Acute Gastroenteritis Associated with Norovirus GII.8[P8], Thailand, 2023.

Acute gastroenteritis associated with human norovirus infection was reported in Phuket, Thailand, in June 2023. We amplified GII.8[P8] from the outbreak stool specimens. Retrospective sample analysis identified infrequent GII.8[P8] in the country beginning in 2018. In all, the 10 whole-genome GII.8[P8] sequences from Thailand we examined had no evidence of genotypic recombination.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Out-of-sync evolutionary patterns and mutual interplay of major and minor capsid proteins in norovirus GII.2.

Human noroviruses are the most common cause of viral gastroenteritis, resulting annually in 219 000 deaths and a societal cost of $60 billion, and no antivirals or vaccines are available. The minor capsid protein may play a significant role in the evolution of norovirus. GII.4 is the predominant genotype of norovirus, and its VP2 undergoes epochal co-evolution with the major capsid protein VP1. Since the sudden emergence of norovirus GII.2[P16] in 2016, it has consistently remained a significant epidemic strain in recent years. In the construction of phylogenetic trees, the phylogenetic trees of VP2 closely parallel those of VP1 due to the shared tree topology of both proteins. To investigate the interaction patterns between the major and minor capsid proteins of norovirus GII.2, we chose five representative strains of GII.2 norovirus and investigated their evolutionary patterns using a yeast two-hybrid experiment. Our study shows VP1-VP2 interaction in GII.2, with critical interaction sites at 167-178 and 184-186 in the highly variable region. In the intra-within GII.2, we observed no temporal co-evolution between VP1 and VP2 of GII.2. Notable distinctions were observed in the interaction intensity of VP2 among inter-genotype (P<0.05), highlighting the divergent evolutionary patterns of VP2 within different norovirus genotypes. In summary, the interactions between VP2 and VP1 of GII.2 norovirus exhibit out-of-sync evolutionary patterns. This study offered valuable insights for further understanding and completing the evolutionary mechanism of norovirus.

Antigenic cartography reveals complexities of genetic determinants that lead to antigenic differences among pandemic GII.4 noroviruses.

Noroviruses are the predominant cause of acute gastroenteritis, with a single genotype (GII.4) responsible for the majority of infections. This prevalence is characterized by the periodic emergence of new variants that present substitutions at antigenic sites of the major structural protein (VP1), facilitating escape from herd immunity. Notably, the contribution of intravariant mutations to changes in antigenic properties is unknown. We performed a comprehensive antigenic analysis on a virus-like particle panel representing major chronological GII.4 variants to investigate diversification at the inter- and intravariant level. Immunoassays, neutralization data, and cartography analyses showed antigenic similarities between phylogenetically related variants, with major switches to antigenic properties observed over the evolution of GII.4 variants. Genetic analysis indicated that multiple coevolving amino acid changes-primarily at antigenic sites-are associated with the antigenic diversification of GII.4 variants. These data highlight complexities of the genetic determinants and provide a framework for the antigenic characterization of emerging GII.4 noroviruses.

Increased circulation of GII.17 noroviruses, six European countries and the United States, 2023 to 2024.

We report an increase in GII.17 norovirus outbreaks and sporadic infections of acute gastroenteritis in Austria, Germany, France, Ireland, the Netherlands, England and the United States during the 2023/24 season. A decrease in GII.4 coincided with GII.17 prevalence increasing to between 17% and 64% of all GII detections. Overall, 84% of the GII.17 strains clustered closely with strains first reported in Romania in 2021 and two new sub-lineages were identified. Norovirus surveillance and molecular characterisation should be prioritised this winter.

Molecular Evolution of GII.P31/GII.4_Sydney_2012 Norovirus over a Decade in a Clinic in Japan.

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013-2016) remained phylogenetically distinct from later strains (2019-2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment.

Emergence of GII.4 Sydney[P16]-like Norovirus-Associated Gastroenteritis, China, 2020-2022.

Newly evolved GII.4 Sydney[P16] norovirus with multiple residue mutations, already circulating in parts of China, became predominant and caused an abrupt increase in diagnosed norovirus cases among children with gastroenteritis in Shanghai during 2021-2022. Findings highlight the need for continuous long-term monitoring for GII.4 Sydney[P16] and emergent GII.4 norovirus variants.

GII.17[P17] and GII.8[P8] noroviruses showed different RdRp activities associated with their epidemic characteristics.

