Genotype GII Research Articles

Wastewater Surveillance for Norovirus, California, USA.

Norovirus is a leading cause of acute gastroenteritis and imposes a substantial disease burden. In California, USA, norovirus surveillance is limited. We evaluated correlations between wastewater norovirus concentrations and available public health surveillance data. Wastewater surveillance for norovirus genotype GII in California provided timely, localized, and actionable data for public health authorities.

Emerging zooanthroponotic risks: Detection of the human norovirus GII.4 Sydney[P31] strain in a domestic dog in Brazil

Recent increases in zoonotic diseases underscore the integration of companion animals into urban environments, posing complex transmission risks and highlighting the necessity of One Health approaches. Respiratory and enteric viruses have been consistently linked to interspecies transmission between humans and animals. This study aimed to assess the circulation of human noroviruses (NoV), human adenoviruses (HAdV), enteroviruses (EV), parechoviruses (PeV-A), human bocaviruses (HBoV), hepatitis A (HAV) and E viruses (HEV), Influenza A and B viruses (Flu A/B), respiratory syncytial virus (RSV), and SARS-CoV-2 in domestic dogs and cats in Brazil to understand potential zooanthroponosis risks. Between 2012 and 2021, 600 fecal samples from dogs and cats (516 and 84, respectively) were collected at small animal clinics in São Paulo state, Brazil. The specimens underwent in-house qPCR screening for HBoV and HAdV, while EV, PeV-A, NoV, and HEV were tested using in-house RT-qPCR. SARS-CoV-2, Flu A/B, and RSV were investigated with a commercial RT-qPCR kit assay. HAV detection utilized conventional nested (RT)-PCR. Positive samples were sequenced for molecular characterization and phylogenetic analysis. NoV was detected in 0.2 % (1/600) of the animals, while all other investigated viruses tested negative. The NoV-positive sample, collected in 2012 from a pet dog, was identified as genotype GII.4_Sydney[P31]. The Dog/BRA/2012/GII.4_Sydney[P31]/IAL-M21 strain exhibited a close genetic relationship to Brazilian human and environmental NoV GII.4_Sydney[P31] strains, with 98.1–99.2 % nucleotide similarity in ORF1 and 99.2–99.6 % in ORF2 sequences, suggesting interspecies transmission. Pet dogs are frequently exposed to human fecal-borne viruses, highlighting the potential for zooanthroponotic transmission due to their close interaction with humans in shared environments. There is an urgent need to enhance surveillance studies in companion animals to better understand the implications of detecting human NoV strains in pets, as NoV could potentially act as a reverse zoonotic disease in households, animal hospitals, or shelters worldwide.

Insights into human norovirus cultivation in human intestinal enteroids.

Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights into this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells that were then transplanted and matured in mice before making enteroids (H9tHIEs), genetically engineered (J4FUT2 knock-in [KI], J2STAT1 knockout [KO]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of genogroup I and II HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.IMPORTANCEHuman noroviruses (HuNoVs) cause global diarrheal illness and chronic infections in immunocompromised patients. This paper reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from (i) different intestinal segments of single donors, (ii) human embryonic stem cell-derived organoids transplanted into mice, (iii) genetically modified lines, and (iv) a patient with common variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment, and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically modified J4FUT2 knock-in (KI) HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

Prevalence and genotype distribution of norovirus in Ningxia Hui Autonomous Region, China, from 2011 to 2022

The norovirus (NoV) genome is diverse. Therefore, this study explored the epidemiological characteristics and genetic features of NoV in Ningxia Hui Autonomous Region, China, from 2011 to 2022 to clarify the genetic diversity in this region. Stool samples were screened for NoV and then sequenced and genotyped. In total, 1,788 of 13,083 specimens were NoV -positive (13.67%); 204 (1.56%) and 1,584 (12.11%) cases were GI and GII, respectively. Additionally, 559 were NoV infection with other viruses (4.27%), primarily with rotavirus (277/559, 49.55%). The NoV incidence rate was the highest among children aged 0–2 years (18.09%, 1054/5,828) and lowest among adults aged 45–64 years (110/1,495, 7.36%); it was also higher in the winter and spring than in the other seasons. GI.3[P3] was the dominant GI genotype. The dominant GII genotype changed roughly every two years. In the GII group, GII.4 was the most common genotype (46.79%), followed by GII.3 (21.34%), GII.2 (12.34%), and GII.17 (9.77%). There were three variants of GII.4 Den Haag, GII.4 New Orleans and GII.4 Sydney identified in the detected GII.4 strains, with GII.4 Sydney dominating. The GII.4 (87.36%), GII.3 (86.35%), and GII.2 (72.92%) strains were primarily detected in children, whereas it was the GII.17 (52.63%) strain in adults. Overall, the NoV genotypes in the Ningxia Hui Autonomous Region were diverse. Primarily, GII groups were dominant, but this changed over time.

