Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis

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Abstract
Norwalk virus (NV) and other small round-structured viruses (SRSVs) are frequent causes of gastroenteritis outbreaks. The recent cloning and sequencing of the NV genome has made it possible to detect NV and Norwalk-related viruses from fecal specimens by reverse transcription (RT)-PCR. We applied this technique to the examination of a total of 139 fecal specimens from 19 outbreaks characterized by NV serology, including 56 samples from 7 NV outbreaks, 36 from 6 Norwalk-related virus outbreaks, and 47 from 6 outbreaks with SRSVs visualized by electron microscopy that were serologically unrelated to NV. Three primer pairs were evaluated: two pairs in the polymerase region of NV and one pair near the 3' end of the genome. When one set of primers (primer pair 51-3) from the polymerase region was used, 40% of all samples were positive by RT-PCR and specimens from the NV outbreaks were more likely to be positive (64%) than those from outbreaks associated with Norwalk-related viruses (44%) or SRSVs (8%). To determine the relationship of the outbreak strains to NV, we compared the sequences of a 145-base portion of the polymerase gene from 10 specimens obtained from five different outbreaks characterized as NV by serology. No two outbreak strains had the same sequence in this 145-base portion of the polymerase gene, and the identities of the nucleotide and amino acid sequences of these products compared with the sequences of the corresponding region of NV ranged from 62 to 79% and 69 to 90%, respectively. Because of sequence diversity in the polymerase region, the successful application of RT-PCR to investigations of outbreaks of suspected NV-associated gastroenteritis will depend on the use of either multiple primer pairs or primers made against regions of the genome that are more conserved.