Equine picornaviruses: Well known but poorly understood

Veterinary Microbiology 167 (2013) 78–85

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Equine picornaviruses: Well known but poorly understood Jacquelyn Horsington a,1, Stacey E. Lynch b,1, James R. Gilkerson a, Michael J. Studdert a, Carol A. Hartley a,* a b

Centre for Equine Infectious Disease, Department of Veterinary Science, The University of Melbourne, Victoria 3010, Australia Department of Primary Industries, AgriBio Centre, LaTrobe University, Bundoora, Victoria 3083, Australia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 December 2012 Received in revised form 29 May 2013 Accepted 31 May 2013

Of the many members that comprise the family Picornaviridae, only two species are known to infect horses: equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV). Each species now occupies a distinct phylogenetic branch within the family, with the single serotype of ERAV grouping with the aphthoviruses, such as foot-and-mouth disease virus (FMDV), and the three serotypes of ERBV as the sole members of the genus Erbovirus. The high seroprevalence of equine picornaviruses in horse populations worldwide contrasts with the relatively few reports of detection of these viruses and poor understanding of their contribution to disease. This review examines the current knowledge regarding the distribution and pathogenesis of these viruses and discusses recent advances in diagnostic methods that may lead to a better understanding of the role of these viruses as contributors to equine respiratory disease. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Equine rhinitis virus ERAV ERBV Picornavirus Aphthovirus Erbovirus

1. Introduction Of the many members that comprise the family Picornaviridae, only two species are known to infect horses: equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV). The equine picornaviruses were first isolated in the 1960s (Plummer, 1962) and 1970s (Hofer et al., 1972) and despite similarities that initially saw them grouped together in the genus Rhinovirus, they separate into two distinct taxonomical groups. Both ERAV and ERBV are associated with respiratory disease in horses; however, their importance as equine pathogens and the specific roles they have in respiratory disease is still unclear. The clinical manifestations of ERAV and ERBV infection remain ill-defined, with isolates recovered from clinically healthy animals, as well as those with signs of respiratory disease (Carman et al., 1997; Flammini and Allegri, 1970;

* Corresponding author. Tel.: +61 3 8344 7375. E-mail address: [email protected] (C.A. Hartley). 1 Each author contributed equally to this work. 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.05.012

Fukunaga et al., 1983; Hofer et al., 1972; Li et al., 1997; Mumford and Thomson, 1978; Plummer and Kerry, 1962; Studdert and Gleeson, 1978). Their frequent detection in combination with other viral and bacterial pathogens suggests they may have a contributing role in enhancing disease length and severity. Equine respiratory disease is of particular importance in the performance horse industry, representing a major burden due to the additional veterinary bills, lost training time and decreased performance. The high prevalence of ERAV and ERBV in horse populations indicates that further studies are required to better understand the pathogenesis and epidemiology of these widespread viruses. The bulk of research on equine picornaviruses was performed in the 1960s–1980s, with more recent work mostly limited to studies of seroprevalence or detection by molecular methods. In more recent years, genome sequencing has resulted in a significant reclassification of the equine picornaviruses and some reassessment of the ERBV serotype groupings. This review aims to collate current knowledge of the fundamental features of the equine picornaviruses, including current approaches for

