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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.

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Genetically distinct genogroup III noroviruses were detected in two sample pools from different geographic regions and bovine coronavirus was detected in a third pool of samples. This is the first report of bovine norovirus infection in Australian cattle and suggests that future work is required to determine the significance of these agents as a cause of bovine enteric disease in Australia.