Metagenomic analysis of viruses in feces from unsolved outbreaks of gastroenteritis in humans.

The etiology of an outbreak of gastroenteritis in humans cannot always be determined, and ∼25% of outbreaks remain unsolved in New Zealand. It is hypothesized that novel viruses may account for a proportion of unsolved cases, and new unbiased high-throughput sequencing methods hold promise for their detection. Analysis of the fecal metagenome can reveal the presence of viruses, bacteria, and parasites which may have evaded routine diagnostic testing. Thirty-one fecal samples from 26 gastroenteritis outbreaks of unknown etiology occurring in New Zealand between 2011 and 2012 were selected for de novo metagenomic analysis. A total data set of 193 million sequence reads of 150 bp in length was produced on an Illumina MiSeq. The metagenomic data set was searched for virus and parasite sequences, with no evidence of novel pathogens found. Eight viruses and one parasite were detected, each already known to be associated with gastroenteritis, including adenovirus, rotavirus, sapovirus, and Dientamoeba fragilis. In addition, we also describe the first detection of human parechovirus 3 (HPeV3) in Australasia. Metagenomics may thus provide a useful audit tool when applied retrospectively to determine where routine diagnostic processes may have failed to detect a pathogen.

Evaluation of human adenovirus and human polyomavirus as indicators of human sewage contamination in the aquatic environment.

Discharge of inadequately treated human wastewater into surface waters used for recreation, drinking water, irrigation and shellfish cultivation may present a public health hazard due to the potential shedding of high concentrations of pathogenic viruses from the human gastrointestinal tract. Human adenovirus (HAdV) and human polyomavirus (HPyV) are ubiquitous in humans and have excellent survival characteristics in the environment, so are potential candidates for indicators of human sewage contamination. Using qPCR assays, the prevalence and quantity of HAdV and HPyV JC and BK were determined in influent and effluent wastewater and receiving waters (river, urban stream, estuarine), then compared with norovirus (NoV) presence, a significant human pathogen which is not necessarily ubiquitously excreted into the environment. HAdV and HPyV were frequently detected in high concentrations in wastewater and wastewater-contaminated waters confirming their use as potential indicators for the presence of human sewage. Overall, there was a correlation between the presence of HAdV and HPyV with NoV but there were some notable exceptions including the higher frequency of NoV compared to HAdV and HPyV in estuarine waters impacted by wastewater overflows. We found that HAdV and HPyV detection by qPCR was a suitable tool for evaluating water quality and that their detection can aid in determining pollution sources, thus providing useful information for health risk assessments.

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.

Influence of wastewater treatment process and the population size on human virus profiles in wastewater.

Human adenovirus (AdV and AdV species F), enterovirus (EV) and norovirus (NoV) concentrations entering wastewater treatment plants (WWTP) serving different-sized communities, and effectiveness of different treatment processes in reducing concentrations were established. Data was combined to create a characteristic and unique descriptor of the individual viral composition and termed as the sample virus profile. Virus profiles were generally independent of population size and treatment process (moving bed biofilm reactors, activated sludge, waste stabilisation ponds). AdV and EV concentrations in wastewater were more variable in small (<4000) and medium-sized (10,000-64,000) WWTP than in large-sized (>130,000 inhabitants) plants. AdV and EV concentrations were detected in influent of most WWTP (AdV range 1.00-4.08 log(10) infectious units (IU)/L, 3.25-8.62 log(10) genome copies/L; EV range 0.7-3.52 log(10) plaque forming units (PFU)/L; 2.84-6.67 log(10) genome copies/L) with a reduced median concentration in effluent (AdV range 0.70-3.26 log(10) IU/L, 2.97-6.95 log(10) genome copies/L; EV range 0.7-2.15 log(10)PFU/L, 1.54-5.28 log(10) genome copies/L). Highest culturable AdV and EV concentrations in effluent were from a medium-sized WWTP. NoV was sporadic in all WWTP with GI and GII concentrations being similar in influent (range 2.11-4.64 and 2.19-5.46 log(10) genome copies/L) as in effluent (range 2.18-5.06 and 2.88-5.46 log(10) genome copies/L). Effective management of WWTP requires recognition that virus concentration in influent will vary - particularly in small and medium plants. Irrespective of treatment type, culturable viruses and NoV are likely to be present in non-disinfected effluent, with associated human health risks dependent on concentration and receiving water usage.

Recurring norovirus transmission on an airplane.

