Genetic characterization of genogroup I norovirus in outbreaks of gastroenteritis.

In this study, we demonstrate that differences within the P2 domain of norovirus genogroup I (GI) strains can be used to segregate outbreaks which are unrelated, whereas complete conservation within this region allows tracking of strains that are part of a single outbreak and likely to have a common source.

Tracking environmental norovirus contamination in a pediatric primary immunodeficiency unit.

Norovirus strains were detected in two patients and in environmental swabs from a pediatric primary immunodeficiency unit in London, United Kingdom, during an infection control incident in November and December 2007. Detailed analyses of the gene encoding the P2 domain demonstrated that the majority of the strains were not related to the patients and that the environmental contamination was most likely due to secondary transfer by the hands of staff or visitors.

Tracking the transmission routes of genogroup II noroviruses in suspected food-borne or environmental outbreaks of gastroenteritis through sequence analysis of the P2 domain.

The aim of this study was to apply sequence analysis of a hyper variable region of the norovirus (NoV) genome in order to identify point source outbreaks associated with suspect food or water. The hyper-variable region of the gene encoding the P2 domain was chosen as small differences in sequence are likely to indicate virus from different sources whereas identical sequence may reveal transmission routes and the source of contamination. Strains with 100% similarity were considered as originating from a common source, whereas, strains with one or more mutations in the hyper variable region sequenced were regarded as representing unrelated transmission events. This study was able to identify a point source outbreak of a dominant strain, GII-4, on a cruise ship but also of a less common strain, GII-2, between two schools. Also identical GII-3 strains were demonstrated in food handlers amongst the same outbreak; however epidemiologically related outbreaks showed different GII-3 strains indicating multiple sources of contamination.

Transmission events within outbreaks of gastroenteritis determined through analysis of nucleotide sequences of the P2 domain of genogroup II noroviruses.

Tracking the spread of noroviruses during outbreaks of gastroenteritis is hampered by the lack of sequence diversity in those regions of the genome chosen for virus detection and characterization. Sequence analysis of regions of the genes encoding the RNA-dependent RNA polymerase and the S domain of the capsid does not provide sufficient discrimination between genotypically related strains of different outbreaks. However, analysis of sequences derived from the region encoding the P2 domain showed 100% similarity among strains from the same outbreak and <100% similarity among strains of different outbreaks. The prolonged nature of some hospital outbreaks, links between hospitals, and the introduction of multiple strains of a single genotype associated with an outbreak aboard a cruise ship were determined using this method. This provides a powerful tool for tracking outbreak strains and the subsequent analysis and validation of interventions in a background of multiple introductions of virus strains of the same genotype or genetic cluster.

Contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season.

The aims of this study were to examine the extent of gastroenteric virus contamination in a pediatric primary immunodeficiency (PPI) ward and a general pediatric ward over a winter season and to determine whether changes to hospital infection control interventions would have an impact on environmental contamination levels within pediatric units. Environmental swabs were collected weekly from 11 sites in both wards from 15 December 2005 to 3 March 2006 and examined for the presence of norovirus (NoV), astrovirus, and rotavirus (RV) by reverse transcriptase PCR. Viruses were detected in 17% and 19% of swabs from both wards. Virus contamination for NoV and RV decreased from 20% to 6% and 15% to 10% of swabs, respectively, in the PPI ward from the 2004 study by Gallimore et al. (C. I. Gallimore, C. Taylor, A. R. Gennery, A. J. Cant, A. Galloway, M. Iturriza-Gomara, and J. J. Gray, J. Clin. Microbiol. 44:395-399, 2006). Overall, changes to cleaning protocols were deemed to have reduced the level of environmental contamination with gastroenteric viruses, but contamination still occurred due to a breakdown in infection control procedures indicated by contamination in areas frequented by parents but used only occasionally by staff.

Evaluation of the Loopamp (loop-mediated isothermal amplification) kit for detecting Norovirus RNA in faecal samples.

