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.

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.

The equine arteritis virus isolate from the 2010 Argentinian outbreak.

A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.

Comparative analysis of ORF5 nucleotide sequences and amino acid sequences of the GP5 protein of equine arteritis virus (EAV) detected in the semen of stallions from Eastern Poland.

The purpose of this study was to conduct a comparative analysis of the ORF5 gene fragment nucleotide sequences and the GP5 protein amino acid sequences formed on this matrix, for the equine arteritis virus (EAV) strains isolated from the semen of infected stallions from Eastern Poland. The study covered 41 stallions whose blood serum tested positive for antigens specific to the EAV. The presence of EAV genetic material was shown in material from 5 horses, in one of which permanent presence of viral RNA was detected over the entire 4-year study period (the material was sampled four times at yearly intervals). The mutual similarity among the ORF5 nucleotide sequences of EAV obtained in our own studies was 90.7-99%, whereas their similarity to a sequence of an isolate of the PL1 virus, determined in Polish horses previously, was 76.6-83%. A comparison of the primary structure of capsid glycoprotein encoded by the analysed section of ORF5 showed that amino acid substitution happens most frequently in region V1 of GP5, between positions 61 and 121. A phylogenetic analysis of our own isolates with sequences of viruses isolated from horses from the USA, Europe and New Zealand (available in the gene bank), made it possible to determine that the majority of the detected strains of the pathogen can be classified into the European group, with the Austrian strain of EAV as its protoplast.

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.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

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.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.

Equine arteritis virus: a new isolate from the presumable first carrier stallion in Argentina and its genetic relationships among the four reported unique Argentinean strains.

Equine arteritis virus (EAV) was isolated from a testicle of the presumable first stallion infected with EAV in Argentina. This virus isolate (named LT-LP-ARG) was confirmed by GP5-specific PCR and indirect immunofluorescence assays. The PCR product was sequenced, and the phylogenetic analysis revealed that the LT-LP-ARG strain of EAV forms a monophyletic group, together with other strains previously isolated in our laboratory (LP02 group). However, all Argentinean EAV strains belong to a polyphyletic group. We believe that the virus isolate presented in this report could be the origin of EAV infection in our country.

Molecular epizootiology of equine arteritis virus isolates from Poland.

Phylogenetic analysis was performed on the sequences of 44 Polish isolates of equine arteritis virus that were isolated from the semen of stallions from national and private studs, collected during 2001--2005. These sequences were also compared with 41 reference strains previously described and commonly used in phylogenesis. On the basis of the nucleotide sequence analysis of the ORF5 gene, encoding the glycoprotein GP5, it was demonstrated that the Polish EAV isolates belonged to two subgroups and showed the closest relationship to the European strains. Similar results were obtained using the nucleotide sequences of the ORF7 gene. The nucleotide identity between the ORF5 and ORF7 sequences of all Polish isolates was in the range of 80.1-99.0% and 93.6-100%, respectively. The analysis of genetic diversity within the ORF5 sequences enabled a retrospective epizootic investigation. This study suggested that some of the EAV shedding stallions were probably infected before they were moved to Poland.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

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.

Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone.

We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions.

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

Phylogenetic characterisation of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark.

The study describes for the first time the phylogenetic relationship between equine arteritis virus (EAV) isolated from asymptomatic virus-shedding stallions and fatal cases of equine viral arteritis (EVA) in an European country. EAV was isolated from three dead foals and an aborted foetus during three different outbreaks of EVA. From these fatalities, the complete open reading frame 5, encoding the EAV G(L) protein, was amplified by reverse transcription-polymerase chain reaction and subjected to nucleotide sequence analysis. Furthermore, DNA sequences were obtained from virus isolated from semen samples of seven virus-shedding, but clinically healthy, Danish stallions. DNA sequence alignment revealed an overall divergence of 0-14 and 0-10% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis including 24 previously published sequences revealed that European as well as North American "types" of EAV were present in the semen of asymptomatic carrier stallions and in fatal cases of EVA. Our results reveal that the presence of EAV-shedding stallions in Denmark represents a potential source of severe EVA.

Nucleotide sequence and genetic analysis of the leader region of Canadian, American and European equine arteritis virus isolates.

The extreme 5' end, the entire leader sequence of the Arvac vaccine strain, and 10 equine arteritis virus (EAV) isolates, including the ATCC Bucyrus reference strain and 5 Canadian field isolates, were determined and compared at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates, including the Bucyrus reference strain, and the leader sequence of the Arvac vaccine strain was determined to be 206 nt in length (not including the putative 5' cap structure-associated nucleotide) whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences, between the different isolates and the Bucyrus reference strain, ranged from 94.2 to 98.5%. Phylogenetic analysis and estimation of genetic distances, based on the leader nucleic acid sequences, showed that all EAV isolates/strains are likely to represent a large phylogenetically-related group. An AUG start codon found at position 14 in all EAV isolates/strains could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Computer-predicted RNA secondary structures were identified in the 11 EAV leader regions analyzed. All EAV isolates/strains showed 3 conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in 7 EAV isolates, including the Bucyrus reference strain. The leader region distal to stem-loop D did not contain conserved sequences or stem-loop structures common to the EAV isolates/strains.

Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses.

Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody response to EAV. The responses of individual animals varied and ELISAs that utilized individual EAV structural proteins were not reliable for detecting antibodies in all sera that contained neutralizing antibodies to EAV. An ELISA based on a cocktail of all three EAV structural proteins, however, was used successfully to detect antibodies in most equine sera that were positive in the standard serum neutralization assay following natural or experimental EAV infection (100% specificity, 92.3% sensitivity). In contrast, this ELISA did not reliably detect antibodies in the sera of vaccinated horses. EAV frequently causes a persistent infection in stallions and all sera from carrier stallions evaluated in this study had obvious reactivity with the N protein, whereas seropositive non-carrier stallions, mares and geldings did not respond consistently to the N protein.

Genetic and amino acid analysis of the GL protein of Canadian, American and European equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) GL protein encoding gene was investigated. Nucleic and deduced amino acid sequences from 7 different EAV isolates, including 4 eastern Canadian field isolates, were compared with those of the Bucyrus reference strain. Nucleotide sequence identities between these isolates and the Bucyrus reference strain ranged from 87.5% (Vienna isolate) to 93.9% (11958 isolate). Amino acid identities with the Bucyrus reference strain varied from 90.2% (Vienna isolate) to 95.1% (19933 isolate). A 2nd potential N-linked glycosylation site was found at position 81 in the GL protein of all EAV isolates. Three amino acid substitutions at residue position 90 (Glu-->Val), position 101 (Ala-->Val or Thr), and position 119 (Val-->Leu, Phe or Ser) were also found in all EAV isolates. Phylogenetic analysis showed that the North American EAV isolates, including the Canadian isolates, and the European prototype Vienna isolate could be classified in 2 distinct groups. Three putative sequential antigenic sites were predicted in EAV GL protein. The 1st antigenic site (TAQRFT) was located at positions 24 to 29, and the 2nd antigenic site (RYDEHTA) at positions 47 to 53. The 3rd antigenic site was predicted to be located at positions 78 to 84 and showed the less conserved amino acid sequence.

Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance.

Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.

Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates.

cDNA copies of the M and N genes of equine arteritis virus (EAV) isolates were synthesized by reverse transcription followed by polymerase chain reaction amplification. The cDNA was subjected to a cycle sequencing strategy using Taq polymerase, and the nucleotide and derived amino acid sequences of 10 virus isolates were compared. The M and N genes of all isolates had the same initiation and termination sites as the prototype Bucyrus strain and the encoded proteins were conserved between viruses. Comparison of nucleotide sequence homologies and phylogenetic tree analysis implied the existence of three EAV variants originating from the U.S.A. (Bucyrus), Austria (Vienna) and Switzerland (Bibuna), and suggested that RNA recombination between EAV isolates may have occurred.

Detection of equine arteritis virus following amplification of structural and nonstructural viral genes by reverse transcription-PCR.

A reverse transcription (RT)-PCR assay was developed for the detection of equine arteritis virus (EAV) in cell culture supernatant and in horse semen. Four different sets of oligonucleotide primers complementary to sequences located in the 3' end of the polymerase gene (open reading frame [ORF] 1b) and to sequences representing the entire ORFs 3, 4, and 7, which encode for nonstructural (ORFs 3 and 4) or viral nucleocapsid (ORF 7) proteins, were compared for their abilities to amplify the targeted EAV sequences by the RT-PCR procedure. The sensitivities of the RT-PCR for amplification of EAV sequences located in the 3' end of ORF 1b and ORF 4 were 2 median tissue culture infective doses (TCID50s) of viral particles in the EAV-infected cell culture supernatant for both ORFs and 20 and 200 TCID50s of viral particles, respectively, in virus-containing horse semen. The sensitivities were much lower when primers complementary to ORFs 3 and 7 were used in the RT-PCR, with a minimum detection limit of only 2 x 10(4) TCID50s of viral particles in virally infected cell culture supernatant, as determined by analyzing the resulting RT-PCR products on ethidium bromide-stained agarose gels. The specificities of the RT-PCR assays for all primer sets tested were confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in the chemiluminescence assays. Although the sensitivity of the RT-PCR for amplification of ORF 3 and 7 sequences was lower, all sets or primers were capable of amplifying several cell culture-adapted EAV field isolates when the virus was present in high enough quanities in the test sample. When horse semen samples were analyzed for the presence of EAV by the RT-PCR with primers specific to the ORF 1b 3' end and ORF 4 sequences and by virus isolation in cell cultures, there was 100% concordance among the assays. The RT-PCR assay targeting the 3' end of ORF 1b and/or ORF 4 EAV RNA may be an alternative to conventional methods for the diagnosis of EAV infection in horses.