Genogroup II Research Articles

Characterization of a novel group I F-specific RNA bacteriophage isolated from human stool.

A novel F-specific RNA bacteriophage (FRNAPH) YM1, affiliating to genogroup I (GI) of Levivirus, is isolated for the first time from human stool samples using double-layer agar plates with the Escherichia coli ATCC700891 as the host. The complete genomic sequence of YM1 is 3551 nt in length, obtained through next-generation sequencing, and contains four genes encoding for maturation protein, coat protein, lysis protein, and RNA-dependent RNA polymerase (RdRp). The genomic sequence of YM1 shares the highest similarity of 95.3% with that of a GI FRNAPH DL16 isolated from surface water of Great Bay. The YM1 possesses a non-enveloped, icosahedral virion of 23 ± 0.45nm in diameter. One-step growth curve analysis shows that the burst time of YM1 is 30min post-infection (p.i.) with the average burst size of 264 PFU/cell. The YM1 lyses only E. coli strains tested, revealing high host specificity. This newly discovered phage may serve as a candidate for viral indicator to monitor human enteric virus, especially norovirus, contamination in the environments.

Comparison of Skimmed Milk and Lanthanum Flocculation for Concentration of Pathogenic Viruses in Water

Concentration of viruses in water is necessary for detection and quantification of the viruses present, in order to evaluate microbiological barriers in water treatment plants and detect pathogenic viruses during waterborne outbreaks, but there is currently no standardised procedure. In this study, we implemented a previously described fast and simple lanthanum-based protocol for concentration of norovirus genogroup I (GI), genogroup II (GII) and hepatitis A virus (HAV) in drinking and surface water. We compared the results with those of a widely used skimmed milk flocculation method, followed by nucleic acid extraction and RT-qPCR detection. Three seeding levels, with intended concentrations 5 × 103, 5 × 104 and 5 × 105 genome copies/10 L, were added to drinking water or surface water. All seed levels were detected with both flocculation methods. Samples extracted with skimmed milk flocculation had on average 1.82, 1.86 and 1.38 times higher measured concentration of norovirus GI, GII and HAV, respectively, than those extracted with lanthanum flocculation, across all seeding levels and water types tested. Mengovirus was used as a positive process control. Mengovirus recovery was higher for skimmed milk (40.7% in drinking water, 26.0% in surface water) than for lanthanum flocculation (24.4% in drinking water, 9.7% in surface water). Together, this indicates that skimmed milk flocculation provides higher viral recovery than lanthanum flocculation. However, lanthanum-based flocculation can be performed much faster than skimmed milk flocculation (1.5 h versus 16 h flocculation time) and thus could be a good alternative for rapid monitoring of viruses in water.

Evaluation of two commercial molecular diagnostic assays: The Xpert Norovirus and the TRCReady NV

IntroductionNorovirus is highly contagious, and a few particles of this virus are sufficient to make people sick. It is desirable to develop quick and accurate laboratory methods to detect norovirus. MethodsWe evaluated two commercial molecular diagnostic assays, the Xpert Norovirus and the TRCReady NV, using clinical fecal samples. A reference method was performed using in-house real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). ResultsThe results of the real-time RT-PCR analysis of 60 suspected cases of norovirus infection showed 5 cases of Genogroup I (GI) positives and 21 cases of GII positives, among which was 1 GI and GII coinfection. The viral titers of the norovirus-positive samples ranged from 1.54 × 101 to 3.14 × 108 copies/μL. Norovirus GII.17 (12 cases, 48%) was the most frequently detected genotype in this study, followed by GII.4 (6 cases, 24%), GII.13 (2 cases, 8%), GI.2 (2 cases, 8%), GI.3 (2 cases, 8%), GI.1 (1 case, 4%), and GII.2 (1 case, 4%). The kappa coefficient was 1.000 (95% CI: 1.000–1.000) for Xpert Norovirus and 0.966 (95% CI: 0.896–1.000) for TRCReady NV, indicating a strong agreement. ConclusionsNorovirus detection using Xpert Norovirus and TRCReady NV is highly useful for diagnosis and infection control because these assays are easy to operate, quick, and exhibit almost the same performance as that of real-time RT-PCR.

