Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA

The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 don keys, with neutralising antibodies against EAV, on the basis of the amplifi cation of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and show ed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequ enced. Comparing the sequence of reference strain Bucyrus, the analysed sam ples were 78-100% identical and showed a 84-97% nucleotide identity with Bu cyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extr acted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that t his could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be s ensitive, specific and above all directly applicable to semen. Additionally , the short time needed for the overall procedure makes this method suitabl e for diagnostic purposes. (C) 1999 Elsevier Science Ltd. All rights reserv ed.