CD83⁺), diverse Ig-secreting plasma cells, with a low number of tissue macrophages expressing CD163 and CD204 scavenger receptors.^{39, 41} Lymphocyte infiltration mainly involved CD3⁺CD8⁺ T cells, with moderate numbers of CD4⁺CD25⁺FOXP3⁺ T lymphocytes.³⁶ Even though glandular epithelial cells of the ampullae were not infected, there was an intimate association of EAV infected intra- and subepithelial lymphocytes with the reproductive epithelium.³⁹ This close association suggests that infectious virus may gain access to seminal fluid via these infected lymphocytes simply migrating across the glandular epithelium. Furthermore, EAV was not detectable in lymphoid tissues including those associated with lymph drainage from the reproductive tract, suggesting that infected T and B lymphocytes exhibit a specific homing pattern to the reproductive tract and restricted migration from reproductive tract tissues to secondary lymphoid organs. Finally, EAV also induces a mucosal antibody response in the reproductive tract with shedding of virus-specific immunoglobulins (IgA, IgM, IgG1, IgG3/5 and IgG4/7) into the seminal plasma as well as homing of plasma cells in the accessory sex glands.⁴¹ However, despite the systemic neutralizing antibody, local inflammatory, and mucosal antibody responses, EAV evades local host immunity mechanisms, and persistence is maintained. The molecular mechanisms and the role of CXCL16S in the maintenance of persistent infection in the reproductive tract are currently under extensive investigation.

The transcriptional profile analysis of CD8⁺ T lymphocyte from the ampullae of carrier stallions has shown that EAV long-term persistent infection is associated with upregulation of T-cell exhaustion-related transcripts and homing chemokines/chemokine receptors (CXCL9-11/CXCR3 and CXCL16/CXCR6), which may be orchestrated by a specific subset of transcription factors (EOMES, PRDM1, BATF, NFATC2, STAT1, IRF1, TBX21) which are associated with the presence of the susceptibility allele (CXCL16S).³⁶ Moreover, it has been shown that long-term EAV persistence is associated with the downregulation of a specific seminal exosome-associated miRNA (eca-mir-128) along with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128.³⁷ This is the first evidence that this miRNA plays a crucial role in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. Thus, there is ample evidence to suggest that complex host-pathogen interactions shape the outcome of EAV infection in the stallion reproductive tract and that EAV employs complex immune evasion mechanisms favouring long-term persistence.

The innate immune response of the mucosal lining of the respiratory and genital tracts constitutes the first line of defense that EAV encounters following natural exposure.  EAV inhibits type I IFN production in infected cells, and this may allow the virus to subvert the initial equine innate immune response⁸² in contrast,  EAV infection in horses induces long-lasting humoral immunity that likely protects against reinfection with most if not all strains of the virus.^{66, 114} The humoral immune response to EAV is characterized by the development of both complement-fixing and virus-specific neutralizing antibodies.^{70, 71, 117} Complement-fixing antibodies develop 1 to 2 weeks after infection, peak after 2 to 3 weeks, and steadily decline to disappear by 8 months, whereas virus neutralizing antibodies are detected within 1 to 2 weeks after exposure, peak at 2 to 4 months, and persist 3 years or more. The appearance of neutralizing antibodies coincides with the disappearance of virus from the circulation of infected horses. However, virus persists in the reproductive tract of the carrier stallion for a variable period despite the presence of high titers of virus neutralizing antibodies in serum. Recent studies demonstrated that the neutralizing antibody response following EAV infection is mediated by only two virus-specific immunoglobulin isotypes, namely short-lived IgM and long-lived IgG1.⁴¹

Foals born to mares that have been immunized against EVA are protected against the disease through colostrum-derived antibodies.¹¹⁵ The passively acquired virus neutralizing antibodies appear a few hours after colostrum feeding, peak at 1 week of age, and gradually decline to extinction between 2 to 6, and rarely 7, months of age.^{95, 115} The cell-mediated immune response to EAV still remains poorly characterized.⁴²

