A liquid-hybridization method for typing the Vp4 and Vp7 genes of bovine rotaviruses.
A simple liquid-hybridization assay was developed which allows assessment of the degree of hybridization between the two serotype-determining genes of the bovine rotavirus strain UK and the homologous genes of the isolate under test. 32P-labelled transcription probes were produced from cloned complementary DNA (cDNA) copies of UK gene segments 4 and 8 and hybridized to double stranded RNA (dsRNA) extracted from rotavirus-positive field samples. Subsequent treatment with ribonuclease A (RNase A), separation of the RNase A-resistant hybrid fragments by polyacrylamide gel electrophoresis (PAGE) and autoradiography yielded a specific, reproducible banding pattern for each isolate. A total of 74 field samples was tested by both the hybridization assay and by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies (Mabs). The results obtained were in excellent agreement and confirmed that serotype G6 rotaviruses predominated. Hybridization of these G6 viruses with the gene 4 probe suggested that viruses with Vp4s related to that of UK rotavirus are also common. The hybridization assay was more sensitive than the ELISA.
Redmond
D L
DL
Moredun Research Institute, Edinburgh, UK.
Inglis
N F
NF
Fitzgerald
T A
TA
Snodgrass
D R
DR
Herring
A J
AJ
D000818:Animals; D000956:Antigens, Viral; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002417:Cattle; D005069:Evaluation Studies as Topic; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015347:RNA Probes; D012367:RNA, Viral / Q000235:genetics; D012401:Rotavirus / Q000145:classification* / Q000235:genetics*; D012703:Serotyping / Q000379:methods; D014773:Virology / Q000379:methods*
D000956:Antigens, Viral; D036022:Capsid Proteins; D015347:RNA Probes; D012367:RNA, Viral; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus