In 2006-2007, equine viral arteritis (EVA) was confirmed for the first time in Quarter Horses in multiple states in the USA. The entire genome of an equine arteritis virus (EAV) isolate from the index premises in New Mexico was 12 731 nt in length and possessed a previously unrecorded unique 15 nt insertion in the nsp2-coding region in ORF1a and a 12 nt insertion in ORF3. Sequence analysis of additional isolates made during this disease occurrence revealed that all isolates from New Mexico, Utah, Kansas, Oklahoma and Idaho had 98.6-100.0 % (nsp2) and 97.8-100 % (ORF3) nucleotide identity and contained the unique insertions in nsp2 and ORF3, indicating that the EVA outbreaks in these states probably originated from the same strain of EAV.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

We investigated the correlation between in vitro susceptibility of CD3 ؉ T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3 ؉ T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P ‫؍‬ 0.0002). Equine arteritis virus (EAV) is a small, enveloped virus with a positive-sense, single-stranded RNA genome that belongs to the family Arteriviridae (genus Arterivirus, order Nidovirales) (4,11). It is the causal agent of equine viral arteritis (EVA), which is a respiratory and reproductive disease of horses and other equids (3,14). While the vast majority of natural infections with EAV are unapparent or subclinical (3, 2, 14), occasional outbreaks of EVA occur that are characterized by influenza-like signs in adult horses, abortion in mares, and the possibility of pneumonia in foals (7,14). Following EAV infection, a variable proportion of stallions (30 to 70%) can become persistently infected and continuously shed the virus in their semen (14; S. M. Neu, P. J. Timoney, and W. H. McCollum, presented at the 5th International Conference Equine Infectious Diseases, Lexington, Kentucky, 1987). Carrier stallions are the natural reservoir of EAV; they ensure the virus is maintained in equine populations between breeding seasons (14, 16). The continued growth in the international trade of horses and semen has served as a significant means of dissemination of EAV strains around the world (1,8,(13)(14)(15)(16). Therefore, identification of carrier stallions is of critical epidemiological importance in the prevention and control of EAV infection in countries worldwide (2,3,(12)(13)(14). While the mechanism of persistence of EAV in the male reproductive tract is not clear, it has been established that viral persistence in the stallion is testosterone dependent (9; T. V. Little, G. R. Holyoak, W. H. McCollum, and P. J. Timoney, presented at the 6th International Conference on Equine Infectious Diseases, Cambridge, England, 1992). Although multiple factors

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5 ], M 1-162 , and N 1-110 ), as well as partial sequences of these structural proteins (GP5 1-116 , GP5 75-112 , GP5 55-98 , M 88-162 , and N 1-69 ) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5 55-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5 55-98 MIA and VNT outcomes correlated significantly (r ‫؍‬ 0.84; P < 0.0001). Although the GP5 55-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses (61). EAV is a small enveloped virus with a positivesense, single-stranded RNA genome of 12.7 kb and belongs to the family Arteriviridae (genus Arterivirus, order Nidovirales), which also includes porcine reproductive and respiratory syndrome virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus of mice (13, 57). The EAV genome includes nine functional open reading frames (ORFs) (55, 57). ORFs 1a and 1b encode two replicase polyproteins (pp1a and pp1ab) (25,55,57), and the remaining seven ORFs (2a, 2b, and 3 to 7) encode the structural proteins of the virus. These include four membrane glycoproteins, GP2 (25 kDa), GP3 (36 to 42 kDa), GP4 (28 kDa), and GP5 (30-44 kDa), encoded by ORFs 2b, 3, 4, and 5, respectively; two unglycosylated membrane proteins, E (8 kDa) and M (17 kDa), encoded by ORFs 2a and 6; and the phosphorylated nucleocapsid protein N (14 kDa), encoded by ORF 7 (23, 58, 64). The major envelope glycoprotein GP5 expresses the known neutralization determinants of EAV. The two major envelope proteins GP5 and M form a disulfide-linked heterodimer in the virus particle, and this association is critical for their maturation and for the expression of some of the neutralization epitopes in authentic form (3,24,56).Serological and clinical studies indicate that EAV is widely distributed in equine populations around the world (35,46,48). Although there is considerable variation in the sequences of...

Two previously developed TaqMan fluorogenic probe-based 1-tube real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays (T1 and T2) were compared and validated for the detection of Equine arteritis virus (EAV) nucleic acid in equine semen and tissue culture fluid (TCF). The specificity and sensitivity of these 2 molecular-based assays were compared to traditional virus isolation (VI) in cell culture. The T1 real-time RT-PCR had a higher sensitivity (93.4%) than the T2 real-time RT-PCR (42.6%) for detection of EAV RNA in semen. However, the T1 real-time RT-PCR was less sensitive (93.4%) than the World Organization for Animal Health (OIE)-prescribed VI test (gold standard). The sensitivity of both PCR assays was high (100.0% [T1] and 95.2% [T2]) for detecting EAV RNA in TCF. In light of the discrepancy in sensitivity between either real-time RT-PCR assay and VI, semen that is negative for EAV nucleic acid by real-time RT-PCR that is from an EAV-seropositive stallion should be confirmed free of virus by VI. Similarly, the presence of EAV in TCF samples that are VI-positive but real-time RT-PCR-negative should be confirmed in a 1-way neutralization test using anti-EAV equine serum or by fluorescent antibody test using monoclonal antibodies to EAV. If the viral isolate is not identified as EAV, such samples should be tested for other equine viral pathogens. The results of this study underscore the importance of comparative evaluation and validation of real-time RT-PCR assays prior to their recommended use in a diagnostic setting for the detection and identification of specific infectious agents.