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their detection that may provide an insight into the pathogenesis of these viruses. 2. Taxonomy Historically, the equine picornaviruses were classified within the genus Rhinovirus, family Picornaviridae, due to their physical properties and their identification as respiratory pathogens (Ditchfield and McPherson, 1965; Fukunaga et al., 1981; Plummer, 1962; Steck et al., 1978b). Four distinct groups were identified based on serology and acid lability: equine rhinovirus-1, equine rhinovirus-2, equine rhinovirus-3 and acid-stable equine picornavirus. Following comparative genomic studies, considerable phylogenic differences between the equine rhinoviruses and other members of the genus Rhinovirus were identified (Li et al., 1996; Wutz et al., 1996; Huang et al., 2001), warranting the reclassification of the four equine picornaviruses into two distinct genera within the Picornaviridae. Equine rhinovirus-1, renamed ‘equine rhinitis A virus’ (ERAV), was reclassified into the genus Aphthovirus, a classification shared with foot and mouth disease virus (FMDV), the prototype aphthovirus (King et al., 2000). The remaining equine rhinoviruses were reclassified as serotypes of ‘equine rhinitis B virus’ (ERBV), the sole species of the newly created genus Erbovirus (King et al., 2000). The three ERBV serotypes differ in acid stability and include the acid-labile ERBV1 and ERBV2 (formally equine rhinovirus 2 and 3, respectively), and the acid-stable ERBV3 (formally acid-stable equine picornavirus). 3. Genomic organisation and function Picornaviruses have single-stranded, positive-sense RNA genomes between 8800 (in erboviruses) and 7209 (in human rhinoviruses) nucleotides long. The 30 polyadenylated genome contains a single open reading frame, flanked at the 50 and 30 terminal ends by untranslated regions (UTR) (reviewed by Racaniello, 2007). The 50 terminus is covalently bound to a small virally encoded protein (VPg) that acts as a protein primer for the initiation of RNA synthesis by the viral polymerase (Ferrer-Orta et al., 2006). The small region at the extreme 50 end of picornavirus genomes has a high degree of secondary structure and this region has not yet been sequenced for ERAV or ERBV (Huang et al., 2001; Li et al., 1996; Wutz et al., 1996). Following this is the highly ordered secondary structure of the internal ribosome entry site (IRES) which facilitates the internal 50 -terminal independent ribosome entry and cap-independent initiation of translation (Ehrenfeld and Teterina, 2002; Pelletier et al., 1988). The equine picornaviruses have a type II IRES, where multiple initiation codons lie adjacent to the IRES elements, facilitating the initiation of polyprotein translation from either site (Hinton et al., 2000; Li et al., 1996; Wutz et al., 1996). Consequently, the synthesis of two forms of the leader (L) protein has been demonstrated for ERAV, but not for ERBV (Hinton et al., 2000, 2002). The three predicted viral proteases (Lpro, 2Apro and 3Cpro) are common to all picornaviruses and are involved in the proteolytic processing of the polyprotein. The

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generic picornavirus polyprotein is divided into four sections (L, P1, P2, P3) based on structure, function and the position of the primary cleavage sites (Rueckert and Wimmer, 1984). The P1 region contains the four structural proteins of the capsid: virion protein (VP) 1, VP2, VP3 and VP4. The P2 region contains three mature non-structural proteins (2A, 2B and 2C) that function in modulation of the host cell and replication of viral RNA, and the P3 region contains four mature non-structural proteins (3A, 3B, 3C and 3D) involved in polyprotein processing and viral replication (Agol, 2002; Rueckert and Wimmer, 1984). 4. Structural proteins and virion assembly The picornaviruses are non-enveloped particles of approximately 30 nm in diameter. The icosahedral capsid is assembled from three external (VP1, VP2 and VP3) and one internal (VP4) viral protein. The capsid proteins VP1, VP2 and VP3 share a similar protein topology consisting of eight-stranded, antiparallel b-barrels arranged as a wedge-like structure with loops connecting the strands (reviewed by Racaniello, 2007). Sixty copies of each capsid protein form the icosahedral virion where the sequence and three-dimensional structure of these capsid proteins determines the antigenic sites, serotype, and the physical properties of the capsid. These proteins also mediate cell binding and entry and are important molecular determinants of virulence (reviewed by Bonnafous et al., 2008; Domingo, 1997; Domingo and Holland, 1997; Mason et al., 2003; McCright et al., 1999; Shiomi et al., 2004). 5. Genomic and antigenic variation among equine picornaviruses Equine rhinitis A virus exists as a single serotype despite nucleotide variation in the capsid-coding region. An analysis of ten geographically distinct isolates revealed 79.6–96.6% nucleotide identity and 96.8–99.3% identity at the amino acid level (Varrasso et al., 2001). The majority of amino acid changes are located within a 95 amino acid region at the 30 end of VP1 (Li et al., 1997). In crossneutralisation experiments, using a panel of equine polyclonal serum, a small proportion of isolates were not neutralised to the same extent as others, suggesting some variation in neutralising epitopes (Varrasso et al., 2001). Despite this, ERAV genomic sequences show considerable stability, since the genome of some currently circulating ERAV strains have a high level of identity to the original ERAV isolate, PERV, first described by Plummer (1962) (Diaz-Me´ndez et al., 2013). A single neutralising site has been identified for ERAV (Kriegshauser et al., 2003; Stevenson et al., 2004). This conformational site exists in the capsid quaternary structure and involves the C-terminus of VP1 from one protomer and the EF loop of VP1 on the adjacent protomer (Kriegshauser et al., 2003). The binding domain to the cell receptor a-2,3 linked sialic acid is contained within this region (Fry et al., 2010), with recombinant VP1 shown to compete with the binding of purified virus to susceptible cells (Warner et al., 2001). Additional non-neutralising B-cell epitopes have been mapped to both the C- and

