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Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract., Author summary A distinctive feature of equine arteritis virus (EAV) is its ability to establish long-term persistent infection in the stallion reproductive tract in the presence of strong immune and inflammatory responses. The data presented herein provides insight into the molecular signature of the inflammatory response during persistent infection in the male reproductive tract, and shows that long-term persistence is associated with the predominance of specific CD8+ T lymphocyte transcription factors that drive the inflammatory process in the reproductive tract, along with the upregulation of inhibitory receptors and CXCL16/CXCR6, a chemokine axis strongly implicated in EAV persistence. Furthermore, the host’s CXCL16 genotype drives the changes in transcriptional factors that favors EAV persistent infection. These findings have a broad translational importance in the immunopathogenesis of EAV and other persistent viral infections in the male reproductive tract of animals and humans, as well as in the prevention and treatment of such infections.

Summary Equine arteritis virus is the causative agent of equine viral arteritis, a respiratory and reproductive disease that affects the members of the family Equidae. The virus was first isolated from the lung of an aborted fetus after an extensive outbreak of respiratory disease and abortion on a Standardbred breeding farm near Bucyrus, Ohio, in 1953. Since then, periodic outbreaks of equine viral arteritis have been reported in a number of countries around the world. This disease may result in significant economic loss to the equine industry due to the occurrence of abortion in pregnant mares, neonatal mortality, and establishment of the carrier state in stallions. This article provides an extensive review on equine arteritis virus, epidemiology, disease, pathogenesis, and prevention and control measures.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10-70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.

Studies in our laboratory have shown that horses can be divided into two distinct groups based on whether they possess a subpopulation of CD3 + T lymphocytes susceptible to in vitro infection by equine arteritis virus (EAV) or not. It has been also demonstrated that stallions with the CD3 + T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Furthermore, experimental EAV infection of horses with either the in vitro CD3 + T lymphocyte susceptibility or resistance phenotype showed a significant difference between the two groups in terms of proinflammatory and immunomodulatory cytokine mRNA expression. A genome-wide association study (GWAS) demonstrated that these phenotypes are associated with the CXCL16 gene located in equine chromosome 11 (ECA11). We demonstrated that the membrane bound form of the protein encoded by EqCXCL16 gene function as a primary receptor molecule for EAV. Moreover, the secretory form of the CXCL16 protein is the ligand for the chemokine receptor CXCR6 that is predicted to be expressed on CD4 + and CD8 + T cells, NKT cells, and NK cells. Thus, equine CXCL16 and CXCR6 may be two major cellular proteins associated with the EAV carrier state in stallions. Finally, analysis of multiple tissues from EAV carrier stallions unequivocally confirmed the ampullae of the vas deferens as the primary site of EAV persistence with dual immunohistochemistry studies showing that EAV is localized in fibrocytes and mononuclear cells (T and B lymphocytes) but not in the glandular epithelium.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene ( CXCL16S ) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion. IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene ( CXCL16S ) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation.

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8 + T and CD21 + B lymphocytes but not glandular epithelium. 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 + ), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P ≤ 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B79 using the World Organization for Animal Health (OIE)–recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians–accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

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, Author Summary A variable proportion of EAV infected stallions (10–70%) may become persistently infected and continuously shed the virus exclusively in their semen after recovery from acute infection. Previous studies in our laboratory have shown that stallions with the CD3+ T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Here genetic and experimental studies were used to demonstrate that CXCL16 in the horse codes for two proteins, one associated with resistance and the other associated with susceptibility of CD3+ T lymphocytes to EAV infection. The two proteins are the result of four nucleotide substitutions in exon 1 of the equine CXCL16 gene. These alleles determine the outcome of in vitro infection of CD3+ T lymphocytes with EAV and are strongly associated with the establishment and maintenance of long-term carrier state in stallions. In vitro studies demonstrated that one form of CXCL16 protein (CXCL16S) is one of the cellular receptors for EAV and has higher scavenger activity and adhesion ability as compared to the form of the protein associated with resistance (CXCL16R).