BACKGROUND: Noroviruses (NoVs) are associated with outbreaks of diarrhoeal illness in hospitals, nursing and residential homes and other institutional settings. NoV strains exhibit wide genetic diversity, and different virus genogroups and genotypes co-circulate in any geographical region at the same time, although most outbreaks of gastroenteritis are predominantly associated with genogroup II. The reverse transcription-polymerase chain reaction (RT-PCR) is the gold standard for detecting NoVs in clinical samples. OBJECTIVES: This study evaluates commercialised Loopamp kits for detecting NoV GI and NoV GII in faecal samples collected from patients with gastroenteritis and compares the results with those obtained using real-time RT-PCR with NoV genogroup sequence-specific detection. STUDY DESIGN: Five hundred and ten faecal samples collected from patients with gastroenteritis were evaluated for the presence of NoV using the gold-standard real-time RT-PCRs and the Loopamp assays. RESULTS: The Loopamp Norovirus GI and GII detection kits performed well compared to genogroup-specific real-time RT-PCR. Although the sensitivity of detection of GI strains (83.3%) was less than that for GII strains (97.4%), this will have little impact on the laboratory diagnosis of NoV, since GII strains are associated with the majority of outbreaks examined. CONCLUSIONS: The Loopamp GII detection kit is a sensitive method for detecting all the commonly circulating GII-4 strains included in the evaluation panel.

Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis.

Coding regions of double stranded RNA molecules from 3 human faecal samples containing Cryptosporidium hominis, C. felis and C. meleagridis were characterised by sequencing and compared with that previously obtained for C. parvum. Sequences outside the coding regions were also obtained. Overall similarities of between 86% and 92% and between 86% and 93% were observed in the nucleotide and amino acid sequences respectively between these species. These larger sequences will allow further molecular tools for detection, identification and characterisation of Cryptosporidium spp.

Predominance of enterovirus B and echovirus 30 as cause of viral meningitis in a UK population.

BACKGROUND/OBJECTIVES: Enteroviruses are the most common cause of aseptic or lymphocytic meningitis, particularly in children. With reports of unusually severe neurological disease in some patients infected with enterovirus D68 in North America, and a recent increase in the number of paediatric enterovirus meningitis cases presenting in this UK Midlands population, a retrospective regional surveillance study was performed. STUDY DESIGN: Cerebrospinal fluid (CSF) samples received were tested using the polymerase chain reaction (PCR) for HSV-1/2, VZV, enteroviruses and parechoviruses. Enterovirus PCR positive CSF samples were sent for further serotyping. A phylogenetic tree was constructed of the echovirus 30 VP1 sequences, where sufficient sample remained for sequencing. RESULTS: The number of enterovirus positive CSFs from each year were: 21 (2008), 7 (2011), 53 (2012), 58 (2013) and 31 (2014). Overall, 163 of the 170 serotyped enteroviruses belonged to the species B (echovirus 5, 6, 7, 9, 11, 13, 16, 17, 18, 21, 25, 30; coxsackie B1, B2, B3, B4, B5, A9), with only 7 belonging to species A (coxsackie A2, A6, A16 and enterovirus 71). Echovirus 30 was the predominant serotype overall, identified in 43 (25.3%) of samples, with a significantly higher proportion in the adult age group (37.3%) compared to the infant age group (12.3%). Phylogenetic analysis showed that these UK Midlands echovirus 30 VP1 sequences clustered most closely with those from Europe and China. CONCLUSION: This study showed a continued predominance of echovirus 30 as a cause of viral meningitis, particularly in adults, though more surveillance is needed.

Multiple norovirus genotypes characterised from an oyster-associated outbreak of gastroenteritis.

The diversity of norovirus (NV) genotypes was investigated in persons who were ill with acute gastroenteritis associated with the consumption of oysters. Initial results from a commercial enzyme immunoassay (EIA) indicated a mixed NV genogroup I (GI) and II (GII) outbreak. A reverse-transcriptase (RT)-PCR for NVs was applied to nucleic acid extracted from faecal specimens collected from symptomatic cases. Using primers that amplified contiguous sequences in the ORF1/2 region of the NV genome and a hemi-nested PCR derived from this assay, three different GII and two GI NV genotypes were detected and the strains were characterised by DNA sequencing. Using this approach a recombinant NV genotype, rGII-3a (recombinant Harrow/Mexico) the predominant strain identified in several symptomatic cases from the outbreak, was detected and characterised. No other gastroenteric viruses, including rotavirus, astrovirus, sapovirus and adenovirus 40/41 were detected by RT-PCR and PCR.

Chronic excretion of a norovirus in a child with cartilage hair hypoplasia (CHH).

We have demonstrated the long-term excretion of a stable recombinant norovirus in a patient with cartilage hair hypoplasia (CHH), with a T cell immunodeficiency, following bone marrow transplantation (BMT). The patient excreted an ARG320/1999/US-like recombinant norovirus (rGII-3) for 156 days during a period of immune reconstitution. The child was symptomatic during the period of virus shedding. It is not known if the child acquired the recombinant strain or if recombination occurred in vivo.

