Detection and nucleotide sequence analysis of human caliciviruses (HuCVs) from samples in non-bacterial gastroenteritis outbreaks in Hokkaido, Japan

Samples of feces and vomit collected from patients during 13 non-bacterial gastroenteritis outbreaks which occurred in Hokkaido between 1995 and 1998 were examined by electron microscopy (ERI) and reverse-transcription polyme rase chain reaction (RT-PCR) for evidence of infection with human calicivir uses (HuCVs). In 6 food-borne outbreaks, oysters were the probable source o f infection, while the origin of HuCVs was not found out for the other 7 ou tbreaks, One-hundred-eleven of 214 stool, vomit and oyster specimens examin ed gave positive results by RT-PCR, while HuCVs were detected by ERI in 36 of 121 stool specimens examined. We determined the nucleotide sequences of 470-bp or 373-bp PCR products amplified from the RNA polymerase region of t he HuCV genomes with primer sets MR3/3 and Yuri22F/R, respectively. The seq uences of different strains revealed great heterogenicity, with a range of 60 to 100% homology among strains. In a few cases, a mixed genotype was fou nd in the same patient or same outbreak by means of nested PCR and cloning of PCR products into an appropriate vector. Of the 19 different strains fou nd, 4 strains could be classified as Norwalk virus (genogroup 1) and the ot her 15 strains as Snow Mountain agent (genogroup 2) based on genotyping wit h homology analysis. Furthermore, the strains belonging to genogroup 2 coul d be classified into 4 subgroups with more than 93% homology in amino acids among strains in the subgroup.