and negative, respectively. Regarding the buffy coat samples, iiRTPCR detected EIAV nucleic acid in 31/56 AGID positive samples, while 108/109 AGID negative samples tested negative by iiRTPCR. Total agreement was 84.24%. When whole blood samples were evaluated, a total of 16 iiRT-PCR positive and 15 iiRT-PCR negative out of 31 AGID positive samples were obtained. Additionally, EIAV nucleic acid was not detected in 25 AGID negative samples .Total agreement was 73.21% for this sample type. When evaluating the accuracy of EIAV iiRT-PCR and qPCR, 28/165 and 130/165 buffy coat samples tested positive and negative by both assays, respectively. Five iiRT-PCR positive samples were negative by qPCR and two qPCR positive samples were negative by iiRTPCR. Thus, EIAV iiRT-PCR showed more than 95% agreement with qPCR results. It has been demonstrated that the absence of clinical signs is correlated with very low, frequently undetectable viremia. Therefore, EIAV iiRT-PCR appears to be a promising tool to identify infected horses including those experiencing low infectivity titers in blood. Furthermore, as initial EIAV replication rates are frequently high, recently infected equids pose a considerable transmission risk long before seroconversion. In our study, a horse was determined seronegative by AGID, but was identified as EIAV infected by iiRT-PCR. Thus, EIAV iiRT-PCR could be considered as an alternative diagnostic tool in the implementation of control strategies during an EIA outbreak in the low prevalence area of our country.

Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from in vitro . The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14 + monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus. IMPORTANCE Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012).

A single-nucleotide polymorphism (A 2254 or G 2254 ) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E 2 ) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E 1 ) was developed by redesigning primers and probes specific to ORF30. The E 1 and E 2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A 2254 or G 2254 ) in all archived isolates plus 168 of the clinical samples. The E 1 assay was 10 times more sensitive than E 2 , with a lower detection limit of 10 infectious virus particles. Furthermore, all A 2254 and G 2254 genotypes along with samples from three cases of dual infection (A 2254 +G 2254 ) were correctly identified by E 1 , whereas E 2 produced 20 false dual positive results with only one actual mixed A 2254 +G 2254 genotype confirmed. Based on these findings, E 1 offers greater sensitivity and accuracy for the detection and A/G 2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3 + T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3 + T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3 + T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3 + T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis.

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.

This study was undertaken to re-evaluate safety aspects of the commercial modified live virus vaccine against equine viral arteritis (Arvac) in stallions. Ten seronegative stallions were administered a single dose of vaccine, whereas three others were used as controls. Presence of vaccine virus in blood, semen, and nasopharyngeal and conjunctival secretions was determined by virus isolation and reverse transcription-polymerase chain reaction assay. No adverse effects were observed after vaccination. Vaccine virus was detected in buffy-coat cultures of all stallions for an average of 4.3 days, and in nasopharyngeal swabs from 40% of the stallions for an average of 3.2 days. No virus was detected in conjunctival swabs. Small quantities of virus were detected in the semen of one stallion on days 4 and 6 postvaccination. Vaccinated stallions showed a marked increase in neutralizing antibody titers within 5 to 8 days after vaccination. The stallions in the control group remained uninfected. Finding very low levels of virus in the semen of one stallion provides justification for withholding semen from a first-time vaccinated stallion for a minimum of 14 days to avoid the potential risk of virus transmission or having vaccine virus detected in semen frozen from a recently vaccinated stallion. There was no evidence of persistence of vaccine virus in the reproductive tract of the vaccinated stallions. Virus isolation proved more sensitive than reverse transcription-polymerase chain reaction for detection of virus in buffy-coat and nasopharyngeal specimens. Peak antibody responses were reached at a little more than a week after vaccination and these remained undiminished for the remainder of the study.

The objective of the current study was to determine the capability of 3 recently described one-step TaqMan real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of H3N8 Equine influenza virus (EIV NP, EIV M, and EIV HA3 assays, respectively) to detect Canine influenza virus (CIV). The assays were initially evaluated with nucleic acid extracted from tissue culture fluid (TCF) containing the A/canine/FL/43/04 strain of Influenza A virus associated with the 2004 canine influenza outbreak in Florida. The EIV NP, EIV M, and EIV HA3 assays could detect CIV nucleic acid at threshold cycle (Ct) values of 16.31, 23.71, and 15.28, respectively. Three assays using TCF or allantoic fluid (AF) samples containing CIV ( n = 13) and archived canine nasal swab samples ( n = 20) originally submitted for laboratory diagnosis of CIV were further evaluated. All TCF and AF samples, together with 10 nasal swab samples that previously tested positive for virus by attempted isolation in embryonated hens' eggs or Madin–Darby canine kidney cells, were positive in all 3 real-time RT-PCR assays. None of the 3 assays detected the H1N1 Swine influenza virus strain in current circulation. These findings demonstrate that previously described real-time RT-PCR assays targeting NP, M, and H3 HA gene segments of H3N8 EIV are also valuable for the diagnosis of CIV infection in dogs. The assays could expedite the detection and identification of CIV.

Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3 + T lymphocytes and CD14 + monocytes. The VB strain primarily infects CD14 + monocytes and a small subpopulation of CD3 + T lymphocytes (predominantly CD4 + T lymphocytes), as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has a significantly reduced ability to infect CD14 + monocytes and has lost its capability to infect CD3 + T lymphocytes. Using a panel of five recombinant chimeric viruses, we demonstrated that interactions among the GP2, GP3, GP4, GP5, and M envelope proteins play a major role in determining the CD14 + monocyte tropism while the tropism for CD3 + T lymphocytes is determined by the GP2, GP4, GP5, and M envelope proteins but not the GP3 protein. The data clearly suggest that there are intricate interactions among these envelope proteins that affect the binding of EAV to different cell receptors on CD3 + T lymphocytes and CD14 + monocytes. This study shows, for the first time, that CD3 + T lymphocytes may play an important role in the pathogenesis of equine viral arteritis when horses are infected with the virulent strains of EAV.

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. Sequence and phylogenetic analysis of several EAV isolates made following an EVA outbreak on another Quarter Horse farm in New Mexico in 2005 provided evidence that this outbreak may well have been the source of virus for the 2006-2007 occurrence of the disease. A virus isolate from an aborted fetus in Utah was shown to have a distinct neutralization phenotype compared with other isolates associated with the 2006-2007 EVA occurrence. Full-length genomic sequence analysis of 18 sequential isolates of EAV made from eight carrier stallions established that the virus evolved genetically during persistent infection, and the rate of genetic change varied between individual animals and the period of virus shedding.

A significant consequence of equine arteritis virus (EAV) infection of horses is persistence of the virus in a variable percentage of infected stallions. We recently established an in vitro model of EAV persistence in cell culture for the purpose of furthering our understanding of EAV biology in general and viral persistence in the stallion in particular. In this study we investigated whether persistently infected HeLa cells could be cured of EAV infection by treatment with an antisense peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) designed to target the 5'-terminal region of the EAV genome. We found that persistently infected HeLa cells passaged three times in the presence of 5-10 microM EAV-specific PPMO produced no detectable virus. The PPMO-cured HeLa cells were free of infectious virus, viral antigen and EAV RNA as measured by plaque assay, indirect immunofluorescence assay and RT-PCR, respectively. Furthermore, when re-challenged with EAV at several passages after discontinuation of PPMO treatments, PPMO-cured HeLa cells were found to be refractory to re-infection and to the re-establishment of viral persistence. While these findings demonstrate that PPMO can be used to eliminate persistent EAV infection in cell culture, the efficacy of PPMO against EAV in vivo remains to be addressed.

Summary Reasons for performing study:A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. Objectives: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. Methods: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF5-specific primers, subjected to sequence and phylogenetic analysis. Results: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stal lions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from be dding or fo mites contaminated with semen. Conclusions: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. Potential relevance: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV-infected semen.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

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.

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 1-255 ], 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.

International audience; Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001-2004) were determined by sequencing open reading frames (ORFs) 2a-7. The ORFs 2a-7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296-11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France.

Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.

Equid herpesvirus 1 (EHV-1) is one of the most economically important equine viral pathogens. Its clinical manifestations in horses vary from acute upper respiratory tract disease, abortion, or neonatal death, to neurological disease termed equine herpesviral myeloencephalopathy, which may lead to paralysis and a fatal outcome. Successful identification of EHV-1 infection in horses depends on a variety of factors such as suitable case selection with emphasis on timing of sample collection, selection of appropriate sample(s) based on the clinical manifestations, application of relevant diagnostic technique(s) and/or test(s), and careful evaluation and interpretation of laboratory results. Several traditional serologic and virus isolation assays have been described; however, these assays have inherent limitations that prevent rapid and reliable detection of EHV-1. The advent of molecular biologic techniques has revolutionized the diagnosis of infectious diseases in humans and animal species. Specifically, polymerase chain reaction (PCR)–based assays have allowed detection of nucleic acid in clinical specimens precisely and rapidly as compared to the traditional methods that detect the agent or antigen, or agent-specific antibodies in serum. The new molecular methods, especially real-time PCR, can be a very useful means of EHV-1 detection and identification. Veterinarians involved in equine practice must be aware of the advantages and disadvantages of various real-time PCR assays, interpretation of viral genetic marker(s), and latency in order to provide the best standard of care for their equine patients.

