Use of non-radioactive probes for VP4 typing of human rotaviruses.
The rotavirus outer capsid proteins VP4 and VP7 determine the P- and G-serotypes, respectively, of the virus. Three types of VP4 protein are commonly found in human rotaviruses (P4, P6 and P8) which are encoded by distinct VP4 gene alleles. We developed a non-radioactive Northern hybridization method for the P-genotyping of rotavirus field isolates. Double-stranded RNA was isolated from faecal specimens of rotavirus positive patients. Digoxigenin (DIG)-labelled cDNA probes derived from the VP4 gene of the standard strains RV5 (P4), ST3 (P6) and RV4 (P8) were used to discriminate between the different alleles. Although the P4 probe exhibited cross-reactivity with some P8 samples, the P6 and P8 probes were found to be type-specific. In addition, the probes did not react with standard strains representative of other defined human and animal rotavirus P-types. Use of these probes on viral RNA of faecal origin allowed approximately 70% of samples to be assigned a P-type. This method complements PCR- and EIA-based P-typing methods, is relatively inexpensive and is readily applicable to large numbers of samples, thus proving useful for epidemiological studies.
Gorrell
R J
RJ
Department of Gastroenterology, Royal Children's Hospital, Parkville, Victoria, Australia. gastro@cryptic.rch.unimelb.edu.au
Palombo
E A
EA
D015152:Blotting, Northern / Q000379:methods*; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D003362:Cost-Benefit Analysis; D015342:DNA Probes; D004076:Digoxigenin / Q000737:chemistry*; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*; D012401:Rotavirus / Q000145:classification* / Q000235:genetics*; D012680:Sensitivity and Specificity
D036022:Capsid Proteins; D015342:DNA Probes; D012367:RNA, Viral; C060339:VP4 protein, Rotavirus; D004076:Digoxigenin