Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis.

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.

Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant.

Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference standard. The detection limit for the Bucyrus EAV strain was 36 PFU, and the method had a good linear relationship between 72-2297 PFU. The AC-ELISA could specifically detect the Bucyrus EAV strain and had no cross-reaction with other equine viruses. The sensitivity of the AC-ELISA was much higher than that of a western blotting assay but lower than that of a real-time PCR method. However, as a quantitative antigen detection method, the sensitivity of the AC-ELISA was approximately 300 times than the western blotting assay. Furthermore, the AC-ELISA assay could be successfully used in quantification of viral content in an in vitro infection assay, such as a one-step growth curve of EAV, as well as in a transfection assay, such as virus rescue from an infectious cDNA clone of EAV. These results show that the AC-ELISA established in this study is a good alternative for antigen detection of EAV, being a simple, convenient and quantitative detection method for EAV antigens.

Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions.

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.

Occurrence of viral diseases in donkeys (Equus asinus) in São Paulo State, Brazil / Ocorrência de enfermidades virais em asininos (Equus asinus) no estado de São Paulo, Brasil

Among the diseases that affect equines, viral diseases play an important role from a health and economic point of view, especially influenza, viral arteritis, herpes infections and vesicular stomatitis. In the Brazilian literature, there is little or no account of the occurrence of infectious diseases in donkeys. Given the importance of donkeys in different activities and the lack of information on infections that may occur in these animals, the aim of this study was to determine the frequency of anti-equine herpesvirus (EHV), anti-equine arteritis virus (EAV), anti-vesicular stomatitis, and anti-equine influenza (H3N8) antibodies in the serum of 85 donkeys bred in some regions of the state of São Paulo. We found the following antibody frequencies: 50.6% (43/85) antibodies against influenza virus subtype H3N8, 47% (40/85) anti-EHV, and 20% (17/85) anti-EAV. The donkeys were not seropositive for vesicular stomatitis. The results suggested that the agents EHV, EAV, and equine influenza subtype H3N8 circulate among donkeys in some regions of the state of São Paulo, Brazil, reinforcing the importance of establishing a routine diagnosis and epidemiological study of this species.(AU)

Dentre as doenças que acometem os equídeos, as enfermidades virais assumem um papel importante do ponto de vista sanitário e econômico, especialmente a influenza, arterite viral, as infecções herpéticas e a estomatite vesicular. Na literatura nacional, existe pouco ou nenhum relato sobre a ocorrência de enfermidades infecciosas nos asininos. Tendo em vista a importância dos asininos para diferentes atividades e a falta de informações sobre as doenças que acometem esses animais, este trabalho teve como objetivo estudar a frequência de anticorpos anti-EHV, antivírus da arterite equina, anti-estomatite vesicular e anti-influenza equina (H3N8) em 85 soros de jumentos criados no estado de São Paulo. Estimou-se que 50,6% apresentavam anticorpos contra o subtipo H3N8 do vírus da influenza; 47% (40/85) apresentavam anticorpos contra o EHV e 20% apresentavam anticorpos contra o vírus da arterite. Os jumentos não foram soro reagentes contra a estomatite vesicular. Os resultados obtidos sugerem que os agentes EHV, vírus da arterite equina e influenza equina subtipo H3N8, circulam entre os jumentos do estado de São Paulo, caracterizando a importância do estabelecimento de uma rotina diagnóstica e estudos epidemiológicos na espécie.(AU)

Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

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.

Enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using Equine arteritis virus purified by anion-exchange membrane chromatography.

In an effort to improve a competitive blocking enzyme-linked immunosorbent assay (cELISA) for antibody detection to Equine arteritis virus (EAV), antigen purified by anion-exchange membrane chromatography capsule (AEC) was evaluated. Virus purification by the AEC method was rapid and easily scalable. A comparison was made between virus purified by the AEC method with that obtained by differential centrifugation based on the following: 1) the relative purity and quality of EAV glycoprotein 5 (GP5) containing the epitope defined by monoclonal antibody 17B7, and 2) the relative sensitivity of a commercial antibody cELISA with the only change being the 2 purified antigens. On evaluation by Western blot using GP5-specific monoclonal antibody 17B7, the AEC-purified EAV contained 86% GP5 monomer whereas the differentially centrifuged EAV contained <29% of the monomer. Improvement of analytical sensitivity without sacrifice of analytical specificity was clearly evident when cELISAs prepared with EAV antigen by each purification method were evaluated using 7 sensitivity and specificity check sets. Furthermore, the AEC-purified EAV-based cELISA had 30-40% higher agreement with the virus neutralization (VN) test than the cELISA prepared with differentially centrifuged EAV based on testing 40 borderline EAV-seropositive samples as defined by the VN test. In addition, the AEC-purified cELISA had highly significant (P = 0.001) robustness indicated by intra-laboratory repeatability and interlaboratory reproducibility when evaluated with the sensitivity check sets. Thus, use of AEC-purified EAV in the cELISA should lead to closer harmonization of the cELISA with the World Organization for Animal Health-prescribed VN test.

