Genogroup II Research Articles

Molecular epidemiology of genogroup II norovirus infections in acute gastroenteritis patients during 2014\u20132016 in Pudong New Area, Shanghai, China

BackgroundNorovirus (NoV), a member of the Caliciviridae, is now recognized as the leading cause of acute gastroenteritis (AGE) worldwide. Globally, the GII.4 Sydney_2012 variant has predominated in NoV-related AGE since 2012, although the novel variant GII.17 has also been reported as responsible for gastroenteritis outbreaks in East Asia since 2014. This study aimed to disclose the recent genotype patterns of NoV genogroup II (GII) presenting in AGE patients in Pudong New Area of Shanghai through a laboratory-based syndromic surveillance system. The study further aimed to delineate the predominant strains circulating in the population.MethodsPudong New Area is located in eastern Shanghai and covers 20.89% of the Shanghai population. The laboratory-based syndromic surveillance system is composed of 12 sentinel hospitals among the 68 general hospitals in this area. AGE patients who sought medical care were sampled following an AGE surveillance protocol. Stool samples were collected from participating patients, and a standardized questionnaire was given to each patient by trained nurses to gain information on the disease profiles and demographics of the patients. Real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to screen the GI nd GII NoV and RT-PCR was used to amplify NoV GII partial capsid protein open reading frame 2 (ORF2). NoV Genotyping Tool (version 1.0, RIVM, MA Bilthoven, Netherlands) was used for genotyping, and a phylogenetic analysis was conducted by MEGA 7.0.ResultsDuring 2014–2016, among the 2069 virus-infected AGE cases, 65.88% were caused by NoV. NoV-AGE occurred most frequently in the periods from October to March. The patients with more severe diarrheal symptoms and vomiting were more likely to be infected by NoV. The main genotypes were GII.17 (44.69%) and GII.4 (39.26%), which dominated the NoV-AGE epidemics jointly or in turn, whereas a slight increase in GII.2 was observed beginning in May 2016. The GII.17 strains tended to cluster more with the Hu/JP/2014/GII.P17_GII.17/Kawasaki323 variants, representing novel prevalent strains. Among the GII.4 strains, the GII.4 Sydney_2012 variant was still the predominant strain.ConclusionsNoV GII has become the main cause of virus-infected AGE in Pudong New Area, Shanghai. The predominant genotypes of NoV GII were GII.17 and GII.4. Comprehensive laboratory-based surveillance is important for clinical diagnosis and treatment. Identification of emerging new genotypes is also crucial for the prevention and control of NoV-infected AGE.

Evaluation of Xpert® norovirus assay performance in comparison with easyscreen™ enteric viral RT-PCR for rapid detection of norovirus genogroup I (GI) and genogroup II (GII) in hospitalised patients with acute gastroenteritis

The rapid identification of noroviruses, a leading cause of epidemic and sporadic gastroenteritis, is crucial for infection control and bed management.

Series of Norovirus Outbreaks Caused by Consumption of Green Coral Lettuce, Denmark, April 2016.

Introduction: In early April 2016, an unusual high number of point-source outbreaks of gastrointestinal disease were reported to occur in Denmark.Methods: Outbreaks were individually investigated. Two analytical studies were performed. Patient stool samples collected and analysed; positive stool samples were sequenced over the polymerase and/or capsid gene areas. Implicated lettuce heads were collected and analysed for the presence of norovirus. Foods were traced-back and traced-forward and international alert systems applied.Results: A total of 23 linked point-source outbreaks occurred over the course of one week. Fresh green coral lettuce (Lollo Bionda lettuce) had been consumed in all settings. In a cohort study including 234 participants a dish containing green lettuce was associated with illness. Norovirus of Genogroup I (GI) was detected in samples from 28 patients comprising eight of the outbreaks. Sequencing showed GI.P2-GI.2. GI norovirus was detected in one of 20 examined lettuce heads. All lettuce consumed was supplied by the same packer who in turn had bought the lettuce from a wholesaler in France. The two lots of lettuce came from two different growers in different parts of France.Discussion: Green coral lettuce produced in France was found to have caused a large series of linked norovirus outbreaks in Denmark as established by a number of lines of evidence. A similar incidence occurred in 2010. Fresh lettuce increasingly appear to be a risk food for norovirus infections.