Insights into Human Norovirus Cultivation in Human Intestinal Enteroids.

HuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from i) different intestinal segments of single donors, ii) human embryonic stem cell-derived organoids transplanted into mice, iii) genetically-modified lines, and iv) a patient with chronic variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically-modified J4 FUT2-KI HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.

Isolation and characterization of porcine epidemic diarrhea virus with mutations in the spike gene in China

Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global swine industry. Due to frequent mutations in the spike (S) gene of PEDV, commercial vaccines used today are gradually losing their protective efficacy against variants. It's significant to monitor the S gene of PEDV variants and understand its evolutionary trend. In this study, we report four novel PEDV strains isolated from Sichuan, Guangdong and Shanxi Provinces and determined their S gene sequences. Phylogenetic analysis showed that they all belong to GII genotype. Amino acid alignment revealed a unique mutation pattern. We also predicted their three-dimensional structures and continuous B-cell epitopes and compared them to those of the vaccine strain. Our study provides references for understanding the evolution of S gene and antigenic change of S protein, which are of great significance for formulating the prevention and control of PEDV.

Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

The viral trends and genotype diversity of norovirus in the wastewater of Shenzhen, China

Norovirus (NoV) is the primary cause of acute gastroenteritis (AGE) on a global scale. Numerous studies have demonstrated the immense potential of wastewater surveillance in monitoring the prevalence and spread of NoV within communities. This study employed a one-step reverse transcription-quantitative PCR to quantify NoV GI/GII in wastewater samples (n = 2574), which were collected once or twice a week from 38 wastewater treatment plants from March 2023 to February 2024 in Shenzhen. The concentrations of NoV GI and GII ranged from 5.0 × 104 to 1.7 × 106 copies/L and 4.1 × 105 to 4.5 × 106 copies/L, respectively. The concentrations of NoV GII were higher than those of NoV GI. Spearman's correlation analysis revealed a moderate correlation between the concentration of NoV in wastewater and the detection rates of NoV infections in sentinel hospitals. Baseline values were established for NoV concentrations in Shenzhen's wastewater, providing a crucial reference point for implementing early warning systems and nonpharmaceutical interventions to mitigate the impact of potential outbreaks. A total of 24 NoV genotypes were identified in 100 wastewater samples by sequencing. Nine genotypes of NoV GI were detected, with the major genotypes being GI.4 (38.6 %) and GI.3 (21.8 %); Fifteen genotypes of NoV GII were identified, with GII.4 (53.6 %) and GII.17 (26.0 %) being dominant. The trends in the relative abundance of NoV GI/GII were significantly different, and the trends in the relative abundance of NoV GII.4 over time were similar across all districts, suggesting a potential risk of cross-regional spread. Our findings underscore the effectiveness of wastewater surveillance in reflecting population-level NoV infections, capturing the diverse array of NoV genotypes, and utilizing NoV RNA in wastewater as a specific indicator to supplement clinical surveillance data, ultimately enhancing our ability to predict the timing and intensity of NoV epidemics.

Genetic diversity of enteric viruses responsible of gastroenteritis in urban and rural Burkina Faso.

