Multiple outbreaks of a novel norovirus GII.4 linked to an infected post-symptomatic food handler.

Multiple norovirus outbreaks following catered events in Auckland, New Zealand, in September 2010 were linked to the same catering company and investigated. Retrospective cohort studies were undertaken with attendees of two events: 38 (24·1%) of 158 surveyed attendees developed norovirus-compatible illness. Attendees were at increased risk of illness if they had consumed food that had received manual preparation following cooking or that had been prepared within 45 h following end of symptoms in a food handler with prior gastroenteritis. All food handlers were tested for norovirus. A recombinant norovirus GII.e/GII.4 was detected in specimens from event attendees and the convalescent food handler. All catering company staff were tested; no asymptomatic norovirus carriers were detected. This investigation improved the characterization of norovirus risk from post-symptomatic food handlers by narrowing the potential source of transmission to one individual. Food handlers with gastroenteritis should be excluded from the workplace for 45 h following resolution of symptoms.

Enteric viruses in New Zealand drinking-water sources.

This study determined whether human pathogenic viruses are present in two New Zealand surface waters that are used as drinking-water sources. Enteric viruses were concentrated using hollow-fibre ultrafiltration and detected using PCR for adenovirus (AdV), and reverse transcription PCR for norovirus (NOV) genogroups I-III, enterovirus, rotavirus (RoV) and hepatitis E virus (HEV). Target viruses were detected in 106/109 (97%) samples, with 67/109 (61%) samples positive for three or more viral types at any one time. AdV, NoV and ROV were detected the most frequently, and HEV the least frequently. Human NoV was not usually associated with animal NOV. Our results suggest that New Zealand would be well served by assessing the ability of drinking-water treatment plants to remove viruses from the source waters, and that this assessment could be based on the viral concentration of AdV-NoV-RoV. The long-term aim of our work is to use this information to estimate the risk of waterborne viral infection.

Inactivation and elimination of human enteric viruses by Pacific oysters.

AIMS: To investigate the comparative elimination of three different human enterically transmitted viruses [i.e. hepatitis A virus (HAV), norovirus (NoV) and poliovirus (PV)] and inactivation of HAV and PV by Pacific oysters. METHODS AND RESULTS: New Zealand grown Pacific oysters (Crassostrea gigas) were allowed to bioaccumulate HAV, NoV and PV. Samples of oyster gut, faeces and pseudofaeces were then analysed by using real-time RT-PCR to determine the amount of viral RNA and cell culture methods to identify changes in the number of plaque forming units. The results suggest that the majority of the PV present in the oyster gut and oyster faeces is noninfectious, while in contrast, most of the HAV detected in the oyster gut are infectious. Depuration experiments identified a large drop in the count of PV in the gut over a 23-h cleansing period, whereas the levels of HAV and NoV did not significantly decrease. CONCLUSIONS: Human enterically transmitted viruses are eliminated and inactivated at different rates by Pacific oysters. SIGNIFICANCE AND IMPACT OF STUDY: The research presented in this article has implications for risk management techniques that are used to improve the removal of infectious human enteric viruses from bivalve molluscs.

Evaluation of murine norovirus as a surrogate for human norovirus and hepatitis A virus in heat inactivation studies.

AIMS: To determine the suitability of murine norovirus (MNV) as a surrogate for human norovirus (HuNoV) in heat inactivation studies. METHODS AND RESULTS: MNV, hepatitis A virus (HAV) and HuNoV genogroup I and II (GI and GII) specific real-time quantitative reverse transcription (qRT)-PCR assays were used to determine the effects of heat exposure (63 and 72 degrees C) for up to 10 min in water and milk. Using culture assays, MNV and HAV showed similar reductions in infectivity over time. Both HuNoV GI and GII showed lower log reductions in qRT-PCR titre following heat exposure than either MNV or HAV. No significant protective effect of milk was observed for any virus. CONCLUSIONS: MNV is as suitable a surrogate for HuNoV as HAV. In heat inactivation studies at 63 and 72 degrees C, qRT-PCR results indicate that HuNoV is less susceptible to heat than either HAV or MNV and so neither virus may be an appropriate surrogate for HuNoV. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be used when extrapolating surrogate virus data for HuNoV. Although not conclusive, our results suggest that HuNoV may be more resistant to heat than either HAV or MNV.