BACKGROUND: Previously reported outbreaks of norovirus gastroenteritis associated with aircraft have been limited to transmission during a single flight sector. During October 2009, an outbreak of diarrhea and vomiting occurred among different groups of flight attendants who had worked on separate flight sectors on the same airplane. We investigated the cause of the outbreak and whether the illnesses were attributable to work on the airplane. METHODS: Information was obtained from flight attendants on demographic characteristics, symptoms, and possible transmission risk factors. Case patients were defined as flight attendants with diarrhea or vomiting <51 hours after the end of their first flight sector on the airplane during 13-18 October 2009. Stool samples were tested for norovirus RNA. RESULTS: A passenger had vomited on the Boeing 777-200 airplane on the 13 October flight sector. Sixty-three (82%) of 77 flight attendants who worked on the airplane during 13-18 October provided information, and 27 (43%) met the case definition. The attack rate among flight attendants decreased significantly over successive flight sectors from 13 October onward (P < .001). Working as a supervisor was independently associated with development of illness (adjusted odds ratio, 5.8; 95% confidence interval, 1.3-25.6). Norovirus genotype GI.6 was detected in stool samples from 2 case patients who worked on different flight sectors. CONCLUSIONS: Sustained transmission of norovirus is likely to have occurred because of exposures on this airplane during successive flight sectors. Airlines should make provision for adequate disinfection of airplanes with use of products effective against norovirus and other common infectious agents after vomiting has occurred.

Viral multiplex quantitative PCR assays for tracking sources of fecal contamination.

Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected humans, pigs, cattle, sheep, deer, and goats, and for the detection of F+ RNA bacteriophage genogroups I to IV, which are associated with human and animal wastes. The sensitivity of this viral toolbox (VTB) was tested against 10-fold dilution series of DNA plasmids that carry the target sequences of the respective viruses and was shown to detect at least 10 plasmid copies for each assay. A panel of human and animal enteric and respiratory viruses showed these assays to be highly sensitive and specific to their respective targets. The VTB was used to detect viruses in fecal and environmental samples, including raw sewage and biosolids from municipal sewage treatment plants, abattoir sewage, and fecally contaminated shellfish and river water, which were likely to contain animal or human viruses.

Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001-2007.

BACKGROUND: Noroviruses (NoVs) are the most common cause of viral gastroenteritis. Their high incidence and importance in health care facilities result in a great impact on public health. Studies from around the world describing increasing prevalence have been difficult to compare because of differing nomenclatures for variants of the dominant genotype, GII.4. We studied the global patterns of GII.4 epidemiology in relation to its genetic diversity. METHODS: Data from NoV outbreaks with dates of onset from January 2001 through March 2007 were collected from 15 institutions on 5 continents. Partial genome sequences (n=775) were collected, allowing phylogenetic comparison of data from different countries. RESULTS: The 15 institutions reported 3098 GII.4 outbreaks, 62% of all reported NoV outbreaks. Eight GII.4 variants were identified. Four had a global distribution--the 1996, 2002, 2004, and 2006b variants. The 2003Asia and 2006a variants caused epidemics, but they were geographically limited. Finally, the 2001 Japan and 2001 Henry variants were found across the world but at low frequencies. CONCLUSIONS: NoV epidemics resulted from the global spread of GII.4 strains that evolved under the influence of population immunity. Lineages show notable (and currently unexplained) differences in geographic prevalence. Establishing a global NoV network by which data on strains with the potential to cause pandemics can be rapidly exchanged may lead to improved prevention and intervention strategies.

Human dietary exposure to heavy metals via the consumption of greenshell mussels (Perna canaliculus Gmelin 1791) from the Bay of Islands, northern New Zealand.

Cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb) and tin (Sn) concentrations were determined using ICP-MS in soft tissues (wet wt.) from whole greenshell mussels (Perna canaliculus) collected from Urapukapuka-Rawhiti Island, Opua Marina, Waitangi Bridge and Opua Wharf from the Bay of Islands, northern New Zealand (NZ). All samples had metal concentrations well below the Food Standards Australia and New Zealand (FSANZ) maximum limits and were comparable to, or less than, concentrations observed in previous NZ studies. Based on the average values detected in the current study, the concentrations of heavy metals ingested in a 'typical diet' containing greenshell mussels are below the provisional tolerable weekly intake (PTWI). However, Maori (indigenous people of New Zealand), Pacific Islanders and Asians consume a far greater quantity of seafood (and therefore heavy metals) than the general public of New Zealand and could potentially consume enough shellfish to exceed the PTWI for Cd (but not for Hg, As, Pb or Sn). Although our results, based on the current PTWIs, indicate no significant health risk to greenshell mussel consumers in this region, PTWIs change over time; concentrations which were thought to be safe are later found to be harmful. Additionally, differences in individual human susceptibilities to various toxins could increase the risk of harm for consumers with low tolerance to heavy metals. We suggest that a survey of the frequency, amount and species consumed by groups whose diet may be largely shellfish-based is required to enable a more comprehensive risk assessment to be made.

