Development of one-step TaqMan？ real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses

Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n？=？11) or ERBV (n？=？10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml).The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.The family Picornaviridae is a large family of viruses classified into several genera with extensive diversity in physical properties, antigenicity and mechanisms of pathogenesis [1]. Although there are many different picornaviruses with various degrees of relatedness, all share several features in common. The picornaviruses have a single-stranded positive-sense RNA genome with a 5'-end covalently linked to a VPg (virion protein genome-linked) protein. The RNA genome contains a 5′ untranslated region (UTR) with an internal ribosome entry site (IRES), a single open reading frame (ORF) encoding the viral capsid proteins and the viral replicase proteins, a 3′ UTR and a 3′ poly(A) tail [2]. The ORF is divided into three regions: P1 enc