Functionality and avidity of norovirus-specific antibodies and T cells induced by GII.4 virus-like particles alone or co-administered with different genotypes

Norovirus (NoV) is the main cause of acute gastroenteritis worldwide across all age groups. Current NoV vaccine candidates are based on non-infectious highly immunogenic virus-like particles (VLPs) produced in cell cultures in vitro. As NoVs infecting human population are highly divergent, it is proposed that the vaccine should contain at least two different NoV genotypes, potentially affecting the immunogenicity of each other. We investigated the immunogenicity of NoV GII.4 VLPs administered by intramuscular (IM) or intradermal (ID) injections to BALB/c mice either alone or co-delivered with genogroup I (GI) and other genogroup GII VLPs. Serum NoV-specific IgG binding antibody titers and antibody functionality in terms of avidity and blocking potential were assessed. Furthermore, the specificity and functional avidity of CD4+ and CD8+ T cell responses were analyzed using synthetic peptides previously identified to contain NoV VP1 P2 domain-specific H-2d epitopes. The results showed that IM and ID immunization induced comparable GII.4-specific antibodies and T cell responses. Similar magnitude and functionality of antibodies and interferon-gamma producing T cells were developed using monovalent GII.4 VLPs or different genotype combinations. For the first time, degranulation assay using multicolor flow cytometry showed that NoV GII.4-specific CD8+ T cells had cytotoxic T lymphocyte phenotype. To conclude, our results demonstrate that there is no immunological interference even if up to five different NoV VLP genotypes were co-administered at the same time. Furthermore, no inhibition of NoV-specific antibody functionality or the magnitude, specificity and affinity of T cell responses was observed in any of the immunized animals, observations relevant for the development of a multivalent NoV VLP vaccine.

Genetic Diversity Among Genogroup II Noroviruses and Progressive Emergence of GII.17 in Wastewaters in Italy (2011-2016) Revealed by Next-Generation and Sanger Sequencing.

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detectedwith the broad-range assay: GII.1 (n=6), GII.2 (n=8), GII.3 (n=3), GII.4 (n=13), GII.6 (n=3), GII.7 (n=2), GII.13 (n=2), and GII.17 (n=3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

Molecular epidemiological characteristics of norovirus outbreaks in Qingdao, 2014-2016

Objective To analyze the molecular epidemiological characteristics of norovirus (NoV) outbreaks in Qingdao between 2014 and 2016. Methods Stool samples were collected from NoV outbreaks between January 2014 and December 2016 and detected by real-time RT-PCR. NoV open reading frame 1 (ORF1) and ORF2 were partially amplified by RT-PCR. The amplified products were further analyzed by gene sequencing and genotyping. Phylogenetic analysis was conducted by using MEGA 6.0 software package. Results A total of 23 NoV outbreaks, involving 260 cases, were reported during 2014 to 2016. Of all collected stool samples, 128 were positive for NoV including 6 of genogroupⅠ (GⅠ) and 122 of genogroupⅡ (GⅡ). All positive samples were genotyped into 6 genotypes, which were GⅡ.P17-GⅡ.17, GⅡ.P12-GⅡ.3, GⅡ.P7-GⅡ.6、GⅡ.P2-GⅡ.2, GⅠ.Pb-GⅠ.6 and GⅡ.Pg-GⅡ.12. The 23 outbreaks included both single infections and mixed genotype infections, which were 11 of GⅡ. 17 single infection, 4 of GⅡ. 3 single infection, 3 of GⅡ. 17 and GⅡ. 3 mixed infection, 2 of GⅡ. 17 and GⅡ. 6 mixed infection, 1 of GⅠ. 6 single infection, 1 of GⅡ. 17 and GⅡ. 2 mixed infection and 1 of GⅡ. 17 and GⅡ. 12 mixed infection. Conclusion NoV was an important pathogen responsible for viral diarrhea outbreaks in Qingdao. Several different genotypes were detected. The newly variant GⅡ.P17-GⅡ.17 was the predominant epidemic strain causing norovirus outbreaks in Qingdao during 2014 to 2016. Key words: Norovirus; Outbreak; Molecular epidemiology

Can Use of Viral Load Improve Norovirus Clinical Diagnosis and Disease Attribution?

Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) is the state-of-the-art diagnostic for norovirus. Cycle threshold (Ct), an indicator of viral load, may be associated with symptomatic disease as well as demographic and outbreak characteristics. Data on (1) outbreak and sporadic cases and (2) asymptomatic controls in the United States and Latin America were analyzed. With multivariate regression models, we assessed relationships between various factors and Ct values, and we calculated odds ratios (ORs) for the presence of symptoms and attributable fractions of norovirus. Receiver-operating characteristic analysis was performed to define an optimal Ct cutoff to identify disease-causing infections. Cycle threshold values were lower (ie, higher viral loads) among symptomatic cases (model-adjusted mean ± standard error: 25.3 ± 1.2) compared with asymptomatic controls (28.5 ± 1.4). Cycle threshold values were significantly different across age groups, norovirus genogroups, timing of specimen collection, outbreak settings, and transmission modes. Genogroup II (GII) Ct values were associated with presence of symptoms (OR = 1.1), allowing us to estimate that 16% of diarrheal disease was attributable to norovirus. The optimized Ct cutoff led to poor sensitivity and specificity for genogroup I and GII. Cycle threshold values were associated with host, pathogen, and outbreak factors. Cycle threshold values may not effectively distinguish disease-causing infection for individual patients, but they are useful for epidemiological studies aiming to attribute disease.

Detection of Norovirus GII.17 Kawasaki 2014 in Shellfish, Marine Water and Underwater Sewage Discharges in Italy.

Norovirus (NoV) is a major cause of non-bacterial acute gastroenteritis worldwide, and the variants of genotype GII.4 are currently the predominant human strains. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been reported as the cause of gastroenteritis outbreaks in Asia, replacing the pandemic strain GII.4 Sydney 2012. The GII.17 Kawasaki 2014 variant has also been reported sporadically in patients with gastroenteritis outside of Asia, including Italy. In this study, 384 shellfish samples were subjected to screening for human NoVs using real-time PCR and 259 (67.4%) tested positive for Genogroup II (GII) NoV. Of these, 52 samples, selected as representative of different areas and sampling dates, were further amplified by conventional PCR targeting the capsid gene, using broad-range primers. Forty shellfish samples were characterized by amplicon sequencing as GII.4 (n=29), GII.2 (n=4), GII.6 (n=2), GII.12 (n=2), and GII.17 (n=3). Sixty-eight water samples (39 seawater samples from the corresponding shellfish production areas and 29 water samples from nearby underwater sewage discharge points) were also tested using the above broad-range assay: eight NoV-positive samples were characterized as GII.1 (n=3), GII.2 (n=1), GII.4 (n=2), and GII.6 (n=2). Based on full genome sequences available in public databases, a novel RT-PCR nested assay specific for GII.17 NoVs was designed and used to re-test the characterized shellfish (40) and water (8) samples. In this second screening, the RNA of GII.17 NoV was identified in 17 additional shellfish samples and in one water sample. Upon phylogenetic analysis, these GII.17 NoV isolates were closely related to the novel GII.17 Kawasaki 2014. Interestingly, our findings chronologically matched the emergence of the Kawasaki 2014 variant in the Italian population (early 2015), as reported by hospital-based NoV surveillance. These results, showing GII.17 NoV strains to be widespread in shellfish samples collected in 2015 in Italy, provide indirect evidence that this strain has started circulating in the Italian population. Notably, using a specific assay, we were able to detect many more samples positive for GII.17 NoV, indicating that, in food and water matrices, broad-range assays for NoV may grossly underestimate the prevalence of some, less common, NoVs. The detection of the GII.17 strain Kawasaki 2014 in clinical, water and food samples in Italy highlights the need for more systematic surveillance for future disease control and prevention.