Viral gastrointestinal infections remain a major public health concern in developing countries. In Burkina Faso, there are very limited updated data on the circulating viruses and their genetic diversity. This study investigates the detection rates and characteristics of rotavirus A (RVA), norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) in patients of all ages with acute gastrointestinal infection in urban and rural areas. From 2018 to 2021, stool samples from 1,295 patients with acute gastroenteritis were collected and screened for RVA, NoV, SaV and HAstV. Genotyping and phylogenetic analyses were performed on a subset of samples. At least one virus was detected in 34.1% of samples. NoV and SaV were predominant with detection rates of respectively 10.5 and 8.8%. We identified rare genotypes of NoV GII, RVA and HAstV, recombinant HAstV strains and a potential zoonotic RVA transmission event. We give an up-to-date epidemiological picture of enteric viruses in Burkina Faso, showing a decrease in prevalence but a high diversity of circulating strains. However, viral gastroenteritis remains a public health burden, particularly in pediatric settings. Our data advocate for the implementation of routine viral surveillance and updated management algorithms for diarrheal disease.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

A quadrivalent norovirus vaccine based on a chimpanzee adenovirus vector induces potent immunity in mice

Norovirus (NoV) infection is a major cause of gastroenteritis worldwide. The virus poses great challenges in developing vaccines with broad immune protection due to its genetic and antigenic diversity. To date, there are no approved NoV vaccines for clinical use. Here, we aimed to develop a broad-acting quadrivalent NoV vaccine based on a chimpanzee adenovirus vector, AdC68, carrying the major capsid protein (VP1) of noroviral GI and GII genotypes. Compared to intramuscular (i.m.), intranasal (i.n.), or other prime-boost immunization regimens (i.m. ​+ ​i.m., i.m. ​+ ​i.n., i.n. ​+ ​i.m.), AdC68-GI.1-GII.3 (E1)-GII.4-GII.17 (E3), administered via i.n. ​+ ​i.n. induced higher titers of serum IgG antibodies and higher IgA antibodies in bronchoalveolar lavage fluid (BALF) and saliva against the four homologous VP1s in mice. It also significantly stimulated the production of blocking antibodies against the four genotypes. In response to re-stimulation with virus-like particles (VLP)-GI.1, VLP-GII.3, VLP-GII.4, and VLP-GII.17, the quadrivalent vaccine administered according to the i.n. ​+ ​i.n. regimen effectively triggered specific cell-mediated immune responses, primarily characterized by IFN-γ secretion. Furthermore, the preparation of this novel quadrivalent NoV vaccine requires only a single recombinant adenovirus to provide broad preventive immunity against the major GI/GII epidemic strains, making it a promising vaccine candidate for further development.

Epidemiology of norovirus infection in Nigeria: a systematic review and meta-analysis.

Human norovirus (HuNoV) is responsible for most cases of gastroenteritis worldwide, but information about the prevalence and diversity of HuNoV infections in lower-income settings is lacking. In order to provide more information about the burden and distribution of norovirus in Nigeria, we systematically reviewed original published research articles on the prevalence of HuNoV in Nigeria by accessing databases, including PubMed, Web of Science, ScienceDirect, Google Scholar, and African Journals Online (AJOL). The protocol for the review was registered on PROSPERO (registration number CRD42022308857). Thirteen relevant articles were included in the review, and 10 of them were used for meta-analysis. The pooled prevalence of HuNoV-associated gastroenteritis among children below 5 years of age in Nigeria, determined using the random-effects model, was 10.9% (95% CI, 6.7-16.7%). Among children below the age of 5 presenting with HuNoV infections, the highest prevalence was in children ≤2 years old (n = 127, 83%). The prevalence of HuNoV infections was seen to decrease with increasing age. In addition, HuNoV was detected in asymptomatic food handlers, bats, and seafoods. A total of 85 sequences of HuNoV isolates from Nigeria have been determined, and based on those sequences, the most prevalent norovirus genogroup was GII (84%). Genotypes GII.4 and GI.3 were the most frequently identified genotypes, with GII.4 constituting 46% of all of the HuNoVs identified in Nigeria. These results suggest a risk associated with cocirculation of emerging variants with known genotypes because of their recombination potential. Larger molecular epidemiological studies are still needed to fully understand the extent and pattern of circulation of HuNoVs in Nigeria.

Diverse genotypes of norovirus genogroup I and II contamination in environmental water in Thailand during the COVID-19 outbreak from 2020 to 2022

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water.

