DETECTION AND IDENTIFICATION OF AQUATIC BIRNAVIRUSES BY PCR ASSAY

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was d eveloped for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and Pro) that we used are sp ecific for regions of cDNA coded by genome segment A of aquatic birnav iruses. PrA identifies a large fragment (1,180 bp) within the pVP2-cod ing region, and PrB identifies a 524-bp fragment within the sequence a mplified by PrA. Primer set PrC frames a genome fragment (339 bp) with in the NS-VP3-coding region, and Pro identifies a 174-bp sequence with in the fragment identified by PrC. PrB and Pro amplified cDNAs from al l nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results in dicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA r outinely produced amplification products from eight serotypes but exhi bited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT -PCR assay was evaluated by comparison of the results with those of ce ll culture isolation assays. With the exception of one sample, the RT- PCR assay with primer Pro was as accurate as cell culture isolation fo r detecting virus in kidney and spleen tissues from naturally infected , asymptomatic carrier fish. These results indicate that the RT-PCR as say can be a rapid and reliable substitute for cell culture methods fo r the detection of aquatic birnaviruses.