Norovirus infections in young children in Lusaka Province, Zambia: clinical characteristics and molecular epidemiology

BackgroundThe burden, clinical features, and molecular epidemiology of norovirus infection in young children in southern Africa are not well defined.MethodsUsing data from a health facility-based surveillance study of children <5 years in Lusaka Province, Zambia presenting with diarrhea, we assessed the burden of norovirus infection. A convenience sample of 454 stool specimens was tested for norovirus using reverse-transcriptase polymerase chain reaction (RT-PCR). RT-PCR positive samples underwent additional nucleotide sequencing for genogroup and genotype identification. Clinical features and severity of diarrheal illnesses were compared between norovirus-positive and -negative subjects using Chi-squared and t-tests.ResultsNorovirus was detected in 52/454 (11.5%) specimens tested. Abdominal pain, fever, and vomiting were the most common presenting features in norovirus-associated illnesses. However, there were no significant differences in the clinical features of norovirus-positive compared to norovirus-negative illnesses. Of 43 isolates that were available for sequencing, 31 (72.1%) were genogroup II (GII) and 12 (27.9%) were genogroup I (GI). The distribution of genotypes was diverse.ConclusionsNoroviruses were detected in approximately 10% of young children with diarrhea in the Lusaka Province of Zambia, with GII representing the majority of infections. These findings support the role of norovirus in symptomatic diarrhea disease in Africa. Further studies are needed to confirm these observations and to evaluate prevention strategies.

Detection and differentiation of norovirus genogroups I and II from clinical stool specimens using real-time PCR

A real-time RT-PCR assay was designed to detect and differentiate norovirus genogroups I (GI) and II (GII), with primers and probes targeting the nonstructural polyprotein gene. Stool samples (n = 100) submitted for routine testing by the BioFire FilmArray® GI panel were also tested by the norovirus GI/GII real-time PCR assays. When compared to the FilmArray GI panel, the norovirus real-time PCR assay demonstrated a sensitivity of 77.5% (62/80) and specificity of 95% (19/20). Specimens yielding discordant results (n = 19) were tested at two outside laboratories for adjudication. Following discordant resolution, the adjusted sensitivity and specificity of the norovirus real-time PCR assays were 96.9% (63/65) and 100% (35/35), respectively. These results suggest that the real-time PCR assays are able to accurately detect and differentiate norovirus GI/GII from clinical stool specimens. Furthermore, our report highlights a potential issue with the specificity of the BioFire FilmArray® norovirus assay, which warrants additional investigation.

Risk factors for norovirus contamination of shellfish water catchments in England and Wales

This study examines the relationships between concentrations of human noroviruses (NoV) genogroups I (GI) and II (GII) and Escherichia coli monitored in oysters from 31 commercial harvesting areas on the coast of England and Wales from May 2009 to April 2011 and demographic, hydrometric, climatic and pollution source characteristics of upstream river catchments using multiple regression techniques. The predictive environmental factors for E. coli contamination in the oysters were rainfall (cumulative 7days before sampling) while the predictive factors for NoV (GI+GII) were water temperature, catchment area and the combined volume of continuous sewage discharges in the catchment. Oysters from cold waters (<5°C) had significantly higher NoV content than those from warmer waters (>10°C). The association with water temperature may be consequential on the seasonal prevalence of the virus in the community or linked with oyster metabolic function. In a group of 10 study sites, mean concentrations of NoV increased as the number of stormwater spills at those sites also increased. The results of this study could be used to evaluate the likely impact of sewerage infrastructure improvements in catchments at risk of NoV contamination and to help identify sites suitable for shellfish farming.

Development of one-step reverse transcription loop-mediated isothermal amplification for norovirus detection in oysters

The aim of this study was to develop a simple and rapid technique for detecting human norovirus (HuNoV). The loop-mediated isothermal amplification (LAMP) technique was evaluated and found to be sensitive, highly specific, and useful for routine oyster testing. Reverse transcription-LAMP (RT-LAMP) primers were designed to detect HuNoVs genogroups I (GI) and GII. One-step RT-LAMP was found to be more sensitive than real-time RT-PCR, semi-nested RT-PCR and real-time RT-LAMP methods and could detect 101 HuNoV genome copies per microliter. The specificity of one-step RT-LAMP for HuNoVs GI and GII was confirmed by restriction fragment length polymorphism analysis. When a field test of oyster samples was performed, one-step RT-LAMP proved to be a rapid and accurate technique for detecting HuNoV. Thus, this could be reliable rapid technique for detecting HuNoV in fishery production fields, so to improve food safety and enable the oyster industry to become more competitive in the world market.

Distribution of Human Norovirus in the Coastal Waters of South Korea.

