Double-stranded cDNA was synthesized from
nucleic acid extracted from Norwalk
virus purified from stool specimens of volunteers. One clone was isolated from a
cDNA library constructed in a pUC-13 vector after amplification of the cDNA. The specificity of this cDNA (pUCNV-953) was shown by hybridization assays. The cDNA reacted with post (but not pre-) infection stool samples from Norwalk volunteers and with highly purified Norwalk
virus, but not with other common enteric viruses such as
hepatitis A
virus and
rotavirus. Finally, the probe detected virus in the same fractions of CsCl gradients in which
viral antigen was detected using a specific Norwalk virus
radioimmunoassay, and particles were detected by immune
electron microscopy. Single-stranded
RNA probes derived from the
DNA clone after
subcloning into an
in vitro transcription vector were also used to show that the Norwalk virus contains a ssRNA
genome of about 8 kb in size. The original clone was also used to detect additional cDNAs which represent at least 7 kb of
nucleic acid of the Norwalk
genome. The availability of a Norwalk-specific cDNA and the first partial
genome sequence information allow rapid
cloning of the entire genome and of establishment of sensitive diagnostic assays. Such assays can be based on detection of Norwalk virus
nucleic acid or Norwalk
viral antigen using polyclonal or
monoclonal antibodies to proteins expressed from the cDNA or to synthetic peptides made based on the knowledge of the genome sequence. Assays using proteins deduced from the Norwlk virus genome and produced in expression systmes can measure
antibody responses. Vaccines made by
recombinant DNA technology are now feasible.