Spatial and temporal distributions of enteric viruses and indicators in a lake receiving municipal wastewater treatment plant discharge.

Although lake water can be used as a source of drinking water and recreational activities, there is a dearth of research on the occurrence and fate of enteric viruses. Over a period of 14months at six points in 2014-2015, we conducted monthly monitoring of the virological water quality of a Japanese lake. The lake receives effluent from three surrounding wastewater treatment plants and retains water for about two weeks. These features allowed us to investigate the occurrence and fate of viruses in the lake environment. Human enteric viruses such as noroviruses and their indicators (pepper mild mottle virus and F-specific RNA bacteriophage [FRNAPH] genogroups) were quantified by PCR-based assays. Additionally, FRNAPH genogroups were quantified by infectivity-based assays to estimate the degree of virus inactivation. Pepper mild mottle virus, genogroup II (GII) norovirus, and GI-FRNAPH were identified in relatively high frequencies (positive in >40% out of 64 samples), with concentrations ranging from 1.3×101 to 2.9×104 copies/L. Human enteric viruses and some indicators were not detected and less prevalent, respectively, after April 2015. Principal component analysis revealed that the virological water quality changed gradually over time, but its differences between the sampling points were not apparent. FRNAPH genogroups were inactivated during the warm season (averaged water temperature of >20°C) compared to the cool season (averaged water temperature of <20°C), which may have been due to the more severe environmental stresses such as sunlight and water temperature. This suggests that the infection risk associated with the use of the lake water may have been overestimated by the gene quantification assay during the warm season.

Prevalence and Genetic Diversity of Norovirus in Acute Gastroenteritis Cases in the Southwest Province of Turkey.

Aims:Noroviruses may cause both epidemic and sporadic acute gastroenteritis globally. Thus, this study evaluated the prevalence of norovirus in stool samples of hospitalized patients with acute gastroenteritis in Aydin, Turkey using enzyme-linked immunoassay (ELISA) and real-time reverse transcription-polymerase chain reaction (rRT-PCR) and genotyped positive samples to detect which genotypes have currently circulated.Methods:This retrospective descriptive study collected 92 stool samples from patients with acute gastroenteritis symptoms from Aydın Adnan Menderes University Hospital from September 2017 to May 2019. The samples were tested using the commercial Third Generation Ridascreen norovirus ELISA and rRT-PCR. Positive samples were genotyped by sequencing of conventional positive RT-PCR products followed by phylogenetic analysis.Results:Of the 92 samples, 5 (5.4%) using ELISA and 12 (13%) using rRT-PCR tested positive for norovirus. All positive samples were genogroup II (GII). Two norovirus positive samples were genotyped successfully using DNA sequencing of the nested conventional PCR products. One sample (GII/Hu/TR/2019/Aydin25) could be categorized as GII.3 and the other (GII/Hu/TR/2019/Aydin20) as GII.13.Conclusion:rRT-PCR testing of stool samples is more sensitive than Ridascreen ELISA. Data from our study provide protocols for how to study norovirus epidemiology.

Involvement of Workers in Closed and Semiclosed Institutions in Outbreaks of Acute Gastroenteritis Due to Norovirus.