A high-passage rabbit kidney RK-13 cell line (HP-RK-13[KY], originally derived from the ATCC CCL-37 cell line) used in certain laboratories worldwide is contaminated with noncytopathic bovine viral diarrhea virus (ncpBVDV). On complete genome sequence analysis, the virus strain was found to belong to BVDV group 1b.

Strains of equine arteritis virus (EAV) differ in their virulence phenotypes, causing anywhere from subclinical infections to severe disease in horses. Here, we describe the in silico design and de novo synthesis of a full-length infectious cDNA clone of the horse-adapted virulent Bucyrus strain (VBS) of EAV encoding mCherry along with in vitro characterization of the progeny virions (EAV sVBSmCherry) in terms of host-cell tropism, replicative capacity and stability of the mCherry coding sequences following sequential passage in cell culture. The relative stability of the mCherry sequence during sequential cell culture passage coupled with a comparable host-cell range phenotype (equine endothelial cells, CD3(+) T cells and CD14(+) monocytes) to parental EAV VBS suggest that EAV-sVBSmCherry-derived virus could become a valuable research tool for identification of host-cell tropism determinants and for characterization of the viral proteins involved in virus attachment and entry into different subpopulations of peripheral blood mononuclear cells. Furthermore, this study demonstrates that advances in nucleic acid synthesis technology permit synthesis of complex viral genomes with overlapping genes like those of arteriviruses, thereby circumventing the need for complicated molecular cloning techniques. In summary, de novo nucleic acid synthesis technology facilitates innovative viral vector design without the tedium and risks posed by more-conventional laboratory techniques.

Equine arteritis virus (EAV) causes a persistent infection of the reproductive tract of carrier stallions. The authors determined the complete genome sequences of viruses (CW96 and CW01) that were present 5 years apart in the semen of a carrier stallion (CW). The CW96 and CW01 viruses respectively had only 85·6 % and 85·7 % nucleotide identity to the published sequence of EAV (EAV030). The CW96 and CW01 viruses had two 1 nt insertions and a single 1 nt deletion in the leader sequence, and a 3 nt coding insertion in ORF1a; thus their genomes included 12 708 nt as compared to the 12 704 nt in EAV030. Variation between viruses present in the semen of stallion CW and EAV030 was especially marked in the replicase gene (ORF1a and 1b), and the greatest variation occurred in the portion of ORF1a encoding the nsp2 protein. The ORFs 3 and 5, which respectively encode the GP3 and GP5 envelope proteins, showed greatest variation amongst ORFs encoding structural EAV proteins. Comparative sequence analyses of CW96 and CW01 indicated that ORFs 1a, 1b and 7 were highly conserved during persistent infection, whereas there was substantial variation in ORFs 3 and 5. Although the variation that occurs in ORF5 results in the emergence of novel phenotypic viral variants as determined by neutralization assay, all variants were neutralized by high-titre polyclonal equine antisera, suggesting that immune evasion is unlikely to be responsible for the establishment of persistent EAV infection of carrier stallions. Northern blot analyses of RNA extracted from cell culture propagated viruses isolated from 10 different persistently infected stallions failed to demonstrate any large genomic deletions, suggesting that defective interfering particles are also unlikely to be important in either the maintenance or clearance of persistent EAV infection of the reproductive tract of carrier stallions.

This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.