Identification of a Novel Conserved B Cell Epitope in the N Protein of Equine Arteritis Virus (Bucyrus Strain).

The nucleocapsid (N) protein is the most conserved structural protein in equine arteritis virus (EAV). This study aimed to identify the minimal conserved B cell epitope on the EAV N protein. The purified N protein was used to immunize mice for preparing monoclonal antibody (mAb). The reactivity of mAb was evaluated by Western blot and immunofluorescence assay. Moreover, 11 overlapping peptides (named MBP-N1 to MBP-N11) were designed to localize the linear antigenic epitope within the N protein. The peptides were identified by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot. The minimal conserved B cell epitope on the EAV N protein was identified. The homology analysis was also performed. An EAV N-reactive mAb was selected and designated as 1C11. Indirect ELISA results showed that overlapping domain between MBP-N10 and MBP-N11 was recognized by the mAb 1C11. Furthermore, the indirect ELISA and Western blot showed that (101)QRKVAP(106) was the minimal linear epitope of the EAV N protein. The homology analysis showed that the identified epitope was conserved among all EAV strains analyzed in this work, with the exception of the ARVAC. One EAV N-specific mAb (1C11) was developed, and a minimal linear peptide epitope ((101)QRKVAP(106)) within the N protein was identified.

In vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2.

Arteriviruses are a family of positive-stranded RNA viruses that includes the prototypic equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). Although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of PRRSV. The ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. We have recently shown that the deubiquitinase (DUB) activity of EAV papain-like protease 2 (PLP2) is important for the inhibition of innate immune activation during infection. A vaccine virus lacking PLP2 DUB activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its DUB-competent counterpart. To test this hypothesis, twenty Shetland mares were randomly assigned to one of three groups. Two groups were vaccinated, either with DUB-positive (n=9) or DUB-negative (n=9) recombinant EAV. The third group (n=2) was not vaccinated. All horses were subsequently challenged with the virulent KY84 strain of EAV. Both vaccine viruses proved to be replication competent in vivo. In addition, the DUB-negative virus provided a similar degree of protection against clinical disease as its DUB-positive parental counterpart. Owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of PLP2 DUB activity could not be detected under these experimental conditions. Taken together, the data obtained in this study warrant further in vivo investigations into the potential of using DUB-mutant viruses for the improvement of arterivirus vaccines.

Membrane proteins of arterivirus particles: structure, topology, processing and function.

Arteriviruses, such as equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), are important pathogens in veterinary medicine. Despite their limited genome size, arterivirus particles contain a multitude of membrane proteins, the Gp5/M and the Gp2/3/4 complex, the small and hydrophobic E protein and the ORF5a protein. Their function during virus entry and budding is understood only incompletely. We summarize current knowledge of their primary structure, membrane topology, (co-translational) processing and intracellular targeting to membranes of the exocytic pathway, which are the budding site. We profoundly describe experimental data that led to widely believed conceptions about the function of these proteins and also report new results about processing steps for each glycoprotein. Further, we depict the location and characteristics of epitopes in the membrane proteins since the late appearance of neutralizing antibodies may lead to persistence, a characteristic hallmark of arterivirus infection. Some molecular features of the arteriviral proteins are rare or even unique from a cell biological point of view, particularly the prevention of signal peptide cleavage by co-translational glycosylation, discovered in EAV-Gp3, and the efficient use of overlapping sequons for glycosylation. This article reviews the molecular mechanisms of these cellular processes. Based on this, we present hypotheses on the structure and variability of arteriviral membrane proteins and their role during virus entry and budding.

Equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein 1 as a main interferon antagonist.

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-ß was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-ß mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-ß promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.

Development of a peptide ELISA for the diagnosis of Equine arteritis virus.

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations.

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

Currently, little is known on the cellular pathogenesis of equine arteritis virus (EAV). The purpose of the present study was to identify the target cells in ponies experimentally inoculated with EAV 08P178 (EU, clade-1). EAV-target organs (respiratory tissues with associated lymphoid tissues and large intestines), collected at 3 and 7 days post inoculation (dpi) and with virus titers≥10(5.0) TCID50/g, were processed with double immunofluorescence staining for the simultaneous detection of EAV N-protein and one of the following cell markers: CD172a (myeloid cells), CD3 (T lymphocytes), IgM (B lymphocytes) and von Willebrand factor (endothelial cells). In the different analyzed organs, 31-58% and 47-63% of the EAV-positive cells were mononuclear leukocytes (mainly CD172a(+) followed by CD3(+)) at 3 and 7 dpi, respectively. EAV-positive endothelial cells were not detected in 3.200 large blood vessels (≥3 endothelial cells/vessel cross section). However, in terminal capillaries (1-2 endothelial cells/vessel cross section) of the different organs, 15-51% of the endothelial cells were EAV-positive. In conclusion, the present study demonstrates that EAV 08P178 (i) has a main tropism for CD172a(+) and CD3(+) mononuclear leukocytes and (ii) infects a large number of endothelial cells in terminal capillaries. EAV 08P178 infection in capillaries is most probably the cause of an increased vascular permeability leading to leakage of fluid (edema-serous exudate) but not to severe vasculitis and hemorrhages.