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.

Epidemics of gastroenteritis during 2006 were associated with the spread of norovirus GII.4 variants 2006a and 2006b.

BACKGROUND: Acute gastroenteritis is commonly associated with norovirus genogroup II (GII) infection. Norovirus GII has 17 classified genotypes (GII.1-GII.17), but only 1 norovirus genotype (GII.4) is associated with global epidemics of gastroenteritis. In 2006, an increase in global norovirus activity was observed. METHODS: During the period from December 2005 through August 2006, a total of 231 fecal samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. Norovirus RNA was amplified and sequenced to determine norovirus genotype and relatedness to known epidemic norovirus GII.4 variants. RESULTS: Two GII.4 variants, designated 2006a and 2006b, were identified in 61.8% and 11.3%, respectively, of the 186 cases investigated. Norovirus 2006a and 2006b have also been implicated as the predominant causes of norovirus-associated gastroenteritis across Europe in 2006. CONCLUSIONS: The global increase in norovirus-associated gastroenteritis in 2006 was linked to the emergence of 2 novel GII.4 variants, 2006a and 2006b.

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.

Effect of heat treatment on hepatitis A virus and norovirus in New Zealand greenshell mussels (Perna canaliculus) by quantitative real-time reverse transcription PCR and cell culture.

Quantitative real-time reverse transcription PCR (qRT-PCR) and cell culture (50% tissue culture infectious dose [TCID50]) were used to determine the effect of heat treatments on norovirus and hepatitis A virus (HAV) in the New Zealand Greenshell mussel (Perna canaliculus). Since it is common practice to cook mussels until the shells open, internal temperatures and opening times of mussels on boiling and steaming were determined at regular time intervals. Fifty mussels in batches of six were exposed to boiling and steaming. A mean internal temperature of 90 degrees C (recommended for virus inactivation when maintained for 90 s) was reached after boiling for 170 s, with all 50 mussels open at 210 s. For steaming, the mean internal temperature achieved was only 83 degrees C after 300 s, and all 50 mussels were open. When mussels were steamed for 180 s (mean internal temperature of 63 degrees C), a significant 1.5-log decrease in the HAV titer (log TCID50) was observed. Following the immersion of mussels in boiling water for 180 s (mean internal temperature of 92 degrees C), no viable HAV was detected. For both boiling and steaming experiments, there was no significant change in the norovirus or HAV qRT-PCR titers compared with the controls. Our results show that when New Zealand Greenshell mussels open on heating, their internal temperature may not reach the parameters required for virus inactivation. Immersion for a minimum of 3 min in boiling water rather than steaming is recommended to reduce the risk of viral foodborne illness from contaminated shellfish.

Survival and persistence of norovirus, hepatitis A virus, and feline calicivirus in marinated mussels.

Noroviruses (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of raw or lightly cooked bivalve shellfish. Marinated mussels are a popular delicacy, but there is no published information on whether enteric viruses survive the marination process. The survival and persistence of HAV, NV, and a surrogate calicivirus, feline calicivirus (FCV), in marinated mussels over time was determined. NV, HAV, and FCV were inoculated into marinated mussels and marinade liquid and then held at 4 degrees C for up to 4 weeks. Survival of HAV and FCV was quantified by determining the 50% tissue culture infectious dose (TCID50), and these results were correlated with those of the reverse transcription (RT)-PCR assay. The persistence of nonculturable NV was determined by RT-PCR assay only. Over 4 weeks, HAV survived exposure to acid marinade at pH 3.75. There was a 1.7-log reduction in HAV TCID50 titer but no reduction in NV or HAV RT-PCR titer after 4 weeks in marinated mussels. FCV was inactivated in acid conditions although it was still detectable by RT-PCR. To simulate preharvest virus contamination and commercial marination processing, experiments using fresh mussels infected with HAV and NV were performed. HAV and NV persistence was determined using semiquantitative real-time RT-PCR, and HAV infectivity was determined by the TCID50 assay. HAV retained infectivity following simulated commercial marination and exposure to acid conditions over 4 weeks. The survival of pathogenic enteric viruses in marinated mussels constitutes a potential health risk and so is of concern to public health authorities.