Tailored wastewater surveillance framework uncovered the epidemics of key pathogens in a Northwestern city of China

Wastewater surveillance enables rapid pathogen monitoring and community prevalence estimation. However, how to design an integrated and tailored wastewater surveillance framework to monitor major health threats in metropolises remains a major challenge. In this study, we first analyzed the historical clinical data of Xi'an city and designed a wastewater surveillance framework covering five key endemic viruses, namely, SARS-CoV-2, norovirus, influenza A virus (IAV), influenza B virus (IBV), respiratory syncytial virus (RSV), and hantavirus. Amplicon sequencing of SARS-CoV-2, norovirus and hantavirus was conducted biweekly to determine the prevalent community genotypes circulating in this region. The results showed that from April 2023 to August 2023, Xi'an experienced two waves of SARS-CoV-2 infection, which peaked in the middle of May-2023 and late August-2023. The sewage concentrations of IAV and RSV peaked in early March and early May 2023, respectively, while the sewage concentrations of norovirus fluctuated throughout the study period and peaked in late August. The dynamics of the sewage concentrations of SARS-CoV-2, norovirus, IAV, RSV, and hantavirus were in line with the trends in the sentinel hospital percent positivity data, indicating the role of wastewater surveillance in enhancing the understanding of epidemic trends. Amplicon sequencing of SARS-CoV-2 revealed a transition in the predominant genotype, which changed from DY.1 and FR.1.4 to the XBB and EG.5 subvariants. Amplicon sequencing also revealed that there was only one predominant hantavirus genotype in the local population, while highly diverse genotypes of norovirus GI and GII were found in the wastewater. In conclusion, this study provided valuable insights into the dynamics of infection trends and predominant genotypes of key pathogens in a city without sufficient clinical surveillance, highlighting the role of a tailored wastewater surveillance framework in addressing public health priorities. More importantly, our study provides the first evidence demonstrating the applicability of wastewater surveillance for hantavirus, which is a major health threat locally.

Prevalence, Clinical Features, and Genotypes of Norovirus-Associated Diarrhea in Wuxi, China, 2013-2020.

Norovirus (NoV) is a common pathogen that can cause infectious diarrhea. This study aimed to determine the prevalence, clinical features, and genotypes of NoV-associated diarrhea in Wuxi, China. A total of 4,416 stool samples were collected from patients with diarrhea at enteric disease clinics of sentinel hospitals in Wuxi from February 1, 2013 to December 31, 2020. Univariate and Akaike information criterion stepwise logistic regression were used to identify differences as integrated within a clinical setting (NoV positive [+] versus NoV negative [-], NoV+ versus rotavirus [RV]+, NoV+ versus bacteria+, genogroup [G] I and GII genotypes). Norovirus was detected in 9.85% of stool samples, which was greater than other tested pathogens. Excluding coinfection of NoV and other viruses or bacteria, patients infected with NoV had a lower chance of acquiring the virus in summer (P < 0.001; odds ratio [OR], 0.257; 95% CI, 0.189-0.36) when compared with patients without NoV. Patients with diarrhea infected with NoV featured nausea and vomiting (P < 0.001; OR, 2.297, 95% CI, 1.85-2.86) and loose stools (P = 0.006; OR, 2.247; 95% CI, 1.30-4.10), but less abdominal cramping (P = 0.001; OR, 0.676; 95% CI, 0.54-0.84). Patients infected with RV (P < 0.001; OR, 0.413; 95% CI, 0.25-0.68) or bacteria (P < 0.001; OR, 0.422; 95% CI, 0.26-0.67) were more vulnerable to fever than those infected with NoV. A total of 379 GII strains were detected concomitant with 48 GI strains, and there was a seasonal difference between the GI and GII genotypes. Strengthening pathogen detection for infectious diarrhea was helpful for understanding the epidemiological characteristics of infections with NoV and, potentially, for preventing disease outbreaks.

Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

Assessment of Gastroenteric Viruses in Marketed Bivalve Mollusks in the Tourist Cities of Rio de Janeiro, Brazil, 2022.