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N-termini of VP1, VP2 and VP3, and the EF and GH loop regions of VP1 (Li et al., 2005; Stevenson et al., 2003). In contrast to ERAV, the erboviruses can be divided into three distinct serotypes and this correlates with the nucleotide sequence of the P1 region of the viral genome. The amino acid identity between these groups is approximately 72% in P1, whereas the non-structural P2 and P3 regions share over 95% identity (Black et al., 2007a; Huang et al., 2001). There is direct correlation between genotype, serotype and acid-stability phenotype with no crossneutralisation observed between the three serotypes (Fukunaga et al., 1981, 1983; Horsington et al., 2011). Previously, a small group of viruses that were genetically and phenotypically similar to ERBV3 were classified on the basis of serology as ‘‘acid-stable ERBV1’’ (Black et al., 2005) however, more recent studies have shown this to be a case of misidentification due to the occurrence of dual serotype infections (Horsington et al., 2011). Linear B cell epitopes of ERBV show VP1 contains serotype-specific epitopes and VP2 is highly cross-reactive across the serotypes. Major B-cell epitopes were further mapped to the C-terminus of VP1 of ERBV1, 2 and 3 and this region was also identified as the location of a major neutralising site in ERBV2 (Horsington et al., 2012). VP1 was also identified as the location of neutralisation epitopes in ERBV1 and 3, although these were not as dominant as in ERBV2, suggesting these viruses may have additional, possibly conformational, neutralising sites (Horsington et al., 2012). 6. Equine rhinitis A virus 6.1. Physical properties Equine rhinitis A virus is resistant to ether and chloroform, and has a CsCl density of 1.45 g/ml (Newman et al., 1977). The VP1 protein dominates the surface profile of the capsid, with the carboxyl terminus and the bE to bF loop associating to form a prominent crown, extending to an outer radius of 159 A˚, and marked depressions at the five-fold axis (Tuthill et al., 2009). The capsid is acid labile, dissociating into pentamers at pH levels between pH 6.5 and 5.5 (Tuthill et al., 2009). 6.2. Epidemiology Equine rhinitis A virus has a broad distribution and is endemic in horse populations worldwide, including the United Kingdom (Rose et al., 1974), Australia (Black et al., 2007b; Studdert and Gleeson, 1978), United Arab Emirates (Wernery et al., 2008), and the United States of America (Holmes et al., 1978; McCollum and Timoney, 1992). Seropositivity is associated with age (Black et al., 2007b; Holmes et al., 1978; McCollum and Timoney, 1992), with primary infection a feature of waning maternal antibodies in foals between 3 and 9 months of age and increased contact with other horses, such as the entry into training stables (Black et al., 2007b; Holmes et al., 1978). In older horses, seropositivity rates close to 100% have been reported (Holmes et al., 1978; McCollum and Timoney, 1992). Transmission of ERAV is likely to occur via

