NoV Infection Research Articles

Production of Norovirus VLPs of the Nine Representative Genotypes Widely Distributed in Japan using the Silkworm-Baculovirus Expression Vector System

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

Prevalence and associated factors of norovirus infections among patients with diarrhea in the Amhara national regional state, Ethiopia

BackgroundNoroviruses (NoVs) are the leading cause of diarrheal disease among all age groups worldwide, with an increased burden in developing countries. As there is no surveillance, epidemiological data is limited in Ethiopia. Hence, this study aimed to investigate the prevalence and associated factors of NoV infection among patients with diarrhea in the Amhara National Regional State, Ethiopia.MethodsA prospective health facility-based cross-sectional study was conducted from May 2021 to November 2021. A total of 550 study participants of all age groups with symptoms of diarrhea were proportionately assigned to the four study areas, area with three health facilities. Study participants were systematically sampled in each health facility. A fecal sample from each case was collected. The RNA was extracted and tested for NoV by one-step RT-PCR. Sociodemographic and other variables were gathered using a pre-tested questionnaire. A descriptive analysis was performed. Both binary and multiple logistic regressions were utilized to identify factors associated with NoV infection. Variables with a p-value < 0.05 in the final model were considered statistically significant.ResultsFive hundred nineteen out of 550 samples were analyzed (94.4% response rate). The overall prevalence of NoV was 8.9% (46/519). The positivity rates were higher among the elderly (33.3%) and under-5 children (12.5%). Both genogroup I and genogroup II (GII) were identified, with GII being the predominant, at 82.6% (38/46). Of all participants, only 20% reported a history of vomiting. Norovirus infection was more prevalent among participants from Debre Tabor (AOR = 4, 95%CI: 1.2–14) and Bahir Dar areas (AOR = 3.6, 95%CI: 1.04–11) compared to Debre Markos. Additionally, older adults (AOR = 7, 95% CI: 2–24) and under-5 children (AOR = 3.5, 95% CI: 2.8–12) were disproportionately affected compared to adults. The previous history of diarrhea (AOR = 3.6, 95% CI: 1.7–7) was a significant factor contributing to NoV infections. Moreover, the odds of NoV infection were higher among individuals with a high frequency of diarrhea (AOR = 15.3, 95%CI: 7.6–43) and vomiting (AOR = 3.5, 95%CI: 1.5-8).ConclusionsThe prevalence of NoV was considerably high, with the predominance of NoV-GII. The positivity rate was higher among the extreme age groups and varied across the study areas. To obtain a comprehensive understanding of the virus`s epidemiology and its genetic diversity, further research is warranted.

Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

Epidemiological and genetic characteristics of norovirus in Hangzhou, China, in the postepidemic era

ObjectiveNorovirus (NoV) is an important human pathogen that can cause severe gastroenteritis in vulnerable populations. This study aimed to analyze the epidemiological and genetic characteristics of 2021–2023 NoV in Hangzhou, China. MethodsThis study enrolled patients aged 0–18 years who underwent NoV RNA detection in the hospital between January 2021 and October 2023 and analyzed the epidemiological characteristics of NoV. Polymerase chain reaction (PCR) was used to detect NoV RNA. Subtype classification and whole-genome sequencing were performed. ResultsThere was a high prevalence of NoV infection in 2023, with NoV-positive samples accounting for 63.10 % of the total number of positive samples collected during the three-year period. The prevalence was abnormally high in summer, and the number of positive samples accounted for 48.20 % of the total positive samples for the whole year, which was much greater than the level in the same period in previous years (2023, 48.20% vs 2021, 13.66% vs 2022, 15.21 %). The GⅡ.4 subtype played a leading role, followed by increased mixed infection with GⅠ.5 and GⅡ.4. Whole-genome sequencing results suggested that GII.P16-GⅡ.4 had R297H and D372N key locus mutations. The evolutionary rate was 4.29 × 10−3 for the RdRp gene and 4.84 × 10−3 for the VP1 gene. The RdRp gene and VP1 gene of NoV GII.P16-GⅡ.4 have undergone rapid population evolution during the COVID-19 epidemic. ConclusionIn the summer of 2023, an abnormally high incidence of NoV appeared in Hangzhou, China. The major epidemic strain GII.P16-GⅡ.4 showed a certain range of gene mutations and a fast evolutionary rate.

