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.

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

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.

Variation in ORF3 of genogroup 2 Norwalk-like viruses.

The nucleotide sequences of the 3'-terminal open reading frame (ORF3) and 3' untranslated region (3'UTR) were determined for four Norwalk-like viruses (NLVs) belonging to genogroup 2. Three of the viruses, isolated in 1995 and 1996, were closely related to Mexico virus (92-93% nucleotide identity in ORF3). The fourth virus, isolated in 1984, was unique, showing only 49-58% nucleotide identity with other NLVs. The variation in sequence of the 3'-terminal ORF of NLVs was greater than that observed for other caliciviruses. This variation was partly due to repeated sequences and frameshifting. To investigate the properties of the ORF3 encoded polypeptide, a signal sequence and N-linked glycosylation sites predicted for Camberwell virus were tested for function by in vitro translation in the presence of microsomes. Membrane insertion, cleavage of an N-terminal signal sequence, or glycosylation were not detected.

Combined infection of Norwalk-like virus and verotoxin-producing bacteria associated with a gastroenteritis outbreak.

Detection of multiple pathogens, particularly a combination of viruses and bacteria, is infrequently documented in outbreaks of gastroenteritis. This paper reports the presence of Norwalk-like virus (NLV) and enterohaemorrhagic verotoxin-producing Escherichia coli in one individual, and NLV and verotoxin-producing Aeromonas sobria in another individual, both part of a large gastroenteritis outbreak. The causes of gastroenteritis in such outbreaks may be more complex than previously thought.

Small round-structured (Norwalk-like) viruses and classical human caliciviruses in southeastern Australia, 1980-1996.

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.

Hospital outbreak of Norwalk-like virus.

An outbreak of gastroenteritis caused by Norwalk-like virus occurred in two areas of the hospital: area 1, consisting of three adjacent and interconnected wards, with mostly elderly patients; and area 22, an acute ward in a separate building with elderly patients. In area 1, 40 patients and 20 staff were affected; in area 2, 18 patients and 14 staff were affected. Infection control measures were instituted in consultation with the government health authority. These measures did not appear to affect the course of the outbreak, but may have prevented spreads to the other wards.