Norovirus outbreaks frequently occur in closed or semiclosed institutions. Recent studies in Catalonia and various countries indicate that, during outbreaks in these institutions, norovirus is detected in between 23% and 60% of workers, and the prevalence of infection in asymptomatic workers involved in outbreaks ranges from 17% to 40%. In this work, we carried out a prospective study to investigate the involvement of workers in closed and semiclosed institutions during outbreaks. The attack rates (ARs) and the rate ratios (RRs) were calculated according to the type of transmission and occupational category. The RRs and 95% confidence intervals (CIs) between workers and users were calculated. The mean cycle of quantification (Cq) values were compared according to the genogroup and the presence of symptoms. ARs were higher in person-to-person transmission than in common vehicle outbreaks, and 38.8% of workers were symptomatic. The RR between workers and users was 0.46 (95% CI 0.41–0.52). The ARs in workers were high, particularly in workers with closer contact with users. The mean Cq was lower in patients than in asymptomatic infected persons, although the difference was only significant for genogroup I (GI). The frequency of asymptomatic infected persons suggests that personal hygiene measures should be followed by all workers in the centers affected.

Hospital-based Surveillance for Pediatric Norovirus Gastroenteritis in Bangladesh, 2012-2016.

Globally, noroviruses are recognized as an important cause of acute gastroenteritis (AGE), but data from low and middle-income countries are limited. To examine the epidemiology and strain diversity of norovirus infections among children hospitalized for AGE in Bangladesh. We implemented active surveillance of children <5 years of age hospitalized with AGE at 8 geographically dispersed tertiary care hospitals in Bangladesh from July 2012 to June 2016. We tested random samples of AGE cases stratified by site and age group for norovirus by real-time RT-PCR. Noro-positive specimens were genotyped. Coinfection with rotavirus was assessed based on prior EIA testing. We enrolled 5622 total AGE cases, of which 1008 were tested for norovirus. Total of 137 (14%) AGE cases tested positive for norovirus (range, 11%-17% by site). Most (94%) norovirus-associated hospitalizations were among children less than 2 years of age. Norovirus was detected year-round, with higher detection from March to June (20%-38%) and November to January (9%-18%). Genogroup II (GII) noroviruses were detected in 96% of cases, and the most frequent genotypes were GII.4 Sydney [P4 New Orleans] (33%), GII.3 [P16] (20%), and GII.4 Sydney [P16] (11%). The proportion of norovirus-positive specimens was significantly greater among rotavirus-negative AGE patients compared with rotavirus-positive AGE patients (27% vs. 5%, P < 0.001). As measured by the Vesikari severity score, a similar proportion of norovirus and rotavirus positive AGE patients were considered severe (68% vs. 70%, P = 0.86). Norovirus is an important cause of AGE hospitalization in Bangladeshi children with most infections caused by GII viruses.

Surveillance of human norovirus in oysters collected from production area in Shandong Province, China during 2017–2018

Human norovirus (HuNoV) is the leading cause of non-bacterial gastroenteritis worldwide. Oysters are known food-related vehicles for HuNoV transmission. However, there are few data available on the viral contamination of shellfish from production areas in China. A one-year monitoring program was carried out to detect and characterize HuNoV contamination in oysters, collected from two shellfish commercial harvesting areas in Shandong province, China. Oysters were sampled every month from July 2017 to August 2018. From the 356 oyster samples, HuNoV was detected in 16.9% (60/356) using reverse transcription-qPCR. Of the 60 positive samples, 43.3% (26/60) were positive for HuNoV genogroup I (GI) only and 31.7% (19/60) were positive for genogroup II (GII) only. Both GI and GII were detected in 25.0% (15/60) samples. The majority (65.0%, 39/60) of positive samples were collected in winter months (November–December). There was no significant correlation between the detection of HuNoV in shellfish and the pH or fecal coliforms levels in seawater. Using next-generation sequencing, several HuNoV genotypes were identified including capsid genotypes GI.5, GI.6, GII.2, GII.3, GII.4_Sydney 2012, GII.13 and GII.14. These Results indicate that monitoring of HuNoV in oyster production area is necessary to ensure the safety of shellfish prior to distribution to consumers.

FUT2, Secretor Status and FUT3 Polymorphisms of Children with Acute Diarrhea Infected with Rotavirus and Norovirus in Brazil.