Estimation of Norovirus infections in Japan: An application of wastewater-based epidemiology for enteric disease assessment

Noroviruses of genogroup I (NoV GI) and NoV GII are the primary causes of acute gastroenteritis (AGE) in developed countries. However, asymptomatic and untested NoV infections lead to an underestimation of AGE cases, and the lack of mandatory viral identification in clinical cases hinders precise estimation of NoV infections. Back estimation of NoV infections in the community using a wastewater-based epidemiology (WBE) approach can provide valuable insights into the disease's extent, progression, and epidemiology, aiding in developing effective control strategies. This study employed a one-step reverse transcription-quantitative PCR to quantify NoVs GI and GII in wastewater samples (n = 83) collected twice a week from June 2022 to March 2023 in Japan. All samples from the Winter-Spring (n = 27) tested positive for NoV GI and GII RNA, while 73 % and 88 % of samples from the Summer-Autumn (n = 56) were positive for NoV GI and NoV GII RNA, respectively. Significantly higher concentrations of NoV GI/GII RNA were found in the Winter-Spring season compared to the Summer-Autumn season. NoV RNA was consistently detected in wastewater throughout the year, demonstrating the persistence of AGE cases in the catchment, suggesting an endemic NoV infection. Estimates of NoV infection incorporated viral RNA concentrations, wastewater parameters, and signal persistence in a mass balance equation using Monte Carlo Simulation. The median estimated NoV GI infections per 100,000 population for Summer-Autumn was 133 and for the Winter-Spring season, it was 881. Estimated NoV GII infections were 1357 for Summer-Autumn and 11,997 for the Winter-Spring season per 100,000 population. The estimated NoV infections exceeded by 3.2 and 23.9 folds than the reported AGE cases in Summer-Autumn and Winter-Spring seasons, respectively. The seasonal trend of estimated NoV infections closely matched that of AGE cases, highlighting the utility of WBE in understanding the epidemiology of enteric infections.

Viruses in Beef, Mutton, Chevon, Venison, Fish and Poultry Meat Products

Beef means meat of cattle, mutton means meat of sheep, chevon means meat of flesh of the goat used as food, venison means meat of dear, fish means meat of fish and shellfish, poultry means chicken , duck, geese, turkey, pigeon and rabbit. Beef, mutton, chevon, venison, fish and poultry meat act as a main sources of animal protein, In recent decades, viruses have been increasingly known as important causes of foodborne diseases mostly due to the improved methods of diagnosis and investigation of viruses. Viruses transmission through consumption of infected beef, mutton, chevon, venison, fish and poultry meat products or contact with contaminated beef, mutton, chevon, venison, fish and poultry meat products and water is now well known. The viruses most frequently involved in foodborne infections are public noroviruses, hepatitis A virus, human rotavirus, and hepatitis E virus. Beef, mutton, chevon, venison, fish and poultry meat act as major sources of animal protein, NoV and RV infections are common cause of acute human gastroenteritis, while hepatitis A virus and hepatitis E virus cause human hepatitis worldwide. Most of the cases remain unreported due to subclinical cases.

High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity.

Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.

Blockade Antibody Responses in Human Subjects Challenged with a New Snow Mountain Virus Inoculum.

Noroviruses (NoVs) are a leading cause of non-bacterial gastroenteritis in young children and adults worldwide. Snow Mountain Virus (SMV) is the prototype of NoV GII genotype 2 (GII.2) that has been developed as a viral model for human challenge studies, an important tool for studying pathogenesis and immune response of NoV infections and for evaluating NoV vaccine candidates. Previous studies have identified blockade antibodies that block the binding of NoV virus-like particles (VLPs) to histo-blood group antigens (HBGAs) as a surrogate for neutralization in human Norwalk virus and GII.4 infections but little is known about SMV blockade antibodies. In this secondary data analysis study, blockade antibodies were characterized in pre-challenge and post-challenge serum samples from human subjects challenged with a new SMV inoculum. The correlation between blockade antibody geometric mean antibody titers (GMTs) and SMV-specific serum IgG/IgA GMTs were examined after stratifying the subjects by infection status. A linear mixed model was applied to test the association between HBGA blockade antibody concentrations and post-challenge days accounting for covariates and random effects. Laboratory results from 33 SMV inoculated individuals were analyzed and 75.7% (25/33) participants became infected. Serum SMV-specific blockade antibodies, IgA, and IgG were all significantly different between infected and uninfected individuals beginning day 15 post-challenge. Within infected individuals, a significant correlation was observed between both IgG and IgA and blockade antibody concentration as early as day 6 post-challenge. Analysis of blockade antibody using the linear mixed model showed that infected individuals, when compared to uninfected individuals, had a statistically significant increase in blockade antibody concentrations across the post-challenge days. Among the post-challenge days, blockade antibody concentrations on days 15, 30, and 45 were significantly higher than those observed pre-challenge. The intraclass correlation coefficient (ICC) analysis indicated that the variability of blockade antibody titers is more observed between individuals rather than within subjects. These results indicate that HBGA-blockade antibody GMTs are generated after SMV challenge and the blockade antibodies were still detectable at day 45 post-challenge. These data indicate that the second-generation of SMV inoculum is highly effective.

