Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2007
Publication Date: 7/14/2007
Citation: McClenahan, S.D., Neill, J.D., Burek, K.A., Beckmen, K.B., Romero, C.H. 2007. Development of a real-time RT-PCR assay for the detection of marine caliciviruses [abstract]. American Society for Virology 26th Annual Meeting. Paper No. #W21-5. p. 126. Interpretive Summary:
Technical Abstract: More than forty different marine caliciviruses (family Caliciviridae, genus Vesivirus) have been identified from marine and terrestrial host since their initial isolation in 1972. Marine vesiviruses have previously infected swine along the Western coast of the United States and produce a disease clinically indistinguishable from foot-and-mouth disease, and other vesicular swine diseases such as vesicular stomatitis and swine vesicular disease. Current methods for identification of marine vesiviruses include RT-PCR and serological assays. These assays may fail to identify all positive samples, due to the high mutation rate of these RNA viruses and the resulting large number of serotypes and genotypes. We have developed a rapid and differential diagnostic real-time RT-PCR assay to identify marine vesiviruses. Primers were designed to amplify a conserved 176 nucleotide fragment of the A region of the capsid gene, based on multiple alignments of Vesivirus sequences in the GeneBank database and two novel genotypes of marine vesiviruses previously identified by our laboratory. A probe, sixteen bases in length, was designed within the amplicon and was labeled with a fluorescent dye for real-time detection. The probe includes locked nucleic acid (LNA) bases to increase the melting temperature and stability of the probe in the assay. This assay successfully identified ten different marine vesiviruses grown in cell culture, these viruses included San Miguel sea lion virus (SMSV) type 1, SMSV-2, SMSV-4, SMSV-5, SMSV-6, SMSV-13, SMSV-14, bovine calicivirus (Bos-1), and both Steller sea lion vesiviruses. This assay was shown to be specific for only the marine vesiviruses by the failure to amplify three isolates of feline calicivirus (FCV), another species within the Vesivirus genus. The assay described here can be used as a diagnostic tool to rapidly identify and differentiate marine vesiviruses from other viruses causing vesicular diseases.