Host susceptibility according to human histo-blood group antigens (HBGAs) is widely known for norovirus infection, but is less described for rotavirus. Due to the variable HBGA polymorphism among populations, we aimed to evaluate the association between HBGA phenotypes (ABH, Lewis and secretor status) and susceptibility to rotavirus and norovirus symptomatic infection, and the polymorphisms of FUT2 and FUT3, of children from southeastern Brazil. Paired fecal-buccal specimens from 272 children with acute diarrhea were used to determine rotavirus/norovirus genotypes and HBGAs phenotypes/genotypes, respectively. Altogether, 100 (36.8%) children were infected with rotavirus and norovirus. The rotavirus P[8] genotype predominates (85.7%). Most of the noroviruses (93.8%) belonged to genogroup II (GII). GII.4 Sydney represented 76% (35/46) amongst five other genotypes. Rotavirus and noroviruses infected predominantly children with secretor status (97% and 98.5%, respectively). However, fewer rotavirus-infected children were Lewis-negative (8.6%) than the norovirus-infected ones (18.5%). FUT3 single nucleotide polymorphisms (SNP) occurred mostly at the T59G > G508A > T202C > C314T positions. Our results reinforce the current knowledge that secretors are more susceptible to infection by both rotavirus and norovirus than non-secretors. The high rate for Lewis negative (17.1%) and the combination of SNPs, beyond the secretor status, may reflect the highly mixed population in Brazil.

A recombinase polymerase amplification-based lateral flow strip assay for rapid detection of genogroup II noroviruses in the field.

Human norovirus is the leading cause of viral gastroenteritis worldwide. Rapid detection facilitates management of disease outbreaks, but field diagnosis is difficult to achieve due to the lack of reliable and portable methods. Recombinase polymerase amplification (RPA) is a robust isothermal amplification method that is capable of rapidly amplifying and detecting nucleic acids using simple equipment. In this study, RPA combined with lateral flow (LF) strips specific for human genogroup II (GII) noroviruses was established and evaluated. The assay specifically detects purified GII noroviruses as well as RNA in boiled human stool samples, with a sensitivity of 50 norovirus genome copies per reaction. The whole detection procedure of the one-step RT-RPA-LF is completed within 20 min, which is eight times faster than that of the standard real-time RT-PCR. The RT-RPA-LF method described here is suitable for rapid field diagnosis of all GII noroviruses in human stool samples.

Attribution of Pediatric Acute Gastroenteritis Episodes and Emergency Department Visits to Norovirus Genogroups I and II

Norovirus is a leading cause of acute gastroenteritis. With vaccines in development, population-based estimates of norovirus burden are needed to identify target populations, quantify potential benefits, and understand disease dynamics. We estimated the attributable fraction (AF) for norovirus infections in children, defined as the proportion of children testing positive for norovirus whose gastroenteritis was attributable to norovirus. We calculated the standardized incidence and emergency department (ED) visit rates attributable to norovirus using provincial gastroenteritis visit administrative data. From 3731 gastroenteritis case patients and 2135 controls we determined that the AFs were 67.0% (95% confidence interval [CI], 31.5%-100%) and 91.6% (88.8%-94.4%) for norovirus genogroups I (GI) and II (GII), respectively. Norovirus GII AF varied by season but not age. We attributed 116 episodes (95% CI, 103-129) and 59 (51-67) ED visits per 10 000 child-years to norovirus GII across all ages, accounting for 20% and 18% of all medically attended gastroenteritis episodes and ED visits, respectively. In children, a large proportion of norovirus GII detections reflect causation, demonstrating significant potential for norovirus GII vaccines. Seasonal variation in the norovirus GII AF may have implications for understanding the role asymptomatic carriage plays in disease dynamics.

