REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION DETECTION AND SEQUENCE-ANALYSIS OF SMALL ROUND-STRUCTURED VIRUSES IN JAPAN

Between 1985 and 1995, mass outbreaks of acute gastroenteritis caused by small round-structured virus (SRSV), occurred in eight prefectures in Japan. Fecal samples from 59 patients ill during these outbreaks we re recently examined in our laboratory by electron microscopy (EM) and by reverse transcription-polymerase chain reaction (RT-PCR). For RT-P CR, we prepared two sets of primers, a set corresponding to the polyme rase region of open reading frame 1 (ORF-1) and a set corresponding to the capsid region of ORF-2 of Norwalk virus (NV). The SRSV nucleic ac id detection rate with these primers was more than double that achieve d with EM. Most samples found by EM to contain virus particles were al so positive by PCR. When the two sets of primers were used separately, the virus detection rate differed depending on the primer used, sugge sting that the viral strains examined were not genetically not homogen eous. We then selected nine strains of the virus, cloned their PCR pro ducts and analyzed their base sequences. The base sequences of these s trains were compared with those of reference strains including prototy pe NV and Snow Mountain agent (SMA). This comparison yielded the follo wing findings: (1) SRSVs that cause mass outbreaks of gastroenteritis in Japan are genetically variable; (2) SRSV strains that are genetical ly similar to SMA and SRSV-OTH25/89/J (OTH25) are dominant in Japan, b ut strains similar to NV are also present in this country; and (3) a s train (MI1/94) which is genetically identical to Southampton virus (SH V) was detected. Detection of SRSV using sensitive RT-PCR and analysis of the sequences of the amplification products seems to provide a use ful means of studying the molecular epidemiology of SRSV.