Application of the heteroduplex mobility assay (HMA) for the investigation of the genomic diversity among noroviruses in environmental samples.

Recent studies have demonstrated the widespread contamination of river and seawater with noroviruses (NV), often with more than one strain. The heteroduplex mobility assay (HMA) in which amplicons from study samples are hybridised (by denaturing and reannealing) to amplicons from reference strains and resolved by electrophoresis, has the potential to provide a simple and rapid means to identify samples containing multiple NV strains and to establish the diversity of strains within that sample. PCR amplicons from environmental samples that were tested directly in the HMA assay were shown to contain more than one strain. In order to evaluate HMA for investigations of NV diversity in environmental samples, amplicons from three representative samples were cloned and, for each, 20 amplicons derived from individual clones were analysed by HMA. Between two and six different HMA profiles were demonstrated among clones from a single sample indicating the extent of NV diversity in the sample. Sequence analysis confirmed the relationship of HMA profile and NV 'genotype'. Far greater diversity was seen among Genogroup (G) II (Ni/E3) amplicons than Genogroup (G) I (Ando/E3) amplicons (generated from the RNA dependent RNA polymerase region of the ORF1 of noroviruses), which often contained only a single strain, which is reflective of the greater prevalence of GII NVs over GI NVs. Overall, four GII and four GI strains were identified in these environmental water/sewage samples.

A rapid method for identifying diversity within PCR amplicons using a heteroduplex mobility assay and synthetic polynucleotides: application to characterisation of dsRNA elements associated with Cryptosporidium.

A 173-bp fragment of the small extra-chromosomal double-stranded RNA (dsRNA) element of Cryptosporidium parvum was generated by reverse transcriptase PCR from nucleic acid extracted from whole faeces of 18 epidemiologically unrelated cases of cryptosporidiosis. Eleven different sequences were detected and two selected as reference DNA in a heteroduplex mobility assay (HMA). Although sequence diversity was detected, this was difficult to characterise because of the similarity in electrophoretic mobility of the homo- and heteroduplex bands. A PCR method was devised to generate synthetic polynucleotides of greater sequence diversity for use in the HMA. The presence of the synthetic 173-bp fragments was enriched by using, as template for the PCR, material excised from the area of the heteroduplex bands in stained electrophoresis gels. Nine novel sequences were generated and evaluated as reference sequences in the HMA. One of these with 20 bp different from the original sequence was selected for use in the HMA for improved resolution of heteroduplex and homoduplex bands and number of patterns easily resolved (nine different patterns corresponding to different DNA sequences). This method may be useful for analysis of DNA where there is limited natural variation or little sequence variation is described.

Food-related norovirus outbreak among people attending two barbeques: epidemiological, virological, and environmental investigation.

BACKGROUND: Norovirus (NoV) is commonly associated with gastrointestinal infection. It is normally transmitted person-to-person or from contaminated surfaces, although food-borne transmission is possible. METHODS: We conducted environmental, epidemiological, and microbiological investigations to ascertain the route of transmission of two linked outbreaks of NoV associated with events where food was consumed. Multivariate logistic regression was used to determine food items independently associated with infection. RESULTS: In outbreak A, 19 of the 26 people who completed the food questionnaire fulfilled the case definition. The highest relative risks (RR) were for chicken kebab (RR 3, 95% confidence interval (CI) 0.9-10.4), pork sausages (RR 2.1, 95% CI 0.5-9.1), pasta salad (RR 1.94, 95% CI 0.9-4.1), cheese (RR 1.6, 95% CI 0.9-2.8), and green leaf salad (RR 1.5, 95% CI 0.9-2.4). In outbreak B, 60 of the 106 people surveyed fulfilled the case definition. Green leaf salad (adjusted odds ratio (aOR) 3.2, 95% CI 1.4-9.9) and coleslaw (aOR 8.2, 95% CI 3-22.2) were independently associated with illness in the multivariate logistic regression model. NoV genogroup II genotype 6 (GII-6) was identified in cases of both outbreaks and a food handler who had prepared salads for both events. CONCLUSION: Because outbreak investigations of small cohorts may not yield epidemiological association to food, most of these outbreaks may be attributed to the person-to-person transmission route. Therefore ascertainment of food-borne NoV infection may be low, underestimating the true prevalence of this route of transmission.