High detection rates and genetic diversity of picobirnaviruses (PBVs) in pigs on St. Kitts Island: Identification of a porcine PBV strain closely related to simian and human PBVs

We report here high rates (75.38%, 49/65) of detection of genogroup I (GI) PBVs in diarrheic pigs on the Caribbean island of St. Kitts. High quality gene segment-2 sequences encoding a significant region (~350 amino acid (aa) residues) of the putative RNA-dependent RNA polymerase (RdRp) were obtained for 23 PBV strains. The porcine PBV strains from St. Kitts exhibited high genetic diversity among themselves (deduced aa identities of 56–100%) and with other PBVs (maximum deduced aa identities of 64–97%), and retained the three domains that are conserved in putative RdRps of PBVs. The nearly complete gene segment-2 sequence (full-length minus partial 3′- untranslated region) of a porcine PBV strain (strain PO36 from St. Kitts) that is closely related (deduced aa identities of 96–97%) to simian and human GI PBVs was determined using a combination of the non-specific primer-based amplification method and conventional RT-PCR. The complete putative RdRp sequence of strain PO36 preserved the various features that are maintained in PBVs from various species. For the first time, several co-circulating PBV strains from pigs were characterized for a significant region (~350 aa) of the putative RdRp, providing important insights into the genetic diversity of PBVs in a porcine population. Taken together, these observations corroborated growing evidence that PBVs can be highly prevalent and show limited correlation globally with host species or geography. This is the first report on detection of PBVs in pigs from the Caribbean region.

Spatial and temporal profiles of enteric viruses in the coastal waters of Tokyo Bay during and after a series of rainfall events

Recreational activities in coastal waters that are polluted by enteric viruses can result in gastroenteritis etc. In this study, the pollution profiles of enteric viruses were examined in the coastal area of Tokyo Bay, Japan, by collecting 57 water samples from three different depths (0.5 m, 3.0 m, and 5.0 m) during and after a series of heavy rainfall events. Vertically spatial and temporal changes in the concentrations of NoV genogroup I (GI) and genogroup II (GII), pepper mild mottle virus (PMMoV), and Aichi virus (AiV) were determined using quantitative reverse transcription-polymerase chain reaction, while those of the bacterial indicator, Escherichia coli, and F-specific RNA bacteriophages (FRNA phages) were monitored using culture methods. PMMoV was highly abundant (1.4 × 104–6.8 × 106 genome copies/L), whereas the concentrations of the other enteric viruses were relatively low (AiV, 1.3 × 102–2.9 × 104; GI, 2.9 × 10–5.6 × 103; GII, 2.5 × 10–1.2 × 104 genome copies/L). All of the viruses showed lower fluctuations in concentration than E. coli, which increased up to 460-fold after the rainfall event and then decreased over the subsequent two weeks. The maximum vertical difference in E. coli concentration was observed immediately after the rainfall. The E. coli reached the surface and then gradually spread down, whereas the virus concentrations exhibited few fluctuations due to the remaining effects of the previous combined sewer overflows. These findings indicate that viruses have a relatively long retention period over fecal indicator bacteria in this coastal area.

Leafy vegetables as potential transmitters of Norovirus in Cordoba city, Argentina.

The Norovirus (NoV) constitute a genus within the viral family Caliciviridae, being the main cause of outbreaks of food origin among humans. Fresh vegetables are susceptible to being contaminated with these pathogens during their cultivation, harvest, transport, processing and handling. So it was intended to determine the frequency of detection of NoV in plant samples of leaves of the City of Córdoba, and adapt a method of viral concentration with polyethylene glycol for the recovery of viral particles from the surface of vegetables and characterize the genogroups of NoV detected. 19 samples of leafy vegetables were taken between June and December 2012. A viral concentration technique previously validated in the laboratory was applied (elution and precipitation with polyethylene glycol). The viral RNA was extracted to the concentrates of the samples using Trizol and precipitation with isopropyl alcohol. The nucleic acid was amplified by Rt-PCR with specific primers to identify genogroups I (GI) and II (GII). The products of the amplification were revealed by polyacrylamide gel electrophoresis and silver staining. We found 57.89% positive samples. Ten of the detected strains belonged to genogroup I (GI) and one to genogroup II (GII). They were identified throughout the study period, particularly during the months of August, September and November. These pathogens were detected with a prevalence of 57.89%. The strains belonged mainly to the GI, representing a potential risk for the population.