Clinical signs

The clinical outcome following exposure to EAV infection is variable and influenced by a variety of virus, host and environmental factors.¹⁸⁰ Most importantly, the majority of naturally infected horses develop no signs of disease and can be considered asymptomatic cases of infection.^{73, 98, 173} EVA can affect horses of any age, although the severity of the disease is likely to be greater in very young or old horses and in debilitated animals.¹⁸¹  The incubation period of 2-14 days (usually 6-8 days following venereal exposure) is followed by pyrexia of up to 41°C that may persist for 2-9 days.^{2, 4, 39} Fever and leukopenia are often the only clinical signs of infection observed. Clinical signs vary in range and severity, and affected animals may exhibit some or most of the following: fever, depression, anorexia, leukopenia, oedema most notably of the limbs, scrotum, prepuce or mammary glands, or other dependent parts of the body, nasal discharge (initially serous becoming mucoid later), conjunctivitis, which may be accompanied by photophobia, epiphora and a variable degree of supraorbital/periorbital oedema, stiffness in gait, skin rash (often localized to the cheeks or sides of the neck but sometimes generalized), abortion and, infrequently, pneumonia or pneumo-enteritis in very young foals. Colic has been infrequently observed in affected foals.^{59, 77, 85, 125} Less commonly described signs include respiratory distress, coughing, diarrhoea, posterior paresis or ataxia, buccal erosions, papular eruptions beneath the mucous membrane lining the interior of  the upper lip, mandibular lymphadenopathy and oedematous swelling beneath the jaws, the sternum or in the shoulder region.^{65, 67, 133, 174}

EAV-induced abortions occur late in the acute phase or early in the convalescent phase of the infection and not, as is sometimes incorrectly reported, months after viral exposure has taken place.^{65, 84, 123} Abortion can be a sequel to clinical or asymptomatic infection with EAV. Abortion is frequently the result of co-mingling a previously uninfected or unvaccinated mare that has very recently been bred with virus-infective semen with one or more pregnant mares. Mares infected with the virus do not experience any short- or long-term virus-related fertility problems.¹⁸¹

Stallions acutely affected with EVA may experience a period of temporary subfertility that can last for up to six to eight weeks.¹⁴¹ Reduced libido has been observed in association with decreased sperm motility and concentration and an increased percentage of morphologically abnormal sperm. No long-term adverse effects on fertility, however, have been reported in recovered stallions or in stallions persistently infected with EAV.^{33, 141, 181}

Aside from abortion and the infrequently encountered fatal cases of EAV infection in very young foals,^{35, 59, 65, 84, 85, 123, 125, 190} the vast majority of horses  that develop EVA make complete and uneventful clinical recoveries, with or without symptomatic treatment.¹⁸¹ Horses in training can experience a period of impaired performance during the acute and early convalescent phases of the infection, but this is short-term in duration.¹⁶³

Pathology

Mortality in adult horses following natural EAV infection is extremely rare.  Unfortunately, many of the gross and histopathological lesions described in the literature are as a result of experimental infection of horses with the highly virulent horse-adapted Bucyrus strain of EAV (VBS; which causes high mortality in healthy adult horses) and   from infection with naturally-occurring, field strains of EAV. The lesions caused by EAV VBS are reflective of the vascular damage caused by the virus, and characterized by oedema, congestion, and haemorrhage of the subcutaneous tissues, lymph nodes and viscera throughout the body.^{58, 101, 157} Body cavities may contain moderate to copious quantities of fluid exudate.⁵⁸ Congestion, oedema, and haemorrhages in the caecum and small and large colon and regional lymphadenomegaly may be present. Haemorrhagic enteritis, especially of the caecum and colon, may be observed. The extent and severity of similar lesions caused by field strains of EAV vary depending on the virulence of the virus strain involved. Aborted fetuses may be delivered autolyzed or non-autolyzed, and most natural cases of EVA-related abortion present with no gross lesions.^{51, 52} However, some may exhibit a variable degree of interlobular pulmonary oedema, pleural and pericardial effusion and petechial and ecchymotic hemorrhages on the serosal and mucosal surfaces of the small intestine.¹⁶⁷ The fact that autolytic changes are present in the foetus in most cases of EVA-related abortion confirms that foetal death occurs in utero and that this, in conjunction with the viral-mediated damage to the placental vasculature, may provide the signal for premature placental separation and expulsion of the foetus. There are typically no characteristic histologic features of EAV infection in the fetus and foetal membranes,^{51, 52} but there have been exceptions, in which severe necrotizing panvasculitis of small vessels in the placenta, brain, liver, spleen, and lungs has been reported.^{40, 51, 52, 58, 59, 99, 107, 110} Affected muscular arteries show foci of intimal, subintimal and medial necrosis, with oedema and infiltration of lymphocytes and neutrophils.^{51, 58, 109} Despite the lack of significant gross and histologic lesions, abundant viral antigen can be detected in several tissues including placenta and other fetal membranes and fetal organs.⁴⁰

