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Noroviruses are recognized as the leading cause of acute gastroenteritis globally. With improved molecular diagnostics developed over the last two decades, archived clinical specimens are increasingly used to investigate the historic prevalence and molecular epidemiology of human norovirus. Yet the impact of long-term storage on viral integrity in clinical specimens has not been evaluated. In this study, we retested 994 stool specimens collected between 1996 and 2017 that originally tested norovirus-positive to quantify the loss of norovirus RT-PCR positivity with increasing sample storage time at 4 °C. In all, 79% of samples tested positive after retesting, but there was an approximate 3% decline in the positivity ratio and 4% decline in the percentage of samples that could be genotyped with each additional year of sample storage. For samples that were originally quantified by real-time RT-PCR (collected between 2003 and 2017), there was an estimated 1-log loss of viral titer occurring every 7 years of sample storage. Few samples contained PCR inhibitors, assessed using a MS2 extraction control, indicating that loss of RT-PCR signal was due primarily to loss of viral RNA integrity after long-term storage of stool samples at 4 °C. Our results indicate that norovirus positive stool samples can be stored with minimal loss in RT-PCR positivity when stored less than a decade. Longer periods of storage may impair norovirus detection, potentially impacting historic estimates of norovirus prevalence and molecular epidemiology if derived by testing archival clinical specimens.

Background Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. Objective To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Study design Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014–2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5′end of the norovirus ORF2 gene. Results A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5–93.5) and 99.1% (98.0–99.6) and for GII was 94.8% (90.8–97.2) and 98.6% (96.9–99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. Conclusions The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Background The family Caliciviridae consists of a genetically diverse group of RNA viruses that infect a wide range of host species including noroviruses and sapoviruses which cause acute gastroenteritis in humans. Typing of these viruses relies on sequence-based approaches, and therefore there is a need for rapid and accurate web-based typing tools. Objective To develop and evaluate a web-based tool for rapid and accurate genotyping of noroviruses and sapoviruses. Methods The Human Calicivirus Typing (HuCaT) tool uses a set of curated reference sequences that are compared to query sequences using a k-mer (DNA substring) based algorithm. Outputs include alignments and phylogenetic trees of the 12 top matching reference sequences for each query. Results The HuCaT tool was validated with a set of 1310 norovirus and 239 sapovirus sequences covering all known human norovirus and sapovirus genotypes. HuCaT tool assigned genotypes to all queries with 100 % accuracy and was much faster (17 s) than BLAST (150 s) or phylogenetic analyses approaches. Conclusions The web-based HuCaT tool supports rapid and accurate genotyping of human noroviruses and sapoviruses.

In June 2017, the Pennsylvania Department of Health (PADOH) was notified of multiple norovirus outbreaks associated with 179 ill individuals who attended separate events held at an outdoor venue and campground over a month period. Epidemiologic investigations were unable to identify a single exposure route and therefore unable to determine whether there was a persistent contamination source to target for exposure mitigation. Norovirus was detected in a fresh recreational water designated swimming area and a drinking water well. A hydrogeological site evaluation suggested a nearby septic leach field as a potential contamination source via ground water infiltration. Geological characterization revealed a steep dip of the bedrock beneath the septic leach field toward the well, providing a viral transport pathway in a geologic medium not previously documented as high risk for viral ground water contamination. The human-associated microbial source tracking (MST) genetic marker, HF183, was used as a microbial tracer to demonstrate the hydrogeological connection between the malfunctioning septic system, drinking water well, and recreational water area. Based on environmental investigation findings, venue management and local public health officials implemented a series of outbreak prevention strategies including discontinuing the use of the contaminated well, issuing a permit for a new drinking water well, increasing portable toilet and handwashing station availability, and promoting proper hand hygiene. Despite the outbreaks at the venue and evidence of ground water contamination impacting nearby recreational water and the drinking water well, no new norovirus cases were reported during a large event one week after implementing prevention practices. This investigation highlights a new application for human-associated MST methods to trace hydrological connections between multiple fecal pollutant exposure routes in an outbreak scenario. In turn, pollutant source information can be used to develop effective intervention practices to mitigate exposure and prevent future outbreaks associated with human fecal contaminated waters.

Highlights • We validated genogroup-specific one-step conventional RT-PCR assays (PC assays) for sequence-based dual typing of GI and GII norovirus strains. • The PC assays use a combination of oligonucleotide primers that target a genomic region spanning the 3’-end of ORF1 and 5’end of ORF2 of GI and GII noroviruses. • The PC assays are sensitive (5 to 50 copies/rx) and detect all currently identified norovirus P-types and capsid genotypes from different geographic regions. • The PC assays have been successfully implemented by CaliciNet USA and CaliciNet China., Background Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. Objective Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. Results The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010–2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. Conclusion The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.