IDENTIFICATION OF GENE-4 ALLELES AMONG HUMAN ROTAVIRUSES BY POLYMERASE CHAIN REACTION-DERIVED PROBES

Four different alleles of the gene encoding VP4, an outer capsid rotavirus protein, have been recognized among human rotaviruses. To investigate the relative frequency of these alleles, hybridization probes corresponding to a hyperdivergent region (nucleotides 205 to 554) of the fourth gene from three human rotavirus strains, Wa, DS1, and M37 (each of which has a distinct gene four allele), were synthesized by the polymerase chain reaction (PCR) method, using as templates either cloned DNA or cDNA. The latter were obtained by reverse transcription of viral RNA from each of the three strains. The probes were hybridized to heat-denatured rotavirus double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA) which was directly dotted onto nylon membranes. Under hybridization conditions of high stringency (50% formamide, 4× SSC, 54°), it was possible to detect this region in as little as 4 ng of total rotavirus RNA. The probes enabled the detection and classification of these three gene 4 alleles in rotavirus strains recovered from field studies. © 1990.