Analysis of amino acid variation in the P2 domain of the GII-4 norovirus VP1 protein reveals putative variant-specific epitopes.

BACKGROUND: Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques. CONCLUSIONS/SIGNIFICANCE: Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution.

European multicenter evaluation of commercial enzyme immunoassays for detecting norovirus antigen in fecal samples.

A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.

Characterization of sapoviruses collected in the United Kingdom from 1989 to 2004.

A fecal archive containing 115 sapovirus (SaV) strains detected in samples collected from 15 outbreaks and 98 sporadic cases of gastroenteritis between 1989 and 2004 in the UK were characterized in order to determine the genomic diversity within SaV co-circulating in the human population. Strains were characterized by partial sequencing of the genes encoding the RNA-dependent RNA polymerase (RdRp) region and/or the polymerase/capsid (Pol/Cap) junction of the open reading frame (Orf) 1. Overall, SaV of genogroup I genotype 1 (GI 1) were the predominant strains circulating in the UK in each year between 1989 and 2004. During 2004, GII 1 was the predominant strain. These two SaV types accounted for 89.5% of the sporadic cases and outbreaks in the UK. The remaining cases were caused by six other SaV genotypes. On the basis of partial sequencing of the RdRp and capsid encoding genes of strains, which did not show sufficient homology to any of the currently recognized genotypes, we propose the inclusion of a presumptive fourth genotype within genogroup I (GI 4).

Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit.

The aim of this study was to determine if gastroenteric viruses were present on surfaces and equipment in a pediatric primary immunodeficiency unit (PPIU) by environmental sampling using swabs and subsequent nucleic acid extraction and reverse transcriptase PCR assays. A PPIU was chosen, and 11 swabs were taken at the same sites every 2 weeks for 6 months. Nested/heminested PCR assays were used to screen for astroviruses (AsV), noroviruses (NoV), and rotaviruses (RV). AsV, NoV, and RV were detected at multiple swab sites during the study period. NoV was the most frequently detected virus on environmental surfaces; however, RV was detected on 79% and NoV on 50% of swabbing dates during the study period. Toilet taps were the most contaminated sites. Fecal samples from selected patients in the unit were also screened during the study period, and patients excreted AsV, NoV, and RV at times during the study. New cleaning schedules and changes in some of the PPIU sanitary furniture have been suggested as a means of reducing environmental contamination.

Characterisation of norovirus strains in rural Ghanaian children with acute diarrhoea.

The incidence of calicivirus infection in Ghana and many other African countries is not known. Thirteen (15.9%) of the 82 diarrhoeic stool samples tested for caliciviruses were positive for noroviruses (NoVs). NoVs were present in all age groups and were detected only during the diarrhoea peak that coincided with the peak rotavirus season. Ten (76.9%) of the NoV detected were genogroup II (GII) NoVs and the remaining three (23.1%) genogroup I (GI) NoVs. The predominant GII detected was GII-4 (60%, 6/10). Three of the GII NoVs were determined to be recombinants of GII-8/GII-14 as deduced from the sequencing of the region spanning the Orf1/2 junction. The GII genotypes formed four clusters with published GII sequences. The data shown enhances understanding of NoV diversity in Ghanaian children and demonstrate the global spread of distinct common genotypes to African countries.

Use of a heminested reverse transcriptase PCR assay for detection of astrovirus in environmental swabs from an outbreak of gastroenteritis in a pediatric primary immunodeficiency unit.

An outbreak of astrovirus gastroenteritis occurred in the Primary Immunodeficiency Unit at Newcastle General Hospital in March 2004. Environmental swabbing of the unit was undertaken after the outbreak, with multiple sites swabbed pre- and postcleaning. Astroviruses were detected in four environmental swabs and from two patient fecal samples using heminested reverse transcriptase PCR. An astrovirus genotype 3 strain was identified in both environmental swabs and fecal specimens and was the strain identified as being responsible for the outbreak. Environmental transmission of the virus was thought to have occurred by contamination of a syringe pump outside the laminar-flow curtain of a patient who was admitted with astrovirus gastroenteritis. This was subsequently transmitted to a cubicle next door and to a television/games console in a parents' room in the ward. Environmental monitoring of surfaces/equipment, using PCR assays for gastroenteric viruses in hospital situations where infection can give rise to serious clinical complications, may have a role in controlling and monitoring cleaning and the subsequent prevention of nosocomial transmission of gastroenteritis.