Norwalk Virus Genome Cloning and Characterization

TLDR

Norwalk and related viruses cause major epidemic outbreaks of acute gastroenteritis. The study aims to use Norwalk‑specific cDNA and genome sequence data to develop sensitive diagnostics and facilitate molecular biology research. Researchers purified Norwalk virus from infected volunteers, constructed recombinant cDNA, and generated clones that specifically hybridized to post‑infection stool and purified virus. Hybridization signals correlated with gastroenteritis symptoms, and analyses revealed a 7.5‑kb positive‑sense, polyadenylated RNA genome with a conserved RNA‑dependent RNA polymerase motif.

Abstract

Major epidemic outbreaks of acute gastroenteritis result from infections with Norwalk or Norwalk-like viruses. Virus purified from stool specimens of volunteers experimentally infected with Norwalk virus was used to construct recombinant complementary DNA (cDNA) and derive clones representing most of the viral genome. The specificity of the clones was shown by their hybridization with post- (but not pre-) infection stool samples from volunteers infected with Norwalk virus and with purified Norwalk virus. A correlation was observed between the appearance of hybridization signals in stool samples and clinical symptoms of acute gastroenteritis in volunteers. Hybridization assays between overlapping clones, restriction enzyme analyses, and partial nucleotide sequence information of the clones indicated that Norwalk virus contains a single-stranded RNA genome of positive sense, with a polyadenylated tail at the 3′ end and a size of at least 7.5 kilobases. A consensus amino acid sequence motif typical of viral RNA-dependent RNA polymerases was identified in one of the Norwalk virus clones. The availability of Norwalk-specific cDNA and the new sequence information of the viral genome should permit the development of sensitive diagnostic assays and studies of the molecular biology of the virus.