Equine herpesvirus type 1 (EHV-1) can cause respiratory disease, abortion, respiratory illness and death in neonatal foals and neurological disease in horses (Allen et al. 2004; Lunn et al. 2009). Primary infection of young foals typically results in establishment of a latent carrier state and the potential for viral reactivation during the life of the infected individual. Reactivation leads to the production of infectious virus that can be shed into the nasopharynx for a limited period of time and also result in a cell-associated viraemia, which may give rise to clinical disease and abortion in mares. Over the past decade, there has been an unexpected increase in incidence of equine herpesvirus neurological disease (equine herpesvirus myeloencephalopathy [EHM]) (Perkins et al. 2009; Vissani et al. 2009; Fritsche and Borchers 2010; Pronost et al. 2010; Smith et al. 2010). Recent studies suggest that EHM is associated with a single nucleotide polymorphism at position 2254 in the EHV-1 DNA polymerase gene (encoded by open reading frame 30 [ORF30]) (Nugent et al. 2006; Goodman et al. 2007; Perkins et al. 2009; van de Walle et al. 2009). Based on these findings, EHV-1 strains possessing guanine (G2254) at this site are considered to have neuropathogenic potential, whereas those strains with adenine (A2254) are thought to be non-neuropathogenic and usually but not invariably associated with abortion and respiratory disease in horses. The nonsynonymous A to G substitution at nucleotide position 2254 in ORF30 results in replacement of asparagine (N) with a negatively charged aspartic acid (D) residue at amino acid position 752 (N752→D752) in the viral DNA polymerase enzyme. EHV-1 strains of the G2254 genotype have been shown to replicate more efficiently in the horse and produce significantly higher viral loads (Allen and Breathnach 2006; Allen 2008). It is believed that this increased replicative capacity enhances the ability of the virus to infect capillary endothelial cells, leading to interference with the blood supply to the central nervous system and the development of neurological signs. The evidence supporting this association between the G2254 substitution and EHM is derived from nucleotide sequence analysis of a relatively small region of ORF30 (251 nucleotides [10% of the DNA polymerase gene]) from 131 field isolates of EHV-1 involving both neurological and non-neurological clinical episodes (Nugent et al. 2006) and subsequent nucleotide substitution experiments conducted using infectious EHV-1 molecular clones (Goodman et al. 2007; van de Walle et al. 2009; Ma et al. 2010). Furthermore, the recently observed increased incidence of EHM correlates with the higher prevalence of viruses with a G2254 genotype currently being isolated in diagnostic laboratories in Europe and the USA (Perkins et al. 2009; Pronost et al. 2010; Smith et al. 2010). Recently, Perkins et al. (2009) performed statistical analysis of ORF30 from a large number of EHV-1 isolates (n = 176) and demonstrated that the odds of neurological disease being associated with the ORF30 G2254 genotype are 162 times greater than those with the A2254 genotype. A comprehensive analysis of a large panel of archived EHV-1 isolates collected from sporadic cases of equine abortion between 1951 and 2006 in Kentucky using a real-time Taq-Man allelic discrimination PCR, revealed that viruses with the G2254 neuropathogenic genotype existed at least as far back as the 1950s (Smith et al. 2010). Furthermore, such isolates increased in prevalence from 3.3% in the 1960s to 14.4% in the 1990s, with indications of an even higher incidence from 2000 onwards. The studies outlined above certainly support an association between EHM and the G2254 genotype. However, there is an increasing body of very compelling evidence to indicate that this nucleotide substitution is not the only determinant of neurological disease. In the Perkins et al. (2009) survey, 24% of isolates from horses with neurological disease possessed the A2254 and not the G2254 genotype. This finding is supported by our own investigations comparing results from the real-time allelic discrimination assay with detailed case histories provided by attending veterinarians (U.B.R. Balasuriya, unpublished data; Pronost et al. 2010). We identified a number of A2254 genotype EHV-1 isolates from cases of neurological disease, as well as G2254 genotype isolates from numerous horses with no evidence of neurological involvement. In addition, we have identified viruses with nonsynonymous nucleotide substitutions in ORF30 besides A→G2254, from horses without signs of neurological disease, which presents the possibility that these may have an attenuating effect on the viral phenotype (Smith et al. 2010). Conversely, if mutations exist within ORF30 that attenuate the phenotype, there may be many other substitutions not associated with position 2254 and outside the small region included in the study of Nugent et al. (2006) with the capability of enhancing viral replication rates in vivo. This could explain, for example, why some viruses with the A2254 genotype have been isolated from cases of neurological disease. The neuropathogenic potential of such strains to this point has not been fully investigated. Furthermore, it should be emphasised that

The N-terminal hydrophilic ectodomain of the G(L) envelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding G(L) envelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 10B4, indicating that they also recognize epitopes on the G(L) protein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified-live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the G(L) protein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1 variable region (amino acids 61-121) within the second half of the N-terminal hydrophilic ectodomain of the G(L) protein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.

Equine arteritis virus (EAV) causes contagious equine viral arteritis, characterized by fever, anorexia, conjunctivitis, nasal discharge, dependent edema, abortion, infrequent death in foals, and establishment of the carrier state in stallions. The World Organization for Animal Health (OIE) defines a horse as seropositive if the serum neutralization (SN) antibody titer is ≥1:4 to EAV. However, determining the SN titer is time-consuming and requires specific laboratory facilities, equipment, and technical expertise to perform. Furthermore, interpretation of the SN titer of some sera can be difficult because of nonspecific cellular cytotoxicity of particular samples. Finally, the problem of interlaboratory variation also exists with SN assays. For these reasons, an alternative serologic test is desirable; however, none of the reported tests have equivalent sensitivity and specificity to the SN to be generally adopted. In an attempt to improve on a previously developed competitive enzyme-linked immunosorbent assay (cELISA) using EAV gp5-specific neutralizing monoclonal antibody (mAb) 4B2, the current study developed a modified protocol substituting the non-neutralizing mAb 17B7 for the neutralizing mAb 4B2; this along with several modifications of the test procedure improved the performance of the test. The relative specificity of the revamped cELISA was 99.8% when evaluated with 2,223 SN-negative sera. The relative sensitivity was 95.5% when evaluated with 246 SN-positive sera. This new cELISA was not affected by the presence of non–EAV-specific cytotoxicity in sera as observed in the SN assay. The results indicate that this new cELISA may be a viable alternative to the SN assay and merit additional validation.