inhalation of aerosols generated from virus laden respiratory secretions or urine. The extremely high levels of virus shed in the urine of infected animals (Lynch et al., 2013; Quinlivan et al., 2010) is likely to contribute to the rapid spread of ERAV within a naı¨ve population. 6.3. Pathogenesis and clinical signs Clinical signs associated with natural exposure to ERAV are variable. Infection with ERAV is associated with acute febrile respiratory disease, characterised by fever (up to 41.4 8C) lasting between 1 and 3 days (Hofer et al., 1972; Studdert and Gleeson, 1978), anorexia, copious serous nasal discharge which later becomes mucopurulent, pharyngitis, bronchitis, coughing and swollen lymph nodes (Burrows, 1969; Carman et al., 1997; Ditchfield and Macpherson, 1965; Li et al., 1997; Studdert and Gleeson, 1978). However, given the high seroprevalence and infrequent reports of detection during clinical respiratory disease, most infections appear to be subclinical (Burrows, 1969; Hofer et al., 1972; McCollum and Timoney, 1992). Infectious ERAV can be isolated from nasopharyngeal and oral secretions, plasma, and urine of infected horses (Hartley et al., 2001; Li et al., 1997; Lynch et al., 2013; McCollum and Timoney, 1992; Plummer and Kerry, 1962; Studdert and Gleeson, 1978). In equine experimental infection models, shedding of infectious virus in respiratory secretions and plasma ceases with the detection of virus neutralising antibodies in the serum between one to two weeks post-infection (Lynch et al., 2013; Plummer and Kerry, 1962). These studies suggest the bulk of ERAV is shed in the urine, with viral loads exceeding 103 TCID50/ml and 107 RNA copies/ml (Lynch et al., 2013). High levels of ERAV have also been detected in the urine of horses in the general population, such as racehorses (Lynch et al., 2013; Quinlivan et al., 2010). Furthermore, chronic urinary shedding of ERAV (up to 143 days) was observed in a proportion of seropositive animals that did not display any overt clinical signs of disease (McCollum and Timoney, 1992). Although the precise mechanism that facilitates the chronic excretion of large quantities of ERAV in the urine of infected horses is unknown, persistence at this site is unlikely to be life-long. The very high loads of ERAV in the urine of infected animals (Lynch et al., 2013) suggest the consequences of infection may not be limited to the respiratory tract. Equine rhinitis A virus infection has also been associated with clinical signs related to systemic disease, such as decreased athletic capacity, temporary immunosuppression (Jensen-Waern et al., 1998), sustained elevated plasma fibrinogen levels and subtle haematological changes such as elevated neutrophils and decreased lymphocytes (Klaey et al., 1998), however reports of the clinical significance of these manifestations remain limited. Experimentally ERAV has a broad host range with guinea pigs, rabbits, mice and cynomolgus monkeys susceptible to intranasal infection (Plummer, 1963). Recently, a murine ERAV infection model demonstrated transient viraemia and urinary shedding following intranasal infection, with active replication in lung tissue

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detected by immunohistochemistry (Lynch et al., 2013). Although ERAV neutralising antibodies have been detected in serum samples from humans (Kriegshauser et al., 2005), the risk of acquiring zoonotic infection appears low, with clinical disease not documented since the earlier experimental infection studies involving a single human volunteer (Plummer, 1963). 6.4. Immune response to infection The immune response to ERAV is characterised by the generation of a high-titre virus neutralising antibody response, that peaks 2–3 weeks following infection (Hartley et al., 2001; Lynch et al., 2013) and is often detected as early as 5 or 7 days post-infection (Campbell et al., 1982; Hartley et al., 2001; Li et al., 2005; Plummer and Kerry, 1962). Serum antibodies react with varying degrees to the viral capsid proteins VP1, VP2 and VP3 in Western blot (Hartley et al., 2001; Li et al., 2005). The reactivity of serum collected from naturally or experimentally infected horses to individual capsid proteins, does not correlate with virus neutralisation titre (Hartley et al., 2001; Li et al., 2005; Lynch et al., 2011) which is consistent with the conformational nature of the immunodominant neutralising site (Kriegshauser et al., 2003). 7. Equine rhinitis B virus 7.1. Physical properties The ERBV genome is one of the largest in the Picornaviridae at approximately 8800 nucleotides in length. Complete particles have a buoyant density that can range from 1.41 to 1.46 g/ml and are resistant to ether and chloroform (Mumford and Thomson, 1978; Newman et al., 1977). Equine rhinitis B virus is inactivated at 50 8C in water but stabilised with the addition of MgCl2 (Mumford and Thomson, 1978). For ERBV1 and ERBV2, infectivity is lost below pH 5.4 (Newman et al., 1977; Steck et al., 1978a), whereas acid-stable ERBV3 viruses only lose infectivity below pH 3.3 (Mumford and Thomson, 1978). 7.2. Epidemiology Seroprevalence surveys for ERBV are conducted using virus neutralisation assays where, although 3 separate serotypes of ERBV are known, many studies have only examined seroprevalence to the ERBV1 prototype virus 1436/71. A high prevalence of antibodies to ERBV in adult horses has been reported world-wide, with neutralising antibodies detected in up to 100% of horses tested in various studies; though this is seldom associated with clinical signs (Black et al., 2007b; Carman et al., 1997; de Boer et al., 1979; Dunowska et al., 2002; Gerhaghty and Mumford, 2004; Holmes et al., 1978; McCollum and Timoney, 1992; Rose et al., 1974; Wernery et al., 1998). Foals usually have their first infection at 4–6 months of age and persistence leading to recrudescence and re-infection with ERBV, particularly in young horses, has been suggested as significant features in the epidemiology of these viruses (Burrows and Goodridge, 1978; Gerhaghty