Epidemiology of major entero-pathogenic viruses and genetic characterization of Group A rotaviruses among children (≤5 years) with acute gastroenteritis in eastern India, 2018-2020.

This study was carried out from January 2018 to March 2020 in Kolkata, eastern India to determine the prevalence rates and epidemiological patterns associated with the major viral agents of gastroenteritis among children ≤5 years of age. Molecular characterization of GARV, the predominant agent of viral gastroenteritis, was done to understand their genotype diversity. 1284 of 3157 stool samples (~40%) from children (≤5 years) with acute gastroenteritis tested positive for one or more enteric viruses with positivity rates 25.11%, 8.74%, 6.62% and 6.11% for GARV, HAdV-F, AstV and NoV respectively. Co-infection was observed in 5.31% of cases. Associated clinical/meteorological variables like age, sex, symptoms, temperature and precipitation were assessed to find any correlation between these and enteric virus infection rates. >70% of viral gastroenteritis cases were observed in 6-24 months' age group. GARV and AstV infection occurred mostly during cooler months while HAdV-F infection mostly occurred during warmer periods. No definite seasonality was observed for NoV infections. Clinical severity associated with GARV infection was higher compared to other enteric viruses. Genotyping of rotavirus positive samples revealed G3P[8] was the predominantly circulating GARV genotype throughout the study period. GARV remained the predominant viral agent of acute gastroenteritis among children though its prevalence rates in this region declined significantly compared to the previous years (2010-2016). The prevalence of other enteric viruses was below 10%. This study provides valuable insights regarding the current burden of viral gastroenteritis in Eastern India. The 2-year study in children will provide the baseline data for future surveillance studies in evaluating the impact of the introduced GARV vaccine on the overall prevalence of viral gastroenteritis.

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

IntroductionNorovirus (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. MethodIn 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. ResultAll 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. ConclusionThis 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.

Clinical significance and intestinal microbiota composition in immunocompromised children with norovirus gastroenteritis.

BackgroundNorovirus (NoV) infection is common in pediatric patients with immunodeficiency and is more likely to cause severe disease. Objective Our study aims to figure out the clinical differences and distribution of intestinal microbiota in immunocompromised children with NoV gastroenteritis.MethodsPediatric patients admitted to Shang-Ho Hospital with diagnosis of acute gastroenteritis including different immune status were enrolled and their medical records were reviewed. NoV gastroenteritis was validated using RT-PCR molecular methods. Viral shedding period was determined by real-time RT-PCR assays. Intestinal microbiota enrichment analysis was carried out by next generation sequencing after fecal DNA extraction and subsequent Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method.ResultsSignificantly higher frequency of diarrhea [mean, (IQR), 3.8 (3–5) /day] and longer viral shedding time [mean, IQR, 8.5 (5–13) days] was found in immunocompromised NoV infections than in immunocompetent patients without NoV infections (p = 0.013*) and immunocompetent patients with NoV infections (p = 0.030**). The fever prevalence was significantly lower in immunocompromised NoV infections than in different immune or infection status. Intestinal microbiota metagenomics analysis showed no significant community richness difference while the LEfSe analysis showed a significant difference in commensal richness at the phylum level, the family level, and the genus level in patients under different immune status.ConclusionWe evaluated the clinical significances and microbiota composition in immunocompromised children with norovirus gastroenteritis. This will further facilitate studies of the interaction between the intestinal microbiota in such patients with precise determination of their bacterial infection control and probiotic supplements strategy.

Epidemiology, Clinical Features, and Unusual Complications of Norovirus Infection in Taiwan: What We Know after Rotavirus Vaccines.