A diffuse, severe, acute interstitial pneumonia with excess fluid in the thoracic cavity, together with oedema and multifocal serosal and mucosal haemorrhages of the small intestine have been reported in fatal cases of EVA in young foals.^{54, 58, 59} The microscopic lesions in young foals are characterized by congestion, interlobular oedema and mononuclear cell infiltration in the lungs, moderate lymphoid depletion and haemorrhage in the thymus, spleen and bronchial, mediastinal and mesenteric lymph nodes, and in cases in which there is an associated enteritis, focal areas of haemorrhage and necrosis of the mucosa in the small intestine.^{58, 59, 85}

Diagnosis

It is not possible to establish a diagnosis of EVA based solely on the nature of clinical signs.¹⁸³ EVA clinically mimics a number of other infectious and non-infectious diseases (see below). Confirmation of a diagnosis is based on laboratory testing and includes virus isolation (VI), detection of viral nucleic acid or viral antigen and/or demonstration of a specific neutralizing antibody response by testing paired (acute and convalescent) sera taken at a 21- to 28-day interval.¹⁸¹ The blood samples for serological diagnosis should be collected in separate tubes and kept refrigerated during transit to the laboratory. If it cannot be submitted immediately, serum should be separated and stored at -20 °C.

Appropriate specimens for detection of virus by VI and detection of viral nucleic acids by reverse-transcription polymerase chain reaction (RT-PCR) based methods (e.g. standard RT-PCR, real-time RT-PCR [RT-qPCR], or reverse transcription insulated isothermal PCR [RT-iiPCR]) from acutely infected horses include nasopharyngeal swabs or washings, conjunctival swabs, and citrated or EDTA blood samples.^{11, 25, 38, 108, 131, 181} The blood samples should not be collected in tubes containing heparin for VI and RT-PCR based methods. Swabs should be placed in a suitable viral transport medium and kept refrigerated or frozen during transit to the laboratory. Blood samples in anticoagulants should be kept refrigerated (not frozen) during transportation to the laboratory.

In suspect cases of EVA-related abortion, VI/detection nucleic acids can be attempted from placental and foetal fluids, and foetal lymphoreticular and other tissues, especially foetal lung and liver.^{75, 181} When a suspected outbreak of EVA is associated with mortality in young foals or older horses, specimens of a wide range of tissues, especially the lymph nodes in the thoracic and abdominal cavities and their related organs, should be collected for VI, detection of viral nucleic acids and histopathological examination. The snap-frozen or formalin fixed tissues can be used to demonstrate viral antigens by immunohistochemical staining (IHC) or to demonstrate viral nucleic acids by traditional in situ hybridization (ISH) or contemporary RNAscope®.⁴⁰

Detection of the carrier state in the stallion initially involves the determination of EAV serological status.¹⁸⁵ Only seropositive stallions (neutralizing antibody titre of 1:4 or greater) without a certified history of vaccination against EVA need to be considered potential carriers of the virus. A sample of semen containing the sperm-rich fraction of the ejaculate should be collected and screened virologically either by VI in susceptible cell lines or viral nucleic acid detection by RT-PCR based techniques (e.g., RT-qPCR).^{25, 74} Care should be exercised to ensure that VI/detection of nucleic acids should only be attempted from the sperm-rich fraction of the ejaculate since testing the pre-sperm fraction will very likely yield a spurious negative result. Presence of the carrier state can also be determined by test breeding a stallion to two seronegative mares and monitoring the latter for seroconversion up to 28 days after breeding.¹⁸⁶