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and Mumford, 2004; Mumford and Thomson, 1978; Steck et al., 1978b). Despite the high seroprevalence, ERBV is not readily isolated (Black et al., 2007a; Dynon et al., 2007). Both ERBV serotypes 1 and 2 have been isolated from horses around the world (Black et al., 2007a; Carman et al., 1997; de Boer et al., 1979; Dunowska et al., 2002; Holmes et al., 1978; Kriegshauser et al., 2005; McCollum and Timoney, 1992; Rose et al., 1974; Wernery et al., 1998), however to date, isolation of ERBV3 has only been reported in Australia, the UK and Japan (Fukunaga et al., 1983; Horsington et al., 2011; Mumford and Thomson, 1978). Concurrent infection with two ERBV serotypes has been identified through virus isolation from clinical samples (Horsington et al., 2012) and antibody detection using ELISA has indicated dual infection is common, and cocirculation of all three serotypes within a horse population can occur (Horsington, unpublished). 7.3. Pathogenesis and clinical signs The clinical manifestation of ERBV infection is currently not well defined. Spread is thought to be by direct and indirect contact with nasal secretions and aerosols (reviewed by Gerhaghty and Mumford, 2004). Infection occurs primarily in the upper respiratory tract of horses and can result in febrile respiratory disease. All three serotypes have been isolated from horses with clinical signs including fever, serous nasal discharge, anorexia, coughing, lymphadenitis, oedema of the legs and pain and swelling of the lymph nodes of the head and neck (Carman et al., 1997; McCollum and Timoney, 1992; Mumford and Thomson, 1978). Sub-clinical infections are frequent and isolation of virus from clinically healthy horses is common (Burrows and Goodridge, 1978; Carman et al., 1997; DiazMendez et al., 2010; Flammini and Allegri, 1970; Fukunaga et al., 1983; Horsington et al., 2011; Mumford and Thomson, 1978; Newman et al., 1977; Rose et al., 1974; Steck et al., 1978b; Willoughby et al., 1992). Persistent infection has also been documented. In one study, ERBV1 was isolated from individual horses for up to two years following infection (Burrows and Goodridge, 1978). In another study, ERBV3 was isolated consistently from a horse with no clinical signs over an 18-month period (Mumford and Thomson, 1978). The sites of ERBV replication are unknown and it is reasonable to suggest they may vary between the acidstable and acid-labile viruses, since other the acid-stable picornaviruses replicate in the gastrointestinal system. All ERBV serotypes have been isolated from nasopharyngeal and oral swabs (Black et al., 2007a; Carman et al., 1997; Fukunaga et al., 1983; Mumford and Thomson, 1978) and ERBV3 has also been isolated from the liver and spleen of an aborted foal (Fukunaga et al., 1983). A number of cases of dual infection with both acidlabile and acid-stable ERBV have been reported (Horsington et al., 2011). Mixed serotype infections are common with other viruses, such as enterovirus, adenovirus and dengue virus. These can significantly impact on population dynamics as a result of competitive suppression of viral replication, and can lead to recombination events with the potential to generate more virulent strains (Abe et al.,