Redesigned Duplex RT-qPCR for the Detection of GI and GII Human Noroviruses

Abstract Human noroviruses (HuNoVs) are major foodborne pathogens that cause nonbacterial acute gastroenteritis worldwide. As the tissue-culture system for HuNoVs is not mature enough for routine detection of the virus, detection is mainly dependent on molecular approaches such as reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The widely used primers and probes for RT-qPCR were established in the early 2000s. As HuNoVs are highly variant viruses, viral genome mutations result in previously designed primers and/or probes that were perfectly matched working less efficiently over time. In this study, a new duplex RT-qPCR (ND-RT-qPCR) was designed for the detection of genogroup I (GI) and genogroup II (GII) HuNoVs based on an analysis of viral sequences added in the database after 2010. Using long transcribed viral RNAs, the results demonstrate that the sensitivity of ND-RT-qPCR is as low as one genomic copy for both GI and GII HuNoVs. The performance of ND-RT-qPCR was further evaluated by a comparison with the commonly used Kageyama primer/probe sets for RT-qPCR (Kageyama RT-qPCR) for 23 HuNoV-positive clinical samples. All five GI samples were registered as positive by ND-RT-qPCR, whereas only two samples were registered as positive by Kageyama RT-qPCR. All 18 GII samples were registered as positive by ND-RT-qPCR, while 17 samples were registered as positive by Kageyama RT-qPCR. The sensitivity reflected by the quantification cycle (Cq) value was lower in ND-RT-qPCR than in Kageyama RT-qPCR. Our data suggest that ND-RT-qPCR could be a good fit for the detection of current strains of HuNoVs.

Quantitative PCR Detection of Enteric Viruses in Wastewater and Environmental Water Sources by the Lisbon Municipality: A Case Study

Current regulations and legislation require critical revision to determine safety for alternative water sources and water reuse as part of the solution to global water crisis. In order to fulfill those demands, Lisbon municipality decided to start water reuse as part of a sustainable hydric resources management, and there was a need to confirm safety and safeguard for public health for its use in this context. For this purpose, a study was designed that included a total of 88 samples collected from drinking, superficial, underground water, and wastewater at three different treatment stages. Quantitative Polimerase Chain Reaction (PCR) detection (qPCR) of enteric viruses Norovirus (NoV) genogroups I (GI) and II (GII) and Hepatitis A (HepA) was performed, and also FIB (E. coli, enterococci and fecal coliforms) concentrations were assessed. HepA virus was only detected in one untreated influent sample, whereas NoV GI/ NoV GI were detected in untreated wastewater (100/100%), secondary treated effluent (47/73%), and tertiary treated effluent (33/20%). Our study proposes that NoV GI and GII should be further studied to provide the support that they may be suitable indicators for water quality monitoring targeting wastewater treatment efficiency, regardless of the level of treatment.

Detection and Molecular Characterization of Picobirnaviruses (PBVs) in the Mongoose: Identification of a Novel PBV Using an Alternative Genetic Code.

We report high rates of detection (35.36%, 29/82) of genogroup-I (GI) picobirnaviruses (PBVs) in non-diarrheic fecal samples from the small Indian mongoose (Urva auropunctata). In addition, we identified a novel PBV-like RNA-dependent RNA polymerase (RdRp) gene sequence that uses an alternative mitochondrial genetic code (that of mold or invertebrate) for translation. The complete/nearly complete gene segment-2/RdRp gene sequences of seven mongoose PBV GI strains and the novel PBV-like strain were obtained by combining a modified non-specific primer-based amplification method with conventional RT-PCRs, facilitated by the inclusion of a new primer targeting the 3′-untranslated region (UTR) of PBV gene segment-2. The mongoose PBV and PBV-like strains retained the various features that are conserved in gene segment-2/RdRps of other PBVs. However, high genetic diversity was observed among the mongoose PBVs within and between host species. This is the first report on detection of PBVs in the mongoose. Molecular characterization of the PBV and PBV-like strains from a new animal species provided important insights into the various features and complex diversity of PBV gene segment-2/putative RdRps. The presence of the prokaryotic ribosomal binding site in the mongoose PBV genomes, and analysis of the novel PBV-like RdRp gene sequence that uses an alternative mitochondrial genetic code (especially that of mold) for translation corroborated recent speculations that PBVs may actually infect prokaryotic or fungal host cells.