Although not always successful in natural cases of EAV infection,^{73, 122} VI should be attempted from specimens using rabbit, equine or monkey kidney cell cultures in accordance with established procedures.¹⁷⁷ The cell system of choice is the rabbit kidney cell line (RK-13). While the majority of isolations of EAV are made in the first or second passage,¹⁸⁵ there have been a few reported instances where additional blind passages in cell cultures were required.⁸⁵

Serological diagnosis of EVA is based on the OIE-prescribed complement-enhanced microneutralization test. EAV infection can be demonstrated by a rise in neutralizing antibody titers (4-fold or greater) in paired sera collected 21-28 days apart as indicated previously.¹⁶⁴ It is a sensitive and highly specific assay of proven reliability in diagnosing acute infections and in seroprevalence studies. A variety of serological tests, including neutralization, complement fixation, indirect fluorescent antibody, agar gel immunodiffusion and ELISA have been used for the detection of antibodies to EAV and their isotypes.^{41, 177} The complement fixation test is less sensitive but can be used to diagnose recent infection. Several ELISAs have been developed, some of which appear to offer comparable sensitivity and specificity to the neutralization test.^{45, 97, 103} A competitive enzyme-linked immunosorbent assay (cELISA) was recently developed and validated for the detection of EAV specific antibodies, and could be considered as an alternative assay for the serological diagnosis of EVA.^{46-48, 154}

Differential diagnosis

A range of viral and bacterial respiratory diseases that commonly affect horses cannot readily be distinguished clinically from EVA.¹⁸¹ Of particular importance are equid herpesvirus 1 and 4 infections, equine influenza, and streptococcal infections with specific reference to purpura haemorrhagica. Other infectious diseases that can cause systemic signs of illness resembling EVA are equine infectious anaemia, dourine, African horse sickness, and Getah virus infection. Equine viral arteritis also bears many clinical similarities to the syndrome caused by hoary alyssum (Berteroa incana) toxicosis.⁷² Abortions or deaths in neonatal foals due to EAV need to be differentiated in particular from those caused by equid herpesvirus 1 as well as other infectious causes of abortion in the mare.

Control

There is no known effective antiviral therapy for the treatment of EAV infected horses.¹⁸³ Although the vast majority of natural cases of EVA completely recover without symptomatic therapy; antipyretic, anti-inflammatory and diuretic treatments are recommended in severe cases of the disease associated with high fever and severe dependent oedema, especially in stallions. Adequate sexual rest and treatment with nonsteroidal anti-inflammatory drugs and a diuretic are indicated in such cases. Currently, there are no means to eliminate the carrier state in the stallion other than surgical castration, which implies the loss of the commercial value of the stallion as a breeding animal. Experimental antiviral compounds have been evaluated for the treatment of carrier stallions,^{198, 210} but none of them can guarantee clearance of persistent infection in stallions or complete elimination of semen infectivity. There is no effective treatment of neonatal foals with bronchointerstitial pneumonia or the pneumoenteric syndrome other than antibiotic treatment to prevent secondary bacterial infections.

Two commercial vaccines are available against EVA. The first is a modified live virus (MLV) vaccine (Arvac®, Zoetis Animal Health Inc, Kalamazoo, MI, USA) that is commercially available in North America since 1985.^{66, 113, 117, 181} This MLV vaccine has been successfully used as part of the EVA control programmes in Kentucky, New York, New Mexico and other states in North America to curtail the spread of EAV during widespread outbreaks of viral arteritis. Extensive field use of this vaccine has confirmed its safety for use in stallions and nonpregnant mares as well as its immunogenicity.¹⁸¹ However, it is not recommended for administration to pregnant mares nor to foals less than six weeks of age unless under circumstances of significant risk of natural exposure to infection. Vaccination confers a high level of protective immunity that persists for at least several years.¹¹³

The second vaccine against EVA is an inactivated, adjuvanted tissue-culture-derived vaccine (Artervac®, Zoetis Animal Health, Kalamazoo, MI, USA).¹⁷⁷ It is currently available for use in Denmark, France, Ireland, and the UK. While safe for pregnant mares, field studies have shown that this vaccine is not strongly immunogenic. Two or more vaccinations are frequently required to stimulate a detectable neutralizing antibody response. The duration of immunity conferred by this vaccine is currently unknown.