Identification of a conserved B-cell epitope in the equine arteritis virus (EAV) N protein using the pepscan technique.

The nucleocapsid (N) gene of equine arteritis virus (EAV) is highly conserved between isolates, and the N protein is an important antigen that induces immunity when horses are infected with EAV. This study describes the identification of a linear B-cell epitope on the N protein using the pepscan technique with a monoclonal antibody (mAb) 2B1 directed against the N protein. The N protein was divided into 11 overlapping peptides, each containing 16 amino acids associated with six overlapping amino acids. The fragments were expressed as MBP fusion proteins that were then used to probe the 2B1 mAb. The minimal epitope sequence was confirmed step-by-step using single amino acid residue deletion. One completely conserved linear epitope ((38)KPPAQP(43)) was identified that matched with EAV-positive serum in Western blots, thereby revealing the importance of these six amino acids of the epitope for antibody-epitope binding activity. This finding not only contributes to our understanding of the antigenic structure of the N protein of EAV but also has potential for the development of diagnostic techniques.

Equine arteritis virus.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. There has been significant recent progress in understanding the molecular biology of EAV and the pathogenesis of its infection in horses. In particular, the use of contemporary genomic techniques, along with the development and reverse genetic manipulation of infectious cDNA clones of several strains of EAV, has generated significant novel information regarding the basic molecular biology of the virus. Therefore, the objective of this review is to summarize current understanding of EAV virion architecture, replication, evolution, molecular epidemiology and genetic variation, pathogenesis including the influence of host genetics on disease susceptibility, host immune response, and potential vaccination and treatment strategies.

Comparison of an improved competitive enzyme-linked immunosorbent assay with the World Organization for Animal Health-prescribed serum neutralization assay for detection of antibody to Equine arteritis virus.

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.

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection.

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.

Genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus 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.

Development of an antigen-capture ELISA for the detection of equine influenza virus nucleoprotein.

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for the detection of the equine influenza virus (EIV), employing monoclonal and polyclonal antibodies against the A/equine/Xingjiang/2007 (H3N8) nucleoprotein (NP). Immunoglobulin G antibodies were purified and used as capture or detector antibodies. The specificity of the optimized AC-ELISA was evaluated using EIV, equine herpesvirus 1 (EHV-1), equine herpesvirus 4 (EHV-4), equine arteritis virus (EAV) and Japanese encephalitis virus (JEV), resulting in only EIV specimens yielding a strong signal. A minimal concentration of 50 ng/ml EIV protein was detected in Nonidet P40-treated virus preparations. Virus from the nasal swabs of equines infected experimentally were detected from days 3 to 7 post-infection using this AC-ELISA, with results confirmed by virus isolation and multi reverse transcription polymerase chain reaction. Both H3N8 and H7N7 EIV subtypes were AC-ELISA positive, indicating that this assay is suitable for the detection of all EIV subtypes.

Evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period.

OBJECTIVE: To determine whether it is safe to vaccinate pregnant or postpartum mares with a commercial modified-live virus vaccine against equine viral arteritis (EVA). Design-Randomized controlled study. Animals-73 mares and their foals. PROCEDURES: Mares were vaccinated during mid gestation, during late gestation, or 2 or 3 days after parturition with a commercial modified-live virus vaccine or were not vaccinated. Foaling outcomes were recorded, and serum, blood, milk, and nasopharyngeal samples were obtained. RESULTS: All mares vaccinated during mid gestation foaled without any problems; 21 of 22 mares in this group had antibody titers against EAV at the time of foaling. Of the 19 mares vaccinated during late gestation, 3 aborted; antibody titers against EAV were detected in 13 of 15 mares from which serum was obtained at the time of foaling. All postparturient vaccinates were seronegative at foaling; all of them seroconverted after vaccination. No adverse effects were detected in any of their foals. CONCLUSIONS AND CLINICAL RELEVANCE: When faced with a substantial risk of natural exposure to EAV, it would appear to be safe to vaccinate healthy pregnant mares up to 3 months before foaling and during the immediate postpartum period. Vaccinating mares during the last 2 months of gestation was associated with a risk of abortion; this risk must be weighed against the much greater risk of widespread abortions in unprotected populations of pregnant mares naturally infected with EAV.

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen.

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.