DETECTION AND DIFFERENTIATION OF ANTIGENICALLY DISTINCT SMALL ROUND-STRUCTURED VIRUSES (NORWALK-LIKE VIRUSES) BY REVERSE TRANSCRIPTION PCR AND SOUTHERN HYBRIDIZATION

Application of reverse transcription (RT)-PCR to detect small round structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polymerase gene of SRSVs belonging to two distinct genogroups represented by Norwalk virus (primers G-1) and Snow Mountain agent (primers G-2). All 22 SRSV strains examined that had been classified previously by solid-phase immune electron microscopy into four antigenic types (UK1, UK2, UK3, and UK4) could be detected by RT-PCR with these two primer sets. The G-l primer set detected 6 UK2 strains, and the G-2 primers detected 16 strains, including 7 UK1, 5 UK3, and 4 UK4 strains. On the basis of nucleotide sequences of 81-bp fragments of the RT-PCR products from 13 strains determined in this study, together with those previously reported for 17 SRSV strains, we designed four sets of internal oligonucleotide probes (P1-A, P1-B, P2-A, and P2-B) for Southern hybridization, using chemiluminescent detection. The P1-A probe hybridized with PCR products from the UK2 strains; the P1-B probe, with products from two of the seven UK1 strains; the P2-A probe, with four of the remaining five UK1 strains; and the P2-B probe, with products from both UK3 and UK4 strains, as well as with one strain originally typed as UR1,which showed cross-reactivity with UK4 upon retesting by solid-phase immune electron microscopy. RT-PCR, with both the G-l and the G-2 primer sets can increase the detection rate of the many antigenically distinct SRSVs and, when combined with Southern hybridization, may predict the antigenic type of the SRSV associated with infection.