Norovirus is the primary foodborne pathogenic agent causing viral acute gastroenteritis. It possesses broad genetic diversity and the prevalence of different genotypes varies substantially. However, the differences in RNA-dependent RNA polymerase (RdRp) activity among different genotypes of noroviruses remain unclear. In this study, the molecular mechanism of RdRp activity difference between the epidemic strain GII.17[P17] and the non-epidemic strain GII.8[P8] was characterized. By evaluating the evolutionary history of RdRp sequences with Markov Chain Monte Carlo method, the evolution rate of GII.17[P17] variants was higher than that of GII.8[P8] variants (1.22 × 10-3 nucleotide substitutions/site/year to 9.31 × 10-4 nucleotide substitutions/site/year, respectively). The enzyme catalytic reaction demonstrated that the Vmax value of GII.17[P17] RdRp was 2.5 times than that of GII.8[P8] RdRp. And the Km of GII.17[P17] and GII.8[P8] RdRp were 0.01 and 0.15 mmol/L, respectively. Then, GII.8[P8] RdRp fragment mutants (A-F) were designed, among which GII.8[P8]-A/B containing the conserved motif G/F were found to have significant effects on improving RdRp activity. The Km values of GII.8[P8]-A/B reached 0.07 and 0.06 mmol/L, respectively. And their Vmax values were 1.34 times than that of GII.8[P8] RdRp. In summary, our results suggested that RdRp activities were correlated with their epidemic characteristics. These findings will ultimately provide a better understanding in replication mechanism of noroviruses and development of antiviral drugs.

Genetic characterizations and molecular evolution of human norovirus GII.6 genotype during the past five decades.

The norovirus genotype GII.6 is circulating in the population with a relatively high prevalence, but in-depth molecular characterization studies of GII.6 are needed. In this study, norovirus GII.6 sequences were retrieved and analyzed to demonstrate the molecular characterizations of norovirus GII.6. The results show that GII.6 VP1 gene can be divided into three variants and all variants cocirculated in humans in the past decades. The intragenotypic showed no growth trend over time. Since the overall evolutionary rate was 3.432 × 10-3 substitutions/site/year, the infer most recent common ancestor was estimated as 1913. Only a few amino acid sites were recognized to be under positive selection pressure. The mean effective population size was stable in the recent years. The variant C (especially 87 GII.P7-GII.6 strains) had a higher evolutionary rate and more sites under positive selection pressure compared to other variants. NS4 protein had the higher diversity than other nonstructural proteins, and VP1 and VP2 genes had the same phylogenetic relationships. This study provides a systematic description of genetic characterizations and molecular evolution of GII.6. Research on norovirus molecular epidemiology should be pursued to enrich the genomic data of the diverse genotypes and improve their analysis.

Rapid two-stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single-tube two-stage nucleic acid amplification method-reverse transcription recombinase-assisted PCR (RT-RAP)-for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase-aided amplification (RT-RAA) and the second stage consisting of qPCR (quantitative PCR). RT-RAP is more sensitive than either RT-RAA or qRT-PCR (quantitative RT-PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT-PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.

Transcriptional response of wolfberry to infestation with the endophytic Fusarium nematophilum strain NQ8GII4.

Fusarium nematophilum NQ8GII4 is an endophytic fungus isolated from the root of healthy wolfberry (Lycium barbarum). Previous studies have reported that NQ8GII4 could dwell in wolfberry roots and enhance the defense responses in wolfberry against root rot, which is caused by F. oxysporum. To further elucidate the molecular mechanism of wolfberry disease resistance induced by NQ8GII4, in the present study, we adopted RNA sequencing analysis to profile the transcriptome of wolfberry response to NQ8GII4 infestation over a time course of 3 and 7 days post-inoculation (dpi). Gene ontology (GO) enrichment analysis revealed that DEGs were enriched related to biological regulation, response to stimulus, signaling, detoxification, immune system process, transporter activity, electron carrier activity, transcription factor activity, nucleic acid binding transcription factor, and antioxidant activity. Through Kyoto encyclopedia of genes and genomes (KEGG) analysis, it was found that many of these DEGs were enriched in pathways related to plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis pathway in wolfberry. This suggests that innate immunity, phytohormone signaling, and numerous phenylpropanoid compounds, which comprise a complex defense network in wolfberry. Chloroplast 50S ribosomal proteins (50S RP) were consistently located at the core position of the response in wolfberry following infestation with NQ8GII4 analyzed by protein-protein interaction (PPI) network. This study elucidated the molecular mechanism underlying the interaction between NQ8GII4 and wolfberry, clarified the wolfberry immune response network to endophytic fungi infestation, identified candidate resistance genes in wolfberry, and provided a fundamental date for subsequent work.

Prevalence and genetic characterization of noroviruses in children with acute gastroenteritis in Senegal, 2007-2010.