The presence of human norovirus in the aquatic environment can cause outbreaks related to recreational activities and the consumption of norovirus-contaminated clams. In this study, we investigated the prevalence of norovirus genogroups I (GI) and II (GII) in the coastal aquatic environment in South Korea (March 2014 to February 2015). A total of 504 water samples were collected periodically from four coastal areas (total sites = 63), of which 44 sites were in estuaries (clam fisheries) and 19 were in inflow streams. RT-PCR analysis targeting ORF2 region C revealed that 20.6% of the water samples were contaminated by GI (13.3%) or GII (16.6%). The prevalence of human norovirus was higher in winter/spring than in summer/fall, and higher in inflow streams (50.0%) than in estuaries (7.9%). A total of 229 human norovirus sequences were identified from the water samples, and phylogenetic analysis showed that the sequences clustered into eight GI genotypes (GI.1, 2, 3, 4, 5, 6, 7, and 9) and nine GII genotypes (GII.2, 3, 4, 5, 6, 11, 13, 17, and 21). This study highlighted three issues: 1) a strong correlation between norovirus contamination via inflow streams and coastal areas used in clam fisheries; 2) increased prevalence of certain non-GII.4 genotypes, exceeding that of the GII.4 pandemic variants; 3) seasonal shifts in the dominant genotypes of both GI and GII.

Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators.

To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity and maximum filterable volume (MFV) were also measured in the water. Higher prevalence of NoV in secondary-W (GI 100%, GII 55·6%) and tertiary-W (GI 91·7%, GII 66·7%) compared with surface-W (GI 58·4%, GII 22·2%) was observed. Nov GI showed positive correlation with E.coli (Spearman's correlation coefficient=0·68, P<0·01), and with some physicochemical parameters such as COD (0·52, P<0·01), turbidity (0·52, P<0·01) and MFV (0·54, P<0·01). Escherichia coli and enteric viruses were not detected in zucchini. There is a potential risk of contamination of crops with NoV when reclaimed water is used for irrigation. Increase the knowledge on the prevalence of enteric viruses in different irrigation water sources, and its consequences for fresh produce safety.

Norovirus outbreak in a restaurant: investigation of the path of infection by sequence analysis of food and human samples

In September 2014, a small gastroenteritis outbreak occurred in a Greek restaurant. Primary investigations by official food surveillance revealed significant hygienic problems in the premises. Food samples and environmental samples were analyzed for the presence of bacterial and viral food pathogens. Norovirus genogroup I (GI) was detected in 2 environmental samples and in mixed salad. At the same time, stool samples from patients were analyzed and norovirus GI was detected. Further investigations revealed the presence of norovirus GI on some of the restaurant employees. Comparison of nucleic acid sequences revealed full sequence homology between norovirus RNA genotype 1.2 in food, environmental and stool samples, suggesting a common source of contamination and infection. Sequence analysis of food and environmental samples was facilitated by application of a system for total RNA amplification. Despite the fact that original source of contamination could be determined doubtlessly, observed weaknesses in the food production that caused this outbreak were discussed. The mixed salad could have been contaminated either by the lettuce contaminated at primary production or by one of the food-handlers. The investigation of the path of infection is necessary for the kind of legal consequences to be directed by authorities and may contribute to measures to eliminate possible sources of food contamination.

Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses.

Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide. High-pressure processing (HPP) is one of the most promising nonthermal technologies for the decontamination of viral pathogens in foods. However, the survival of HuNoVs after HPP is poorly understood because these viruses cannot be propagated in vitro In this study, we estimated the survival of different HuNoV strains within genogroup II (GII) after HPP treatment using viral receptor-binding ability as an indicator. Four HuNoV strains (one GII genotype 1 [GII.1] strain, two GII.4 strains, and one GII.6 strain) were treated at high pressures ranging from 200 to 600 MPa. After treatment, the intact viral particles were captured by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) that contained histo-blood group antigens, the functional receptors for HuNoVs. The genomic RNA copies of the captured HuNoVs were quantified by real-time reverse transcriptase PCR (RT-PCR). Two GII.4 HuNoVs had similar sensitivities to HPP. The resistance of HuNoV strains against HPP ranked as follows: GII.1 > GII.6 > GII.4, with GII.4 being the most sensitive. Evaluation of temperature and matrix effects on HPP-mediated inactivation of HuNoV GII.4, GII.1, and GII.6 strains showed that HuNoV was more easily inactivated at lower temperatures and at a neutral pH. In addition, phosphate-buffered saline (PBS) and minimal essential medium (MEM) can provide protective effects against HuNoV inactivation compared to H2O. Collectively, this study demonstrated that (i) different HuNoV strains within GII exhibited different sensitivities to high pressure, and (ii) HPP is capable of inactivating HuNoV GII strains by optimizing pressure parameters. Human norovirus (HuNoV) is a leading cause of foodborne disease worldwide. Noroviruses are highly diverse, both antigenically and genetically. Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivable in vitro Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix.

Molecular evolution of the capsid gene in human norovirus genogroup II.

Capsid protein of norovirus genogroup II (GII) plays crucial roles in host infection. Although studies on capsid gene evolution have been conducted for a few genotypes of norovirus, the molecular evolution of norovirus GII is not well understood. Here we report the molecular evolution of all GII genotypes, using various bioinformatics techniques. The time-scaled phylogenetic tree showed that the present GII strains diverged from GIV around 1630CE at a high evolutionary rate (around 10−3 substitutions/site/year), resulting in three lineages. The GII capsid gene had large pairwise distances (maximum > 0.39). The effective population sizes of the present GII strains were large (>102) for about 400 years. Positive (20) and negative (over 450) selection sites were estimated. Moreover, some linear and conformational B-cell epitopes were found in the deduced GII capsid protein. These results suggested that norovirus GII strains rapidly evolved with high divergence and adaptation to humans.

Detection and molecular characterization of norovirus from oysters implicated in outbreaks in the US

Human noroviruses are the leading cause of non-bacterial shellfish associated gastroenteritis. Here we report on the detection and characterization of norovirus (NoV) in shellfish associated outbreaks. Requests were received from state and federal officials for technical assistance in the analysis of shellfish for NoV and male specific coliphage (MSC; an enteric virus surrogate) during the years 2009 thru 2014. In outbreaks where NoV was detected, genogroup II (GII) levels ranged from 2.4 to 82.0 RT-qPCR U/g of digestive diverticula (DD) while NoV genogroup I (GI) levels ranged from 1.5 to 29.8 RT-qPCR U/g of DD. Murine norovirus extraction efficiencies ranged between 50 and 85%. MSC levels ranged from <6 to 80 PFU/100 g. Phylogenetic analysis of the outbreak sequences revealed strains clustering with GI.8, GI.4, GII.3, GII.4, GII.7, and GII.21. There was 100% homology between the shellfish and clinical strains occurring in 2 of 8 outbreaks. Known shellfish consumption data demonstrated probable infectious particles ingested as low as 12. These investigations demonstrate effective detection, quantification, and characterization of NoV in shellfish associated with illness.

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50μM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR.

Norovirus molecular detection in Uruguayan sewage samples reveals a high genetic diversity and GII.4 variant replacement along time.

To determine the prevalence and molecular epidemiology of norovirus (NoV) genogroup I (GI) and GII in Uruguay. One hundred and sixteen sewage samples were collected in six cities (Bella Unión, Salto, Paysandú, Fray Bentos, Melo and Treinta y Tres) from March 2011 to April 2013, viruses were concentrated by ultracentrifugation and NoV studies were performed by semi-nested RT-PCR (partial capsid region). NoV were detected in samples from all the cities and detected in 72% (84/116) of the samples with nine of them belonging to GI, 48 to GII and 27 to both genogroups. Remarkably, a high genetic diversity was identified: GII.2 (n = 13), GII.4 (n = 13), GI.1 (n = 5), GI.4 (n = 5), GI.8 (n = 4), GII.13 (n = 4), GII.1 (n = 3), GII.6 (n = 3), GI.3 (n = 1), GI.5 (n = 1), GI.6 (n = 1), GII.3 (n = 1), GII.17 (n = 1). Interestingly, a complete replacement of GII.4 New Orleans 2009 by GII.4 Sydney 2012 variants during 2012 was evidenced. This study reveals a high circulation of different NoV GI and GII genotypes in sewage evidencing a replacement of GII.4 variants. This approach can be used as an indicator of the presence of a new GII.4 variant which can originate an increase in acute gastroenteritis outbreaks worldwide.