Application of a multiplex salivary immunoassay to detect sporadic incident norovirus infections

Norovirus is one of the most common causes of gastroenteritis. Following infection, anti-norovirus salivary immunoglobulin G (IgG) rises steeply within 2 weeks and remains elevated for several months; this immunoconversion can serve as an indicator of infection. We used a multiplex salivary immunoassay to study norovirus infections among 483 visitors to a Lake Michigan beach in 2015. Saliva was collected on the day of the beach visit (S1); after 10–14 days (S2); and after 30–40 days (S3). Luminex microspheres were coupled to recombinant antigens of genogroup I (GI) and II (GII) noroviruses and incubated with saliva. Immunoconversion was defined as at least 4-fold increase in anti-norovirus IgG antibody response from S1 to S2 and a 3-fold increase from S1 to S3. Ten (2.1%) immunoconverted to either GI (2) or GII (8) norovirus. Among those who immunoconverted, 40% reported at least one gastrointestinal symptom and 33% reported diarrhea, compared to 15% (p = 0.06) and 8% (p = 0.04) among those who did not immunoconvert, respectively. The two participants who immunoconverted to GI norovirus both swallowed water during swimming (p = 0.08). This study demonstrated the utility of a non-invasive salivary immunoassay to detect norovirus infections and an efficient approach to study infectious agents in large cohorts.

645. Singulex Clarity Norovirus Assay (In Development) Provides Ultrasensitive Detection of Norovirus Genogroups I and II

BackgroundCommercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen.MethodsThe Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®).ResultsFrom all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures).ConclusionThe ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico

To determine the seasonal occurrence and diversity of norovirus (NoV) and human adenovirus (HAdV) in groundwater from sinkholes, and brackish water used for recreational activities in the karst aquifer of the Yucatan Peninsula, Mexico. Hollow fibre ultrafiltration was used to concentrate viruses and standard plaque assay methods were used to enumerate somatic and F+ specific coliphages as viral indicators. Real-time quantitative polymerase chain reaction assays were used to estimate the number of genome copies for NoV strains GI, and GII, and HAdVs. The predominant NoV genotypes and HAdV serotypes were identified by comparative sequence analysis. Somatic and male F+ specific coliphages were detected at concentrations up to 94 and 60 plaque-forming units per 100ml respectively. The NoV genogroup I (GI) was associated with 50% of the sampled sites during the rainy season only, at concentrations ranging from 120 to 1600 genome copies per litre (GCl-1 ). The NoV genogroup II (GII) was detected in 30 and 40% of the sampled sites during the rainy and dry seasons, respectively, at concentrations ranging from 10 to 290GCl-1 . During the rainy and dry seasons, HAdVs were detected in 20% of the sites, at concentrations ranging from 24 to 690 GCl-1 . Identification of viral types revealed the presence of NoV GI.2, GII.Pe, GII.P16 and GII.P17, and HAdV F serotypes 40 and 41. These findings demonstrate that NoVs and HAdVs are prevalent as virus contaminants in the karst aquifer, representing potential health risks particularly during the rainy season, in one of the most important areas used for tourism in Mexico. This is one of the few studies conducted in karst aquifers that provide a foundational baseline of the distribution, concentrations and diversity of NoVs and HadVs in these particular environments.