This study investigated the prevalence and genetic diversity of gastroenteric viruses in mussels and oysters in Rio de Janeiro, Brazil. One hundred and thirty-four marketed bivalve samples were obtained between January and December 2022. The viral analysis was performed according to ISO/TS 15216, and the screening revealed the detection of norovirus GII/GI (40.3%), sapovirus (SaV; 12.7%), human mastadenovirus (7.5%), and rotavirus A (RVA; 5.9%). In total, 44.8% (60) of shellfish samples tested positive for one or more viruses, 46.7% (28/60) of the positive samples tested positive for a single viral agent, 26.7% (16) tested positive for two viral agents, 8.3% (5) for three viral agents, and 13.3% (8) for four viral agents. Additionally, three mussel samples were contaminated with the five investigated viruses (5%, 3/60). Norovirus GII showed the highest mean viral load (3.4 × 105 GC/g), followed by SaV (1.4 × 104 GC/g), RVA (1.1 × 104 GC/g), human mastadenovirus (3.9 × 103 GC/g), and norovirus GI (6.7 × 102 GC/g). Molecular characterization revealed that the recovered norovirus strains belonged to genotypes GII.2, GII.6, GII.9, GII.17, and GII.27; SaV belonged to genotypes GI.1 and GIV.1; RVA to genotypes G6, G8, P[8]-III, and human mastadenovirus to types F40 and F41. The GII.27 norovirus characterized in this study is the only strain of this genotype reported in Brazil. This study highlights the dissemination and diversity of gastroenteric viruses present in commercialized bivalves in a touristic area, indicating the potential risk to human health and the contribution of bivalves in the propagation of emerging pathogens.

Norovirus Epidemiology and Genotype Circulation during the COVID-19 Pandemic in Brazil, 2019-2022.

Norovirus stands out as a leading cause of acute gastroenteritis (AGE) worldwide, affecting all age groups. In the present study, we investigated fecal samples from medically attended AGE patients received from nine Brazilian states, from 2019 to 2022, including the COVID-19 pandemic period. Norovirus GI and GII were detected and quantified using RT-qPCR, and norovirus-positive samples underwent genotyping through sequencing the ORF1/2 junction region. During the four-year period, norovirus prevalence was 37.2%, varying from 20.1% in 2020 to 55.4% in 2021. GII genotypes dominated, being detected in 92.9% of samples. GII-infected patients had significantly higher viral concentrations compared to GI-infected patients (median of 3.8 × 107 GC/g and 6.7 × 105 GC/g, respectively); and patients aged >12-24 months showed a higher median viral load (8 × 107 GC/g) compared to other age groups. Norovirus sequencing revealed 20 genotypes by phylogenetic analysis of RdRp and VP1 partial regions. GII.4 Sydney[P16] was the dominant genotype (57.3%), especially in 2019 and 2021, followed by GII.2[P16] (14.8%) and GII.6[P7] (6.3%). The intergenogroup recombinant genotype, GIX.1[GII.P15], was detected in five samples. Our study is the first to explore norovirus epidemiology and genotype distribution in Brazil during COVID-19, and contributes to understanding the epidemiological dynamics of norovirus and highlighting the importance of continuing to follow norovirus surveillance programs in Brazil.

920. A Phase 2 Multi-Center, Prospective, Randomized, Double-Blind Study to Assess the Clinical and Antiviral Efficacy and Safety of Nitazoxanide for the Treatment of Norovirus in Hematopoietic Stem Cell and Solid Organ Transplant Recipients