Localization of norovirus and poliovirus in Pacific oysters.

AIMS: To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters (Crassostrea gigas). METHODS AND RESULTS: Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp. CONCLUSIONS: The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.

Internationally distributed frozen oyster meat causing multiple outbreaks of norovirus infection in Australia.

BACKGROUND: Between November 2003 and January 2004, outbreaks of norovirus in 3 Australian jurisdictions involving 83 cases of illness were associated with imported oyster meat. METHODS: Cohort studies were conducted in 2 jurisdictions to identify relative risks of illness for the consumption of oysters. A case series was conducted in the third jurisdiction. RESULTS: The cohort studies conducted in the first 2 jurisdictions identified relative risks of illness of 17 (95% confidence interval, 5-51) and 35 (95% confidence interval, 5-243), respectively, for the consumption of oysters. Multiple strains of norovirus were detected in fecal specimens from 8 of 14 patients and in 1 of the 3 batches of implicated oyster meat using seminested reverse-transcriptase polymerase chain reaction methods. Traceback investigations revealed that all oyster meat was harvested from the same estuary system in Japan within the same month. CONCLUSIONS: These outbreaks demonstrate the potential of foodborne disease to spread internationally and the need for national and international collaboration to investigate such outbreaks. Foodborne illness related to norovirus is underestimated because of underreporting of human cases and challenges in laboratory detection of viruses in foods, both of which can delay public health action.

Bacteriophages as biocontrol agents in food.

Bacteriophages possess attributes that appear to be attractive to those searching for novel ways to control foodborne pathogens and spoilage organisms. These phages have a history of safe use, can be highly host specific, and replicate in the presence of a host. Campylobacter, Salmonella, and Listeria monocytogenes and various spoilage organisms have responded to phage control on some foods. However, the use of phages as biocontrol agents is complicated by factors such as an apparent requirement for a threshold level of host before replication can proceed and by suboptimal performance, at best, at temperatures beneath the optimum for the host. This review is a summary of the information on these issues and includes brief descriptions of alternative phage-based strategies for control of foodborne pathogens.

An outbreak of hepatitis A associated with consumption of raw blueberries.

This report describes the epidemiology, investigation and control of a hepatitis A (HAV) outbreak in New Zealand. Descriptive and analytical epidemiology, virology, product traceback and an orchard investigation were carried out. A case-control study revealed that 56% of 39 cases had consumed raw blueberries, compared with 14% of 71 controls (odds ratio 7.6; 95% confidence intervals 2.6-22.4). Traceback of product through retailers and wholesalers implicated a single commercial orchard. Hepatitis A virus was detected by reverse transcriptase polymerase chain reaction in faecal specimens from cases as well as a blueberry product from the orchard. Presence of hepatitis A virus was confirmed by DNA hybridization and sequencing of PCR products. Sanitary audit of the orchard revealed multiple opportunities for contamination of blueberries by pickers. This outbreak highlights the need for food safety programmes in the berry fruit industry.

Evaluation of integrated cell culture-PCR (C-PCR) for virological analysis of environmental samples.

AIMS: The aims of this study were to establish an integrated culture-polymerase chain reaction (C-PCR) method for detection of enteric viruses in environmental samples, and to evaluate it for sensitivity, speed and provision of virus infectivity data. METHODS AND RESULTS: C-PCR, direct reverse transcription (RT)-PCR, PCR and plaque assay methods were used to detect enteroviruses and adenoviruses in seeded and naturally contaminated environmental samples. Using C-PCR, infectious enterovirus presence was confirmed in 3 d and adenovirus presence in 5 d, compared with up to 10 d required by conventional cell culture methods. CONCLUSIONS: C-PCR was the preferred method for detection of enteric viruses in environmental samples containing high viral concentrations. It was less successful for samples with low viral concentrations or containing toxic materials or inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: C-PCR provides sensitive, specific results within 2-5 d and is useful as a rapid screen for environmental samples of low toxicity.

