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.

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.

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.

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.

Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the USA.

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.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported.

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

Persistent equine arteritis virus infection in HeLa cells.

The horse-adapted virulent Bucyrus (VB) strain of equine arteritis virus (EAV) established persistent infection in high-passage-number human cervix cells (HeLa-H cells; passages 170 to 221) but not in low-passage-number human cervix cells (HeLa-L cells; passages 95 to 115) or in several other cell lines that were evaluated. However, virus recovered from the 80th passage of the persistently infected HeLa-H cells (HeLa-H-EAVP80) readily established persistent infection in HeLa-L cells. Comparative sequence analysis of the entire genomes of the VB and HeLa-H-EAVP80 viruses identified 16 amino acid substitutions, including 4 in the replicase (nsp1, nsp2, nsp7, and nsp9) and 12 in the structural proteins (E, GP2, GP3, GP4, and GP5). Reverse genetic studies clearly showed that substitutions in the structural proteins but not the replicase were responsible for the establishment of persistent infection in HeLa-L cells by the HeLa-H-EAVP80 virus. It was further demonstrated that recombinant viruses with substitutions in the minor structural proteins E and GP2 or GP3 and GP4 were unable to establish persistent infection in HeLa-L cells but that recombinant viruses with combined substitutions in the E (Ser53-->Cys and Val55-->Ala), GP2 (Leu15-->Ser, Trp31-->Arg, Val87-->Leu, and Ala112-->Thr), GP3 (Ser115-->Gly and Leu135-->Pro), and GP4 (Tyr4-->His and Ile109-->Phe) proteins or with a single point mutation in the GP5 protein (Pro98-->Leu) were able to establish persistent infection in HeLa-L cells. In summary, an in vitro model of EAV persistence in cell culture was established for the first time. This system can provide a valuable model for studying virus-host cell interactions, especially virus-receptor interactions.

Identification of an additional neutralization determinant of equine arteritis virus.

We recently established an in vitro model of equine arteritis virus (EAV) persistence in HeLa cells. The objective of this study was to determine whether viral variants with novel neutralization phenotypes emerged during persistent EAV infection of HeLa cells, as occurs during viral persistence in carrier stallions. Viruses recovered from persistently infected HeLa cells had different neutralization phenotypes than the virus in the original inoculum, as determined by neutralization assays using EAV-specific monoclonal antibodies and polyclonal equine antisera raised against different strains of EAV. Comparative sequence analyses of the entire structural protein genes (ORFs 2a, 2b, and 3-7) of these viruses, coupled with construction of chimeric viruses utilizing an infectious cDNA clone of EAV, confirmed that the alterations in neutralization phenotype were caused by amino acid changes in the GP5 protein encoded by ORF5. Site-directed mutagenesis studies unequivocally confirmed that amino acid 98 in the GP5 protein was responsible for the altered neutralization phenotype of these viruses. Amino acid 98 in the GP5 protein, which has not previously been identified as a neutralization determinant of EAV, should be included in an expanded neutralization site D (amino acids 98-106).

The efficacy of a commercial ELISA as an alternative to virus neutralisation test for the detection of antibodies to EAV.

Infection with equine arteritis virus is a notifiable disease with sporadic occurrence in the UK. As stallions may harbour the virus after infection, horses are screened for exposure by serological testing prior to breeding. The virus neutralisation test is considered the 'gold standard' serological screening test, but it is time-consuming and labour intensive; consequently there is a move towards more rapid screening methodology. In this study, a commercially available EVA antibody ELISA is assessed. The ELISA performed poorly with a specificity [corrected] of 26% and a sensitivity [corrected] of 96% in the samples analysed. It was concluded that this ELISA would be of little value for reducing sample turnaround time. The study emphasises the need for in-house validation of commercially available kits.

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.

[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

[Case report: equine arteritis virus (EAV) as the cause of abortion in alpacas?]. / Fallbericht: Equines Arteritisvirus (EAV) als Aborterreger bei Alpakas?

Here we report a case of a late abortion of a primiparous alpaca where genome fragments of the equine viral arteritis virus (EAV) could be detected in fetal tissues using reverse transcription polymerase chain reaction (RT-PCR). All five alpacas of the herd had virus neutralizing antibodies against EAV. EAV thus must be regarded as a potential agent for abortions in alpacas. Possible routes of introduction of the virus are discussed.

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.

Antibodies against equine herpesviruses and equine arteritis virus in Burchell's zebras (Equus burchelli ) from the Serengeti ecosystem.

A total of 51 sera from a migratory population of Burchell's zebras (Equus burchelli) were collected in the Serengeti National Park (Tanzania) between 1999 and 2001 to assess levels of exposure to equine herpesvirus types 1, 2, 4, 9 (EHV-1, -2, -4, -9), EHV-1 zebra isolate T965, and equine arteritis virus (EAV). Using virus-specific neutralizing antibody tests, seroprevalence was high for EHV-9 (60% of 45), moderate for EAV (24% of 51), and lower for the EHV-1-related zebra isolate (17% of 41), EHV-1 (14% of 49), and EHV-4 (2% of 50). No evidence for exposure to EHV-2 was found (0% of 51). The high level of exposure to EHV-9 is interesting because evidence of infection with this virus has not been previously described in any wild equine population. Although the epidemiology of EHV-9 in Burchell's zebras is presently unknown, our results suggest that in East Africa, this species may be a natural host of EHV-9, a neuropathogenic virus that was only recently isolated from captive Thomson's gazelles (Gazella thomsoni) in Japan. There is currently no evidence that EHV-9 induced mortality in Burchell's zebras in the Serengeti, but because of the reported virulence of this virus for more susceptible species such as Thomson's gazelles, viral transmission from infected zebras to ungulates may result in mortality.

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.

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.

Expression cloning and antigenic analysis of the nucleocapsid protein of equine arteritis virus.

A series of recombinant fusion proteins derived from equine arteritis virus (EAV) open reading frame (ORF) 7 have been used to define the immunoreactive region of the viral nucleocapsid (N) protein. Reactivities of recombinant N fusion proteins with post-infection equine sera in immunoblots and ELISAs indicate that the major nucleocapsid protein epitope is located within amino acid residues 1-69. In ELISAs two recombinant nucleocapsid fusion proteins containing residues 1-69 (rN1-69) and 1-28 (rN1-28) discriminated between pre- and post-infection, and pre- and post-vaccination serum samples. Additionally rN1-69 and rN1-28 detected seroconversions following vaccination with a killed virus preparation, even in the absence of a detectable virus neutralising response. Although a good correlation existed between virus neutralising antibody and rN1-69 ELISA positive values in post-infection sera, all the rN proteins failed to induce any virus neutralising response in immunised rabbits.

Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus.

A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post-EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rGL 55-98 as a diagnostic antigen.