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2009; Bharaj et al., 2008; Kroes et al., 2007; Lorono-Pino et al., 1999; Palmenberg et al., 2009; Pepin et al., 2008; Rakoto-Andrianarivelo et al., 2007). At present, the clinical disease implications of ERBV multi-serotype infections are unclear. The lack of comprehensive information about the pathogenesis of ERBV and immune response to ERBV infection reflects the few attempts made at experimental infection in the natural host. Infection of gnotobiotic and conventional foals by nasal instillation has been reported (Mumford and Thomson, 1978), and while the infected horses developed an ERBV-specific antibody response, no clinical signs were observed and virus could not always be re-isolated after infection. In a subsequent study, minimal clinical signs of respiratory disease were observed in horses exposed to ERBV1; however, these animals were ERBV positive by serum neutralisation and virus isolation prior to the experimental exposure (Willoughby et al., 1992). In common with other viral respiratory infections, ERBV infected animals have an increased susceptibility to more severe secondary infection with bacteria (Carman et al., 1997; Ditchfield, 1969; Steck et al., 1978a) and co-infection with ERBV and other viruses including equine herpesvirus-1 and -4 (Carman et al., 1997) and equine influenza virus (Diaz-Mendez et al., 2010) has been reported. While ERBV may act alone in the development of respiratory disease in some cases, ERBV may also influence the host’s immune response and extend the duration or increase the severity of infections with other pathogens. 7.4. Acid stability phenotype Acid stability, related to the pH at which virus infectivity is lost, was historically an important biological characteristic used in picornavirus grouping and classification. The acid-stable and acid-labile phenotypes can be important determinants of viral tropism, uncoating and infection, although for ERBV the implications of stability at low pH remain undefined. The effect of low pH on capsid structure varies between picornaviruses and for many is an essential component of cell entry. The erboviruses, similar to the aphthoviruses and cardioviruses dissociate into pentameric subunits below their pH threshold of stability (Baxt and Bachrach, 1980; Horsington et al., 2011; Phelps et al., 2000; Tuthill et al., 2009). The acid-stable phenotype of one serotype in a species of respiratory viruses is atypical and the significance of this in ERBV pathogenesis and epidemiology remains to be elucidated. Most acid-stable picornaviruses infect or replicate in the gastrointestinal system, yet this has not been confirmed for ERBV3. Other picornaviruses (for example, the human enterovirus ECHO-11) associated with respiratory disease are known to infect both the upper respiratory and the intestinal tracts (Saliba et al., 1968). While there are no reports of ERBV detection in urine or faeces, only minimal attempts at virus isolation from these samples have been reported (Mumford and Thomson, 1978) and detection by molecular methods has not yet been attempted. More thorough investigations may reveal an association between the acid-stable phenotype and enteric infection.