Raw oyster consumption and outbreaks of viral gastroenteritis in New Zealand: evidence for risk to the public's health.

OBJECTIVE: To investigate the relationship between consumption of raw Pacific half shell oysters and outbreaks of Norwalk-like virus (NLV) gastroenteritis in Auckland in the last third of 1999. METHOD: Ten outbreaks were investigated as retrospective cohorts using standardised questionnaires relating to food and drink exposures. Trace back of oysters and site inspections of implicated commercial growing areas were performed. Virological analyses compared oysters linked to outbreaks and faecal samples from cases. RESULTS: Eighty-six cases were identified, of whom 32 (37.2%) were confirmed NLV positive on faecal analysis. The summary risk estimate for illness among oyster consumers for all outbreaks was RR 8.23 (95% CI 4.55-14.90; p< 0.001) and in five of seven outbreaks permitting statistical analysis, the risk for those consuming raw oysters was greater than five-fold that of non-consumers. NLVs were identified in two batches of oysters from different growing areas, implicated in four outbreaks. In both the strain (Genogroup II/3 'Mexico-like virus') from cases matched that in the oysters from the same harvest batch. CONCLUSION: The epidemiological and virological evidence implicates oysters as the source of a number of outbreaks of NLV gastroenteritis. This is the first time NLVs have been identified in commercially farmed Pacific oysters in New Zealand. Sewage effluent from recreational boats was the likely source of faecal contamination of growing waters in one site. IMPLICATIONS: Combined use of virological and epidemiological methods have proved invaluable in investigating NLV outbreaks. Further research is needed into enteric viral contamination of commercial oyster farms.

Survival of poliovirus in New Zealand green-lipped mussels, Perna canaliculus, on refrigerated and frozen storage.

Poliovirus survival in live and frozen mussels during storage was assessed by both viral culture and molecular methods. Live New Zealand green-lipped mussels were incubated overnight at 20 degrees C in an aerated tank of filtered seawater seeded with the poliovirus 2 (PV2) vaccine strain. An extraction and concentration method that preserved viral infectivity was used to recover PV2 taken up by the mussels at day 0, at day 2 after storage at 4 degrees C, and at days 7, 14, and 28 after storage at -20 degrees C. This method allowed both culture and molecular analysis to be carried out. Presence of intact PV2 in each batch of mussels was determined by a pan-enterovirus specific reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by dot-blot hybridization. Survival of infectious PV2 was determined by the monolayer plaque assay. After 48 h at 4 degrees C, infectious PV2 levels were 81% of the original level detected in the mussels. Infective virus levels then declined to 66, 53, and 44% after storage at -20 degrees C for 7, 14, and 28 days, respectively. Generic RT-PCR methods were 10 times more sensitive than cell culture techniques for virus detection but did not give information on virus infectivity. The survival of infectious pathogenic viruses in fresh and frozen mussels on storage constitutes a potential health risk and so is a major concern for public health authorities.

Molecular epidemiology of 'Norwalk-like viruses' associated with gastroenteritis outbreaks in New Zealand.