The open reading frame 2 (ORF2) of three laboratory strains, the live attenuated vaccine virus, and 18 field isolates of equine arteritis virus (EAV) from Europe and North America was sequenced. The ORF2 of EAV encodes the Gs protein that is abundantly expressed in infected cells but constitutes less than 2% of the virion protein mass. Variation of ORF2 among the isolates facilitated phylogenetic analysis that largely confirmed results of an earlier study based on sequence divergence of ORF5 of the same isolates of EAV, despite exposure of the proteins encoded by ORF2 (Gs) and ORF5 (GL) to potentially different selective pressures in vivo. The data indicate that the Gs protein is highly conserved between isolates, considerably more so than the GL protein, consistent with an important role of the Gs protein in virus replication.

Summary The clinical, virological and serological responses of seven female donkeys ( Equus asinus ) to inoculation with the KY-84 strain of equine arteritis virus (EAV), a strain that causes moderate to severe clinical signs in horses, was investigated. In the donkeys, the only clinical signs observed were fever (mainly 3–9 days after inoculation), mild depression in four animals, and a slight nasal or ocular discharge in three. All of the donkeys became infected with EAV as shown by recovery of the virus for periods of up to 14 days from the nasopharynx and buffy coat and, in three out of four donkeys sampled, from the cervix or vagina. Virus replication in the donkey appeared to mirror that previously described for the horse. The donkeys had “seroconverted” to EAV by the 10th day after inoculation. Additional studies are needed to obtain a better understanding of the pathogenesis of EAV in donkeys.

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV.

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).

A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay.In vitrostudies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells.In vivostudies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.

Three strains of mice, two susceptible to Ehrlichia risticii induced disease ( balb /c and C3H/HeJ) and one resistant ( akr/n ), were evaluated for the development of Immoral and cell mediated immune responses following infection with E risticii . The production of serum antibody was determined by indirect fluorescent antibody testing of sera from mice of each strain challenged with one of three different dose levels of E risticii . Antibody was assayed on days 7, 9, 12, 15 and 20 after inoculation. Cell mediated immune responses were evaluated by measuring the blastogenesis response of spleen cells from E risticii infected mice 28 days after inoculation. All three strains of mice at the high challenge level responded with the production of antibody by day 9 after inoculation. Overall, the antibody response occurred earlier and was of greater magnitude in the susceptible balb /c and C3H/HeJ strains. A marked blastogenesis response occurred in splenocytes from E risticii infected mice of all three strains upon re-exposure to ehrlichial antigen. The findings of this study indicate that susceptibility to E risticii induced disease was not the result of deficient or delayed humoral immune responses and that E risticii infection induced the development of strong cell mediated immunity.

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3 + T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3 + T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3 + T cells in vitro correlate with the outcome and severity of clinical signs in vivo . Thus, horses were divided into two groups based on their CD3 + T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3 + T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.

Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses ( n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions ( n = 4) that were persistently infected with EAV, and from horses ( n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.

Equine viral arteritis is reviewed with specific reference to clinical features, etiology, transmission, diagnosis, epidemiology, and current methods for the control of this disease. There is evidence of variation in pathogenicity among strains of equine arteritis virus. Virus transmission occurs primarily by the respiratory and venereal routes during the acute phase of the infection. The long-term carrier stallion appears to play a major epidemiological role in dissemination and perpetuation of the virus. Unlike the stallion, the carrier state has yet to be demonstrated in the mare or foal. A commercial modifiedlive equine arteritis virus vaccine has been shown to be safe and efficacious for stallions and mares. The disease can be controlled by identification and isolation of carrier stallions, immunization of seronegative stallions, and by restricting the breeding of equine arteritis virus-shedding stallions to equine arteritis virus vaccinated or seropositive mares.

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.