8. Detection of equine picornavirus infection Equine picornavirus infections are demonstrated by direct detection in clinical samples or by an increase in virus-specific antibodies between acute and convalescent serum samples. Equine picornaviruses are detected in nasopharyngeal swab samples either through virus isolation or by the detection of viral RNA using reverse transcription PCR (RT-PCR). Given the high levels of ERAV shed in the urine of infected animals, this sample, if available, should be tested in conjunction with respiratory samples to detect ERAV (Lynch et al., 2013; Quinlivan et al., 2010). The acid-stable phenotype of ERBV3 is unusual for a respiratory virus and further studies into other infection sites may be required to identify the most appropriate sample for detection of this virus. Accurate diagnosis and evidence linking ERAV or ERBV to respiratory disease can be confounded by a number of factors, including the young age at which horse become infected and the presence of maternal antibodies, the high seropositivity in the horse population, and the occurrence of persistent and multiple serotype infections. The presence of antibodies may reduce viral load, however, the establishment of a low level, chronic infection unable to be detected by cell culture or molecular methods may facilitate further spread in the horse population. 8.1. Virus isolation The primary isolation of equine picornaviruses from clinical samples can be difficult. Rabbit kidney 13 (RK13), equine foetal kidney (EFK) and African green monkey kidney (Vero) cells are commonly used for isolation (Li et al., 1997; McCollum and Timoney, 1992; Studdert and Gleeson, 1978), although the susceptibility of the cell line differs between viruses and isolates (McCollum and Timoney, 1992). The addition of magnesium to the culture media increases the susceptibility of cells to various picornaviruses, including poliovirus and rhinovirus (Fiala and Kenny, 1966; Wallis and Melnick, 1962), and this can also improve the recovery of ERBV (Black et al., 2007a). Propagation of non-cytopathic ERAV may complicate diagnosis relying solely on virus isolation in cell culture, however, the detection of viral antigen in the cytoplasm of infected cells can be achieved using virus specific antiserum coupled with fluorescent or colourmetric-based staining protocols (Li et al., 1997). The occurrence of multiple serotype ERBV infections can also complicate diagnosis by virus isolation (Horsington et al., 2011) and a more rigorous approach combining molecular, serological and cell biological techniques are required for definitive diagnosis. 8.2. Serological methods Standard virus neutralisation assays have been used historically to detect ERAV- and ERBV-specific antibodies in serum samples. A recent focus has been the development of enzyme-linked immunosorbent assays (ELISAs) that provide a more rapid, high-throughput assay that does not require tissue culture facilities.

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The suitability of an ELISA for the detection of ERAV antibodies using whole virus or a series of recombinant bacterial derived viral capsid fusion proteins has been investigated (Kriegshauser et al., 2009; Li et al., 2005). These assays appear less sensitive in detecting seropositivity than when a combination of a virus neutralisation assay and Western blotting analysis using purified virus is used (Li et al., 2005). This is most likely due to the frequent detection of very high virus neutralisation titres (>1000 are common) in serum, and the conformational nature of the immunodominant neutralising epitope (Kriegshauser et al., 2003). ELISAs for the detection of ERBV-specific antibody have been developed using fusion proteins expressed in bacteria. In one report, the denatured VP1 protein was used as antigen in an ERBV1 specific ELISA (Kriegshauser et al., 2008). More recently, the identification of viral proteins containing serotype-specific epitopes has led to the development of a serotyping ERBV antibody-detection ELISA (Horsington, unpublished). 8.3. Molecular methods Conventional RT-PCR assays and quantitative RT-PCR (RT-qPCR) assays have been developed to detect both ERAV and ERBV in clinical samples, targeting the 3Dpol, 30 -UTR, 50 -UTR or VP1 regions (Black et al., 2007a; Lu et al., 2012; Mori et al., 2009; Quinlivan et al., 2010). The use of these sensitive molecular techniques has demonstrated that the diagnosis of ERAV and ERBV as causes of respiratory disease may have been previously underestimated due to difficulties in virus isolation (Klaey et al., 1998; Li et al., 1997), however detection rates are still much lower than might be expected from seroprevalence studies. Furthermore, the currently available methods do not detect multiple ERBV serotype infections and the ideal sample site for each equine picornavirus remains to be established. 9. Conclusion Equine respiratory disease remains a significant economic and welfare burden for both performance and pleasure horses worldwide, though there is great potential to reduce this burden with the identification of causative agents and the development of methods for their control. While respiratory viruses such as equine influenza virus, and equine herpesvirus-1 and -4 are known to contribute significantly to this disease burden, in many cases the causative agents of infectious upper respiratory tract disease remain undiagnosed. The high seroprevalence of equine picornaviruses in horse populations worldwide contrasts with the relatively few reports of direct detection of these viruses leading to a poor understanding of their contribution to disease. Indeed, many studies examining ERBV seroprevalence overlook ERBV2 and ERBV3, resulting in prevalence data related only to serotype 1. Further studies into the pathogenesis of ERAV and ERBV to better understand their replication sites will enable more informed sampling, increased success in detection in infected animals and improved understanding of their associations with respiratory or other diseases.

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