Norovirus is the leading cause of sporadic and epidemic acute gastroenteritis (AGE) in children and adults around the world. We investigated the molecular diversity of noroviruses in a pediatric population in Senegal between 2007 and 2010 before the rotavirus vaccine implementation. Stool samples were collected from 599 children under 5 years of age consulting for AGE in a hospital in Dakar. Specimens were screened for noroviruses using the Allplex™ GI-Virus Assay. Positive samples were genotyped after sequencing of conventional reverse transcription-polymerase chain reaction products. Noroviruses were detected in 79 (13.2%) of the children, with GII.4 (64%) and GII.6 (10%) as the most frequently identified genotypes. Our study describes the distribution of genotypes between 2007 and 2010 and should be a baseline for comparison with more contemporary studies. This could help decision-makers on possible choices of norovirus vaccines in the event of future introduction.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.

BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/µL within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

An increase in prevalence of recombinant GII.3[P12] norovirus in sporadic acute diarrhea in children in Nizhny Novgorod, Russia, 2018-2021.

Noroviruses are important etiological agents causing acute intestinal infection in humans. In the last decades, the most common norovirus genotype was GII.4 despite a significant genetic diversity among strains, while the active circulation of noroviruses with other genotypes was observed periodically. This study shows an increase in the detection rate of recombinant GII.3[P12] norovirus in Nizhny Novgorod, Russia, from 6.8% in 2018-2019 to 34.9% in 2020-2021. We performed a phylogenetic analysis based on the nucleotide sequences of noroviruses possessing this genotype obtained in this work, as well as presented in the GenBank database. It has been shown that the circulation of GII.3[P12] noroviruses in the study area was the result of several independent introductions, either directly from the Western Pacific region, or through the Asian part of Russia. The polyphyletic origin, the geographical expansion, and the growth of the epidemic significance of the recombinant GII.3[P12] noroviruses were noted.

Emerging norovirus GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan, during COVID-19, 2021.

INTRODUCTION: Norovirus (NoV) is the most common agent causing outbreaks and sporadic cases of acute gastroenteritis among all ages, especially children under 5 years old. During the coronavirus disease 2019 (COVID-19) pandemic, NoV infection has decreased drastically in Japan due to school closures and no outbreak related to NoV infection had been reported. METHOD: In mid-September 2021, NoV outbreak occurred in kindergarten and nursery schools in Maizuru, Kyoto prefecture, Japan. Twenty-six stool samples collected from patients who were diagnosed of NoV gastroenteritis from the outbreak by an immunochromatographic (IC) kit at a pediatric outpatient clinic in Maizuru city during 3 weeks from September 13 to October 8, 2021 were examined for the presence of NoV GII by reverse transcriptase-polymerase chain reaction (RT-PCR), genome sequencing, and phylogenetic analysis. RESULT: All 26 samples were confirmed positive to NoV GII and their genotypes were identified as GII.4 Sydney[P31]. The amino acid substitutions in open reading frame1 (ORF1) and ORF2 genes were found when compared with previously detected sporadic NoV GII.4 Sydney[P31] strains isolated in Japan. The clinical characterization of infected children was described. Most of the children were mild cases and vomiting was the most frequent clinical symptom. CONCLUSION: This study reported a recent emergence of NoV GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan during the COVID-19 pandemic and suggests a need for further monitoring of NoV GII.4 variants.

Homotypic and Heterotypic Protection and Risk of Reinfection Following Natural Norovirus Infection in a Highly Endemic Setting.

BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide, yet there is limited information on homotypic or heterotypic protection following natural infection to guide vaccine development. METHODS: A total of 6020 stools collected from 299 Peruvian children between 2010 and 2014 were tested by norovirus real-time reverse-transcription polymerase chain reaction followed by sequence-based genotyping. Cox proportional hazards models were used to derive adjusted hazard ratios (HRs) of infection among children with vs without prior exposure. RESULTS: Norovirus was detected in 1288 (21.3%) samples. GII.4 (26%), GII.6 (19%), and GI.3 (9%) viruses accounted for 54% of infections. Homotypic protection for GI.3 (HR, 0.35; P = .015), GI.7 (HR, 0.19; P = .022), GII.4 (HR, 0.39; P < .001), and GII.6 (HR, 0.52; P = .006) infections was observed. Hazard analysis showed that children with prior GII.4 infection exhibited heterotypic protection with a 48% reduction of subsequent GI.3 infection (HR, 0.52; P = .005). Prior exposure to GI.3, GII.2, and GII.17 infections enhanced susceptibility to subsequent infections with several other norovirus genotypes. CONCLUSIONS: Children up to 2 years of age infected with GII.4 noroviruses demonstrated both homotypic and heterotypic protection to reinfection with other genotypes. These data support the need for ongoing vaccine development efforts with GII.4 as the main component and caution the inclusion of genotypes that may enhance susceptibility to infections.

