Abstract
A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited 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 cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.
Full Text
The Full Text of this article is available as a PDF (243.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Caswell-Reno P., Lipipun V., Reno P. W., Nicholson B. L. Use of a group-reactive and other monoclonal antibodies in an enzyme immunodot assay for identification and presumptive serotyping of aquatic birnaviruses. J Clin Microbiol. 1989 Sep;27(9):1924–1929. doi: 10.1128/jcm.27.9.1924-1929.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dobos P., Hill B. J., Hallett R., Kells D. T., Becht H., Teninges D. Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J Virol. 1979 Nov;32(2):593–605. doi: 10.1128/jvi.32.2.593-605.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duncan R., Dobos P. The nucleotide sequence of infectious pancreatic necrosis virus (IPNV) dsRNA segment A reveals one large ORF encoding a precursor polyprotein. Nucleic Acids Res. 1986 Jul 25;14(14):5934–5934. doi: 10.1093/nar/14.14.5934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heppell J., Berthiaume L., Tarrab E., Lecomte J., Arella M. Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles. J Gen Virol. 1992 Nov;73(Pt 11):2863–2870. doi: 10.1099/0022-1317-73-11-2863. [DOI] [PubMed] [Google Scholar]
- Håvarstein L. S., Kalland K. H., Christie K. E., Endresen C. Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae. J Gen Virol. 1990 Feb;71(Pt 2):299–308. doi: 10.1099/0022-1317-71-2-299. [DOI] [PubMed] [Google Scholar]
- Manning D. S., Leong J. C. Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus. Virology. 1990 Nov;179(1):16–25. doi: 10.1016/0042-6822(90)90268-v. [DOI] [PubMed] [Google Scholar]
- McAllister P. E., Schill W. B. Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses. J Wildl Dis. 1986 Oct;22(4):468–474. doi: 10.7589/0090-3558-22.4.468. [DOI] [PubMed] [Google Scholar]
- Rimstad E., Hornes E., Olsvik O., Hyllseth B. Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments. J Clin Microbiol. 1990 Oct;28(10):2275–2278. doi: 10.1128/jcm.28.10.2275-2278.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolf K., Quimby M. C. Fish viruses: buffers and methods for plaquing eight agents under normal atmosphere. Appl Microbiol. 1973 Apr;25(4):659–664. doi: 10.1128/am.25.4.659-664.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]