Outbreaks of gastroenteritis are a major public health problem in New Zealand. The introduction of molecular detection methods has now shown that the 'Norwalk-like viruses' (NLVs) are the major cause of food and waterborne nonbacterial gastroenteritis. Reverse transcription and polymerase chain reaction (RT-PCR) were used to determine the presence of NLVs in faecal specimens from 83 nonbacterial gastroenteritis outbreaks occurring in New Zealand between August 1995 and July 1999. Further characterisation of the NLVs for epidemiological purposes was carried out by dot blot DNA hybridisation and DNA sequencing of representative outbreak strains. The majority of NLV strains occurring in New Zealand since August 1995 are similar to those occurring overseas. The predominant New Zealand strain is genetically similar to the Bristol/Lordsdale virus group. Several New Zealand outbreaks were attributed to Auckland virus, a Mexico-like NLV strain identified as the most likely cause of gastroenteritis after consumption of contaminated oysters in 1994. A new strain, designated Napier virus, has been identified in six outbreaks since 1996. A number of strains closely resembling internationally recognised strains, including Southampton virus, Saratoga virus; Desert Shield virus and Melksham virus have been associated with gastroenteritis outbreaks across New Zealand. Application of these typing methods has provided information on disease transmission for epidemiological investigations of public health significance.

A new papillomavirus of possums (Trichosurus vulpecula) associated with typical wart-like papillomas.

A previously unknown, cutaneous papillomavirus (Papovaviridae) in a brushtail possum (Trichosurus vulpecula) was demonstrated. This represents one of the first viruses reported in this species. Possum papillomas were identified by typical wart-like appearance and histology. Papillomavirus particles were detected by electron microscopy in tissue homogenates following purification and negative staining. The polymerase chain reaction amplified a conserved portion of the L1 gene which was purified and sequenced. Comparison of the DNA and deduced amino acid sequence from the possum papillomavirus with other papillomavirus sequences, together with phylogenetic analysis, indicated that this was a new papillomavirus.

Influence of environmental factors on virus detection by RT-PCR and cell culture.

The effects of clay, humic acid, u.v. light and shellfish tissue residues on the detection of poliovirus type 2 from environmental samples by culture and RT-PCR were investigated. RT-PCR showed 10-100 times greater sensitivity for PV2 detection in the absence of sample contaminants than did culture by plaque assay in BGM cell monolayers. Bentonite clay (100-1000 mg l-1) and shellfish tissue residues reduced virus detection by plaque assay, but the effect of bentonite was mitigated by simple elution procedures. Bentonite clay, humic acid (5-150 mg l-1) and mussel tissue reduced virus detection by RT-PCR by between 1 and 8 logs, although this was mitigated in part by elution and Sephadex filtration of extracts. Sephadex filtration of samples reduced culturable PV2 by 32-50%. Exposure of PV2 in water to u.v. light reduced culturability of PV2 but not detection by RT-PCR. This study demonstrates that virus detection in environmental samples is strongly influenced by naturally occurring substances and disinfection approaches. The accuracy of results of viral analyses of this nature should be carefully scrutinized with respect to sample constituents.

RT-PCR and chemiluminescent ELISA for detection of enteroviruses.

Reverse transcription followed by polymerase chain reaction amplification (RT-PCR) is now used commonly to detect the presence of enteric RNA viruses in environmental samples. A sensitive, non-isotopic microtitre plate hybridisation assay was developed and applied for detection of enteroviruses in environmental samples. Following reverse transcription, viral cDNA was labelled with digoxigenin (DIG)-dUTP during the PCR amplification step. The labelled PCR products were then hybridised with enterovirus-specific biotinylated oligonucleotide probe and captured in streptavidin-coated microtitre wells. Hybridised enteroviral PCR products were detected by an anti-digoxigenin peroxidase conjugate using either a colourimetric or a chemiluminescent substrate and automated measurement. Standard curves were established for poliovirus and other enteroviruses. The chemiluminescent assay was more sensitive than the colourimetric assay for detection of poliovirus, and was specific for enteroviruses. The chemiluminescent ELISA assay was used to confirm the presence of enteroviruses in environmental water samples.

Gallstone obstruction of duodenal cap (Bouveret's syndrome) diagnosed by endoscopy.

A patient presenting with signs and symptoms of gastric outlet obstruction and acute oliguric renal failure underwent early endoscopic examination. A large gallstone was seen obstructing the duodenal cap. Bouveret's syndrome and the role of endoscopy in early diagnosis are discussed.