Noroviruses (NoVs) are one of the emerging and rapidly spreading groups of pathogens threatening human health. A reduction in sporadic NoV infections was noted following the start of the COVID-19 pandemic, but the return of NoV gastroenteritis during the COVID-19 pandemic has been noted recently. Research in recent years has shown that different virus strains are associated with different clinical characteristics; moreover, there is a paucity of research into extraintestinal or unusual complications that may be associated with NoV. The genomic diversity of circulating NoVs is also complex and may vary significantly. Therefore, this short narrative review focuses on sharing the Taiwan experience of NoV infection including epidemiology, clinical features, and complications following suboptimal rotavirus immunization in Taiwan (after October 2006). We also highlight the unusual complications associated with NoV infections and the impacts of NoV infection during the COVID-19 pandemic in the literature for possible future research directions. To conclude, further research is needed to quantify the burden of NoV across the spectrum of disease severity in Taiwan. The evidence of the connection between NoV and the unusual complications is still lacking.

700. Risk Factors and Molecular Epidemiology of Acute and Chronic Norovirus Infection at a Large Tertiary Care Cancer Center

BackgroundNorovirus (NoV) is the leading cause of viral diarrhea in patients with cancer. In this study, we describe risk factors associated with acute and chronic NoV infection in this patient population. MethodsWe identified 132 patients with NoV diarrhea (using stool RT PCR) between 2016-2020 at University of Texas MD Anderson Cancer Center (MDACC). Patient data, including demographics, clinical characteristics, NoV treatments, and complications were retrospectively extracted from charts. Stool samples were analyzed for NoV genogroups and genotypes. We compared characteristics and outcomes of patients with acute diarrhea (< 14day; AD) versus chronic diarrhea ( >14day or recurrences within 12 weeks; CD) and analyzed the data using Pearson Chi square or Fisher’s exact for categorical variables and Wilcoxon rank-sum test for continuous variables. ResultsOf 132 patients identified, 124 had an underlying cancer (39 solid tumor, 85 hematological malignancies, Table 1). On univariate analysis, CD patients were more likely to have a hematological malignancy (p=0.002), be a hematopoietic stem cell recipient (p= 0.013), have a history of gastrointestinal graft versus host disease (p= 0.011), or have received immunosuppressants or steroids in the 90 days before diarrhea onset (p=0.001, Table 2). CD patients had significantly lower white blood cell counts (p=0.038), absolute neutrophil counts (p=0.049), IgG levels (p= 0.001), and serum albumin levels (p=0.002) at the time of NoV diagnosis (Table 3). Patients with CD more often received symptomatic or NoV targeting treatment, including anti-diarrheal (p=0.005), nitazoxanide (p< 0.001), intravenous immune globulin (p=0.017), and oral IgG (p=0.042). CD patients more often had diarrheal recurrence in the first 4 weeks (p=0.001) or the second month (p< 0.001) after initial diagnosis and needed enteral or parenteral nutrition (p=0.004). We genotyped NoV in 67 patients (Figure 1), resulting in identification of the following genogroups: GI (n=9, 13%), GII.4 (n=23, 34%), and other types of GII (n=35, 52%). Genotype diversity was higher in patients with CD than AD (Figure 1). ConclusionIn patients with cancer, CD from NoV is associated with severe immunosuppression, is refractory to therapy and can be caused by a variety of NoV genotypes/genogroups. Disclosures Robert L. Atmar, MD, Takeda Vaccines, Inc. (Grant/Research Support) Mary Estes, PhD, Takeda Vaccines (Consultant, Grant/Research Support) Pablo C. Okhuysen, MD, FACP, FIDSA, Deinove Pharmaceuticals (Grant/Research Support)Ferring Pharmaceuticals (Consultant)Melinta Therapeutics (Grant/Research Support)Merck & Co. (Grant/Research Support)Napo Pharmaceuticals (Consultant, Scientific Research Study Investigator, Research Grant or Support)Singulex (Consultant)Summit Therapeutics (Grant/Research Support)

Molecular epidemiology and genetic diversity of norovirus among hospitalized children with acute gastroenteritis in Tianjin, China, 2018\u20132020

