Nucleotide sequence analysis and development of consensus primers of RT-PCR for detection of Norwalk-like viruses...

A total of 177 different nucleotide sequences of the RNA polymerase region of Norwalk-like viruses (NLVs) genomes, collected via a nation-wide survey project in Japan between 1989 and 1998, were examined by reverse transcription-polymerase chain reaction (RT-PCR) employing various primer pairs. The nucleotide sequences of different strains showed great diversity, with a range of 57 to 100% identities among strains. The strains could be classified into five clusters: Norwalk (NV), Snow Mountain agent/Bristol virus (SMA/BV), Toronto virus/Mexico virus (TV/MX), and Japan specific cluster 1 and 2 (JP-1 and JP-2). Within each cluster there is greater than 85% identity of amino acid sequence (more than 75% identity of nucleotide sequences), based on sequence homology analysis. We believe that two of the five clusters, JP-1and JP-2, define new specific clusters found in Japan according to phylogenetic and pair-wise comparison studies. An RT-PCR procedure was designed using new consensus primer pairs, P1/P2, P1/P3, and Y1/Y2 based on multiple alignment of collected nucleotide sequences, that are expected to detect nearly all NLVs prevailing in Japan. The usefulness of the primers was tested by ten different laboratories in Japan using a panel of ten fecal samples containing different virus strains. The identification of these primer pairs will facilitate routine diagnosis of NLV infection by RT-PCR and offers the potential for their direct detection in food and environmental samples.