Abstract Background Norovirus (NoV) results in potentially severe, relapsing, remitting diarrhea in immunocompromised hosts (ICH). A number of interventions, including nitazoxanide (NTZ), have been tried with unclear success in managing cases of NoV in ICH. Methods We conducted a NIH-sponsored multi-center, prospective, randomized, double-blind study of NTZ for the treatment of Norovirus in adult HSCT and SOT recipients between 2018 and 2021. Subjects with a positive Norovirus test within 14 days of enrollment and active GI symptoms were randomly assigned (1:1) to NTZ 500 mg twice daily or placebo (P) for 56 consecutives doses and were followed for 6 months, including patient reported outcomes (PRO) diary assessments. Primary endpoint was to determine the clinical efficacy, assessed as the time from randomization until symptoms resolution for at least 48 hours. Secondary endpoints included virologic efficacy assessed as the time from randomization to first negative viral load and safety through frequency of adverse events. Results 31 subjects (16 NTZ, 15 P) were enrolled and had balanced demographics (See Table 1). Early withdrawal was documented in 5 subjects from each group. Thirty (30) had received solid organ transplants. Most had chronic ( &amp;gt; 14 days) symptoms (77%). In the mITT population, the median time to initial clinical resolution was 19.0 days (95% CI: 1.0, 31.0) for the Nitazoxanide group and 11.0 days (95% CI: 2.0, 14.0) for the placebo group (p-value=0.459). The difference between time to first negative viral load for the Norovirus GII genotype was not significant (p-value=0.873). Patients appear to have clinical improvement based on PRO results while on active therapy. No serious adverse event related to the study treatment was documented. One severe unsolicited adverse event, abdominal pain, was reported on the day of first dose NTZ. Hospitalization and non-serious or laboratory adverse events were not significantly different between the two arms. Analysis of PK and viral genetics is ongoing and will be reported at the meeting. Conclusion NTZ did not shorten time to clinical resolution or viral shedding duration but may have resulted in transient symptom improvement. Although NTZ appears safe, its role is likely limited in the setting of chronic NoV among ICHs. Disclosures Daniel Kaul, MD, Medscape: Honoraria|Nobelpharma: Grant/Research Support|Takeda: Grant/Research Support Robin K. Avery, MD, Aicuris: Grant/Research Support|Astellas: Grant/Research Support|Astra-Zeneca: Grant/Research Support|Chimerix: Grant/Research Support|Merck: Grant/Research Support|Oxford Immunotec: Grant/Research Support|Qiagen: Grant/Research Support|Regeneron: Grant/Research Support|Takeda: Grant/Research Support Ajit Limaye, Professor/MD, MedPace: DSMB member|merck: Advisor/Consultant|merck: Grant/Research Support|moderna: Advisor/Consultant|moderna: site investigator|syneos: DSMB member Steven A. Pergam, MD, MPH, Cidara: Investigator in clinical trials|F2G: Investigator in clinical trials|Global Life Technologies: Grant/Research Support|Symbio: Investigator in clinical trials Michael D. Green, MD, MPH, ADMA: Advisor/Consultant|Allovir: Advisor/Consultant|Bristol Myers Squibb: Advisor/Consultant|ITB-MED: Advisor/Consultant Marian G. Michaels, MD, MPH, Merck: Grant/Research Support|Viracor: Grant/Research Support Lara A. Danziger-Isakov, MD, MPH, Aicuris: Contracted Clinical Research|Ansun Biopharma: Contracted Clinical Research|Astellas: Contracted Clinical Research|GSK: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Contracted Clinical Research|Pfizer: Contracted Clinical Research|Roche Diagnostics: Advisor/Consultant|Takeda: Advisor/Consultant|Takeda: Contracted Clinical Research Michael P. Angarone, DO, Abbvie Pharmeciuticals: Advisor/Consultant|DKBMed Inc: Advisor/Consultant|DKBMed Inc: Honoraria

Metabarcoding of Hepatitis E Virus Genotype 3 and Norovirus GII from Wastewater Samples in England Using Nanopore Sequencing

Norovirus is one of the largest causes of gastroenteritis worldwide, and Hepatitis E virus (HEV) is an emerging pathogen that has become the most dominant cause of acute viral hepatitis in recent years. The presence of norovirus and HEV has been reported within wastewater in many countries previously. Here we used amplicon deep sequencing (metabarcoding) to identify norovirus and HEV strains in wastewater samples from England collected in 2019 and 2020. For HEV, we sequenced a fragment of the RNA-dependent RNA polymerase (RdRp) gene targeting genotype three strains. For norovirus, we sequenced the 5′ portion of the major capsid protein gene (VP1) of genogroup II strains. Sequencing of the wastewater samples revealed eight different genotypes of norovirus GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.9, GII.13 and GII.17). Genotypes GII.3 and GII.4 were the most commonly found. The HEV metabarcoding assay was able to identify HEV genotype 3 strains in some samples with a very low viral concentration determined by RT-qPCR. Analysis showed that most HEV strains found in influent wastewater were typed as G3c and G3e and were likely to have originated from humans or swine. However, the small size of the HEV nested PCR amplicon could cause issues with typing, and so this method is more appropriate for samples with high CTs where methods targeting longer genomic regions are unlikely to be successful. This is the first report of HEV RNA in wastewater in England. This study demonstrates the utility of wastewater sequencing and the need for wider surveillance of norovirus and HEV within host species and environments.