Detection of Norovirus Variant GII.4 Hong Kong in Asia and Europe, 2017-2019.

We report a new norovirus GII.4 variant, GII.4 Hong Kong, with low-level circulation in 4 Eurasia countries since mid-2017. Amino acid substitutions in key residues on the virus capsid associated with the emergence of pandemic noroviruses suggest that GII.4 Hong Kong has the potential to become the next pandemic variant.

Molecular epidemiology and genetic diversity of norovirus infection in children with acute gastroenteritis in Bangladesh, 2014-2019.

Acute gastroenteritis (AGE) is one of the most common diseases in children, and it continues to be a significant cause of morbidity and mortality worldwide. Norovirus is one of the major enteropathogens associated with both sporadic diarrhea and outbreaks of gastroenteritis. This study aims to investigate genotype diversity and molecular epidemiology of norovirus in Bangladesh. A total of 466 fecal specimens were collected from January 2014 to January 2019 from children below 5 years old with AGE in Bangladesh. All samples were analyzed by reverse transcriptase polymerase chain reaction to detect norovirus, and sequence analysis was conducted if found positive. Norovirus was detected in 5.1% (24 of 466) fecal specimens. Norovirus genotype GII.7 was predominant (62.5%, 15 of 24), followed by GII.3 (37.5%, 9 of 24). Coinfection between rotavirus and norovirus was found in 7 of 24 positive cases. Diarrhea (93.7%) and dehydration (89%) were the most common symptoms in children with AGE. About 80% of the positive cases were detected in children aged under 24 months. One seasonal peak (87.5% infection) was detected in the winter. This study suggests that norovirus continues to be one of the major etiologies of children AGE in Bangladesh. This study will provide a guideline to assess the burden of norovirus infection in Bangladesh, which will assist to combat against AGE.

Norovirus GII.2[P16] strain in Shenzhen, China: a retrospective study.

BACKGROUND: Norovirus (NoV) is the main cause of non-bacterial acute gastroenteritis (AGE) outbreaks worldwide. From September 2015 through August 2018, 203 NoV outbreaks involving 2500 cases were reported to the Shenzhen Center for Disease Control and Prevention. METHODS: Faecal specimens for 203 outbreaks were collected and epidemiological data were obtained through the AGE outbreak surveillance system in Shenzhen. Genotypes were determined by sequencing analysis. To gain a better understanding of the evolutionary characteristics of NoV in Shenzhen, molecular evolution and mutations were evaluated based on time-scale evolutionary phylogeny and amino acid mutations. RESULTS: A total of nine districts reported NoV outbreaks and the reported NoV outbreaks peaked from November to March. Among the 203 NoV outbreaks, 150 were sequenced successfully. Most of these outbreaks were associated with the NoV GII.2[P16] strain (45.3%, 92/203) and occurred in school settings (91.6%, 186/203). The evolutionary rates of the RdRp region and the VP1 sequence were 2.1 × 10-3 (95% HPD interval, 1.7 × 10-3-2.5 × 10-3) substitutions/site/year and 2.7 × 10-3 (95% HPD interval, 2.4 × 10-3-3.1 × 10-3) substitutions/site/year, respectively. The common ancestors of the GII.2[P16] strain from Shenzhen and GII.4 Sydney 2012[P16] diverged from 2011 to 2012. The common ancestors of the GII.2[P16] strain from Shenzhen and previous GII.2[P16] (2010-2012) diverged from 2003 to 2004. The results of amino acid mutations showed 6 amino acid substitutions (*77E, R750K, P845Q, H1310Y, K1546Q, T1549A) were found only in GII.4 Sydney 2012[P16] and the GII.2[P16] recombinant strain. CONCLUSIONS: This study illustrates the molecular epidemiological patterns in Shenzhen, China, from September 2015 to August 2018 and provides evidence that the epidemic trend of GII.2[P16] recombinant strain had weakened and the non-structural proteins of the recombinant strain might have played a more significant role than VP1.

One-step duplex RT-droplet digital PCR assay for the detection of norovirus GI and GII in lettuce and strawberry.

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/µL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/µL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 106 to 1.70 × 108 copies and 4.80 × 105 to 2.50 × 107 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 104 copies/25g (NoV GI) and 2.36 × 104 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 104 copies/25g (NoV GI) and 2.64 × 104 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV.