Molecular epidemiology of norovirus gastroenteritis outbreaks in New Zealand from 2002-2009.

Noroviruses are the most common cause of acute non-bacterial gastroenteritis outbreaks worldwide, including New Zealand. New Zealand has a population of 4.4 million, which allows for centralized outbreak surveillance and a Norovirus Reference Laboratory, which facilitates efficient diagnosis, surveillance, and tracking of norovirus outbreaks. Norovirus outbreak strains are identified, sequenced, and compared with international reference strains. Between January 2002 and December 2009, 1,206 laboratory-confirmed norovirus outbreaks were recorded. The predominant outbreak settings were healthcare institutions for the elderly and acute care patients. Other outbreak settings included catering establishments, cruise ships, homes, community events, school camps, child-related settings, and consumption of contaminated shellfish. Of the 1,206 outbreaks, 105 (8.7%) were caused by norovirus genogroup I (GI) strains, 1,085 (89.9%) were caused by genogroup II (GII) strains, and both GI and GII strains were detected in 9 (0.8%) outbreaks. The genogroup was not identified in 7 (0.6%) outbreaks. A range of norovirus genotypes, including GI genotypes 1-6, GII genotypes 2-8, and GII.12, were associated with these outbreaks. The predominant genotype was GII.4, which was identified in 825 (68.4%) outbreaks. Norovirus GII.4 variant strains, including 2002 (Farmington Hills), 2004 (Hunter), 2006a (Laurens, Yerseke), 2006b (Minerva), and 2010 (New Orleans) implicated in overseas outbreaks also occurred in New Zealand, providing evidence of global spread.

Molecular detection of norovirus in sheep and pigs in New Zealand farms.

Human norovirus (NoV) is reportedly the major cause of non-bacterial gastroenteritis outbreaks worldwide and is commonly associated with water- and food-borne transmission via the faecal-oral route. Aside from humans, norovirus has been detected in pigs, cattle and mice. The close relatedness of some human and animal noroviruses has raised concerns about potential zoonotic transmission. Our laboratory recently reported the development of a multiplex real-time RT-PCR for the detection and genotyping of norovirus of genogroups I-III. Here we report a study of 56 faecal specimens from pigs and sheep that were collected and screened for noroviruses using this assay. Norovirus was found in 2/23 (9%) of porcine specimens (all were genogroup II) and in 8/33 (24%) of ovine specimens (all were genogroup III). Samples tested positive for norovirus were verified by conventional RT-PCR with different primer sets. Genomes of representative porcine and ovine norovirus strains underwent partial sequence analysis (343 and 2045 bases, respectively). This is the first report describing norovirus in sheep.

Detection and characterization of F+ RNA bacteriophages in water and shellfish: application of a multiplex real-time reverse transcription PCR.

Genotyping of F+ RNA bacteriophages has been used to distinguish between human and animal contributions to contaminated water and food. There are four genetically distinct genogroups of F+ RNA bacteriophages. Genogroups I and IV predominate in animal wastes and genogroups II and III in wastes of human origin. In this study, a multiplex real-time RT-PCR-based method was developed to detect and genotype F+ RNA bacteriophages. The assay was shown to be broadly reactive against a wide spectrum of F+ RNA bacteriophage strains, including MS2, GA, Q beta, MX1, SP and FI, and was able to detect and genotype F+ RNA bacteriophages in shellfish and river water. The assay is highly sensitive, with detection limits <10 PFU/reaction and <10 copies/reaction of the target sequences carried in plasmids, respectively. The applications of this assay include F+ RNA semi-quantitation and microbial source tracking.

Gastroenteritis outbreak caused by waterborne norovirus at a New Zealand ski resort.

In July 2006, public health services investigated an outbreak of acute gastroenteritis among staff and visitors of a popular ski resort in southern New Zealand. The source of the outbreak was a drinking water supply contaminated by human sewage. The virological component of the investigation played a major role in confirming the source of the outbreak. Drinking water, source stream water, and 31 fecal specimens from gastroenteritis outbreak cases were analyzed for the presence of norovirus (NoV). Water samples were concentrated by ultrafiltration, and real-time reverse transcription-PCR (RT-PCR) was used for rapid detection of NoV from both water and fecal samples. The implicated NoV strain was further characterized by DNA sequencing. NoV genogroup GI/5 was identified in water samples and linked case fecal specimens, providing clear evidence of the predominant pathogen and route of exposure. A retrospective cohort study demonstrated that staff who consumed drinking water from the resort supply were twice as likely to have gastroenteritis than those who did not. This is the first time that an outbreak of gastroenteritis in New Zealand has been conclusively linked to NoV detected in a community water supply. To our knowledge, this is the first report of the use of ultrafiltration combined with quantitative real-time RT-PCR and DNA sequencing for investigation of a waterborne NoV outbreak.

Sensitive multiplex real-time reverse transcription-PCR assay for the detection of human and animal noroviruses in clinical and environmental samples.

In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishes between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.