Norovirus mixed infection in an oyster-associated outbreak: an opportunity for recombination

We describe an outbreak of gastroenteritis in which the nucleic acid of three distinct noroviruses was amplified from the same fecal sample. To enable the separate amplification of each virus, an inclusion/exclusion RT-PCR primer design strategy was developed. This paired a virus-specific exclusion primer (designed with the exact sequence of one virus in a region displaying low conservation among the three viruses) with a virus-nonspecific inclusion primer (designed in a conserved region). Thus, in each reaction the exclusion primer provided specificity for a single virus, and the inclusion primer increased the sensitivity and allowed hybridization in a region of unknown sequence. Analysis of the partial genomic sequences of the three viruses (3.6-3.8 kb) indicated that each virus belonged to a separate genogroup II cluster, and each displayed evidence of a potential recombination event when the sequences were compared with other published norovirus sequences. Our results, which show a mixed norovirus infection in a single individual, confirm the need to be aware of the possibility of mixed norovirus infections, and of the possibility of genomic recombination causing anomalies in phylogenetic analyses in such instances.