BackgroundNorovirus (NoV) is a major cause of viral acute gastroenteritis (AGE) in children worldwide. Epidemiological analysis with respect to the virus strains is limited in China. This study aimed to investigate the prevalence, patterns, and molecular characteristics of NoV infection among children with AGE in China.MethodsA total 4848 stool samples were collected from children who were admitted with AGE in Tianjin Children’s Hospital from August 2018 to July 2020. NoV was preliminarily detected using real-time reverse transcription polymerase chain reaction (RT-PCR). Partial sequences of the RNA-dependent RNA polymerase (RdRp) and capsid genes of positive samples were amplified by conventional RT-PCR and then sequenced. The NoV genotype was determined by online Norovirus Typing Tool Version 2.0, and phylogenetic analysis was conducted using MEGA 6.0.ResultsThe prevalence of NoV was 26.4% (1280/4848). NoV was detected in all age groups, with the 7–12 months group having the highest detection rate (655/2014, 32.5%). NoV was detected during most part of the year with higher frequency in winter than other seasons. Based on the genetic analysis of RdRp, GII. Pe was the most predominant genotype detected at 70.7% (381/539) followed by GII.P12 at 25.4% (137/539). GII.4 was the most predominant capsid genotype detected at 65.3% (338/518) followed by GII.3 at 26.8% (139/518). Based on the genetic analysis of RdRp and capsid sequences, the strains were clustered into 10 RdRp–capsid genotypes: GII.Pe-GII.4 Sydney 2012 (65.5%), GII.P12-GII.3 (27.2%), GII.P16-GII.2 (1.8%), GII.P12-GII.2 (0.2%), GII.P17-GII.17 (1.1%), GII.Pe-GII.3 (1.8%), GII.Pe-GII.2 (1.1%), GII.Pe-GII.1 (0.4%), GII.16-GII.4 Sydney 2012 (0.7%), and GII.P7-GII.6 (0.2%). The predominant NoV genotypes changed from GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 between August 2018 and July 2019 to GII.Pe-GII.4 Sydney 2012 and GII.P16-GII.2 between August 2019 and July 2020. The patients with GII.Pe-GII.4 Sydney 2012 genotype were more likely to suffer from vomiting symptom than those with GII.P12-GII.3.ConclusionsNoV is an important pathogen responsible for viral AGE among children in China. GII.Pe-GII.4 Sydney 2012 and GII.P12-GII.3 were major recombinant genotypes. Knowledge of circulating genotypes and seasonal trends is of great importance for disease prevention and surveillance.

Simvastatin Reduces Protection and Intestinal T Cell Responses Induced by a Norovirus P Particle Vaccine in Gnotobiotic Pigs.

Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.

Linear B-Cell Epitopes in Human Norovirus GII.4 Capsid Protein Elicit Blockade Antibodies.

Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis worldwide with the GII.4 genotype accounting for over 80% of infections. The major capsid protein of GII.4 variants is evolving rapidly, resulting in new epidemic variants with altered antigenic potentials that must be considered for the development of an effective vaccine. In this study, we identify and characterize linear blockade B-cell epitopes in HuNoV GII.4. Five unique linear B-cell epitopes, namely P2A, P2B, P2C, P2D, and P2E, were predicted on the surface-exposed regions of the capsid protein. Evolving of the surface-exposed epitopes over time was found to correlate with the emergence of new GII.4 outbreak variants. Molecular dynamic simulation (MD) analysis and molecular docking revealed that amino acid substitutions in the putative epitopes P2B, P2C, and P2D could be associated with immune escape and the appearance of new GII.4 variants by affecting solvent accessibility and flexibility of the antigenic sites and histo-blood group antigens (HBAG) binding. Testing the synthetic peptides in wild-type mice, epitopes P2B (336–355), P2C (367–384), and P2D (390–400) were recognized as GII.4-specific linear blockade epitopes with the blocking rate of 68, 55 and 28%, respectively. Blocking rate was found to increase to 80% using the pooled serum of epitopes P2B and P2C. These data provide a strategy for expanding the broad blockade potential of vaccines for prevention of NoV infection.

Molecular epidemiology and genetic diversity of norovirus infection in children hospitalized with acute gastroenteritis in East Java, Indonesia in 2015-2019.