It has been possible to develop effective programmes for the prevention and control of EVA based on what is known about the biology of the causal agent and epidemiology of the disease. Dissemination of EAV can be restricted by eliminating direct or indirect contact of susceptible horses with various secretions, excretions or tissues of infected animals and through immunization during outbreaks. The carrier stallion is the natural reservoir of the virus, and thus, EVA control and prevention measures are primarily based on the identification of persistently in stallions, and the vaccination of susceptible horses.

Current control programmes in the US are focused primarily on curtailing the spread of the virus in breeding horse populations in order to prevent outbreaks of EVA-related abortion and/or illness and death in young foals, and to minimize the risk of establishing the carrier state in the stallions.¹⁸³ These programmes are based on the major epidemiological role that the carrier stallion plays in maintaining the virus in various horse populations worldwide.¹⁸⁸ The guidelines for EVA prevention and control in breeding stallions and mares are described in details in Balasuriya and Carossino (2017) and Balasuriya et al., (2018).^{18, 19}

EVA can be effectively controlled on breeding farms through observance of sound management and biosecurity practices similar to those recommended for the prevention of equid herpesvirus and other respiratory virus infections, and the implementation of a selective vaccination programme against the disease. Crucial to the success of such programmes is the identification of any carrier stallions in a breeding stallion population.^{181, 183} Breeding stallions that are seronegative for EAV should be maintained on an annual vaccination programme against EVA to protect them from becoming carriers of the disease. This also eliminates the possibility of venereal transmission of EAV and the need for extra precautions when breeding mares that are considered to be high risk. Carrier stallions should be physically isolated and strict hygienic precautions observed when breeding or collecting semen from them to avoid the risk of inadvertent transfer of infection to other previously uninfected or unvaccinated horses on the premises. Under satisfactory conditions of breeding farm management, carrier stallions can continue to be used commercially subject to meeting certain safeguards, i.e., they should only be bred to naturally seropositive mares or mares that have been appropriately vaccinated against EVA. Immunization of stallions and sexually immature colts between 6 and 12 months of age provides a mechanism of preventing the establishment of the carrier state and reducing the natural reservoir of EAV in breeds in which the infection is widespread.^{92, 181} If rigorously implemented over a period of years, such a vaccination programme would lead to a significant reduction in the number of carrier animals and thereby largely eliminate the primary reservoir of EAV.

The risk of introducing EAV into a susceptible horse population through the use of infective fresh-cooled or frozen semen and frozen embryos can be considerable. Consequently, it is very important to establish the virus infectivity of semen used for artificial insemination, especially if imported from abroad.^{175, 189} The same precautions should be observed with mares inseminated with EAV infective semen or mares receiving contaminated frozen embryos as those recommended for mares bred by live cover. The risk of EVA-related abortion in mares can be minimized through observance of sound biosecurity measures. All horses returning from other farms, sales, a veterinary clinic or racecourse, or newly purchased animals should be isolated from the resident horses for four weeks. Pregnant mares should be segregated and maintained in small groups until they have foaled. Vaccination of non-pregnant mares against EVA is not routinely recommended unless under circumstances of high-risk exposure to infection. At the present time, certain countries still debar the entry of mares that are seropositive as a result of vaccination or natural infection with EAV. In light of such trade restrictions, wholesale vaccination of all categories of horses, including mares, should be subjected to careful evaluation. It is imperative to maintain good vaccination records for horses that are moving around the world for breeding and various equestrian events.

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