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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.

Norovirus was identified in 30 of 59 gastroenteritis outbreaks occurring in the state of Victoria, Australia in 2001 by RT-PCR and/or electron microscopy (EM). Norovirus outbreaks occurred in hostels/nursing homes (27%), hospitals (13%), youth refuges (3%), social gatherings associated with food consumption (27%), school outings/camps (13%), and pre-school/child-minding centers (17%). Norovirus outbreaks tended to occur in the warmer months. Phylogenetic analysis identified six clusters, one within genogroup 1 (G1) and five within genogroup 2 (G2). Cluster 1, which incorporates the G2 Camberwell/Lordsdale strains, was the most common (39% of outbreaks). In 2 of 27 outbreaks, strains from two G2 clusters, 1 and 5, occurred. Norovirus G2 was more common in the young and very old than in those in intermediate years. Norovirus G2 detection rate was higher in females than in males for adults (>15 years) and the susceptibility of adult females to norovirus G2 infection relative to males increased with age. In one outbreak analyzed, some sequences had a single base substitution, but this did not result in an amino acid (aa) change. The two most common norovirus clusters (G2 clusters 1 and 4) occurred in the capital of Victoria, Melbourne, and regional Victoria, but the least common clusters (G2 clusters 2 and 3 and G1 cluster 8) only occurred in inner Melbourne. Norovirus was occasionally detected by EM but not by RT-PCR. The occurrence of norovirus outbreaks is modulated by a large group of factors, which will have to be considered in any epidemiological model.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

A total of 6,226 fecal samples collected from 1980 to 1996 in the Australian states of Victoria, New South Wales, and Tasmania from individuals with gastroenteritis were tested for small round-structured viruses (SRSVs) and classical human caliciviruses (ClHuCVs) by electron microscopy. There were 223 samples positive for SRSVs, and nine positive for ClHuCVs. SRSVs were detected in individuals of all ages and were commonly associated with gastroenteritis outbreaks in nursing homes and hospitals. SRSVs were detected throughout the year, but were more common in the period from late winter to early summer in Australia (August to December). There were peaks of virus activity in the early 1980s and more recently in 1995 and 1996. Analyses by RT-PCR and sequencing of a segment of ORF1 encoding the putative RNA polymerase for SRSVs and ClHuCVs showed the presence of viruses belonging to several genogroups. Viruses of genogroup 1 (Norwalk/Southampton-like) and genogroup 3 (ClHuCVs) were relatively rare. Viruses of genogroup 2 (Snow Mountain-like) were common, and could be divided into two subgroups, one containing Toronto/Mexico-like viruses, the other Lordsdale/Camberwell-like viruses. The majority of viruses detected belonged to this latter subgroup.