Noroviruses are recognized as a leading cause of outbreaks and sporadic cases of acute gastroenteritis (AGE) among individuals of all ages worldwide, especially in children <5years old. We investigated the epidemiology of noroviruses among hospitalized children at two hospitals in East Java, Indonesia. Stool samples were collected from 966 children with AGE during September 2015-July 2019. All samples were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for the amplification of both the RNA-dependent RNA polymerase (RdRp) and the capsid genes of noroviruses. The genotypes were determined by phylogenetic analyses. In 2015-2019, noroviruses were detected in 12.3% (119/966) of the samples. Children <2years old showed a significantly higher prevalence than those ≥2years old (P=0.01). NoV infections were observed throughout the year, with the highest prevalence in December. Based on our genetic analyses of RdRp, GII.[P31] (43.7%, 31/71) was the most prevalent RdRp genotype, followed by GII.[P16] (36.6%, 26/71). GII.[P31] was a dominant genotype in 2016 and 2018, whereas GII.[P16] was a dominant genotype in 2015 and 2017. Among the capsid genotypes, the most predominant norovirus genotype from 2015 to 2018 was GII.4 Sydney_2012 (33.6%, 40/119). The most prevalent genotype in each year was GII.13 in 2015, GII.4 Sydney_2012 in 2016 and 2018, and GII.3 in 2017. Based on the genetic analyses of RdRp and capsid sequences, the strains were clustered into 13 RdRp/capsid genotypes; 12 of them were discordant, e.g., GII.4 Sydney[P31], GII.3[P16], and GII.13[P16]. The predominant genotype in each year was GII.13[P16] in 2015, GII.4 Sydney[P31] in 2016, GII.3[P16] in 2017, and GII.4 Sydney[P31] in 2018. Our results demonstrate high detection rates and genetic diversity of norovirus GII genotypes in pediatric AGE samples from Indonesia. These findings strengthen the importance of the continuous molecular surveillance of emerging norovirus strains.

Norovirus-Specific CD8+ T Cell Responses in Human Blood and Tissues.

Background & AimsNoroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites.MethodsWe screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8+ T cells in peripheral, lymphoid, and intestinal tissues. Tetramer+ cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation.ResultsWe defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8+ T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer+ cells varied across donors and epitopes.ConclusionsOur findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.

High-Resolution Mapping of Human Norovirus Antigens via Genomic Phage Display Library Selections and Deep Sequencing.

Norovirus (NoV) infections are a leading cause of gastroenteritis. The humoral immune response plays an important role in the control of NoV, and recent studies have identified neutralizing antibodies that bind the capsid protein VP1 to block viral infection. Here, we utilize a NoV GI.1 Jun-Fos-assisted phage display library constructed from randomly fragmented genomic DNA coupled with affinity selection for antibody binding and subsequent deep sequencing to map epitopes. The epitopes were identified by quantitating the phage clones before and after affinity selection and aligning the sequences of the most enriched peptides. The HJT-R3-A9 single-chain variable fragment (scFv) antibody epitope was mapped to a 12-amino-acid region of VP1 that is also the binding site for several previously identified monoclonal antibodies. We synthesized the 12-mer peptide and found that it binds the scFv antibody with a KD (equilibrium dissociation constant) of 46 nM. Further, alignment of enriched peptides after affinity selection on rabbit anti-NoV polyclonal antisera revealed five families of overlapping sequences that define distinct epitopes in VP1. One of these is identical to the HJT-R3-A9 scFv epitope, further suggesting that it is immunodominant. Similarly, other epitopes identified using the polyclonal antisera overlap binding sites for previously reported monoclonal antibodies, suggesting that they are also dominant epitopes. The results demonstrate that affinity selection and deep sequencing of the phage library provide sufficient resolution to map multiple epitopes simultaneously from complex samples such as polyclonal antisera. This approach can be extended to examine the antigenic landscape in patient sera to facilitate investigation of the immune response to NoV.IMPORTANCE NoV infections are a leading cause of gastroenteritis in the United States. Human NoVs exhibit extensive genetic and antigenic diversity, which makes it challenging to design a vaccine that provides broad protection against infection. Antibodies developed during the immune response play an important role in the control of NoV infections. Neutralizing antibodies that act by sterically blocking the site on the virus used to bind human cells have been identified. Identification of other antibody binding sites associated with virus neutralization is therefore of interest. Here, we use a high-resolution method to map multiple antibody binding sites simultaneously from complex serum samples. The results show that a relatively small number of sites on the virus bind a large number of independently generated antibodies, suggesting that immunodominance plays a role in the humoral immune response to NoV infections.

Norovirus risk at a golf course irrigated with reclaimed water: Should QMRA doses be adjusted for infectiousness?

About 25 golf courses in Ontario, Canada have environmental compliance approvals to use reclaimed water for irrigation, where disinfection is confirmed through E. coli limits. A previous study at five Ontario municipal wastewater treatment plants (WWTPs) confirmed that enteric viruses are less susceptible to disinfection than E. coli, when plants provided conventional (secondary or tertiary) treatment and routine (chlorine or UV) disinfection. Here we query whether these four treatment-disinfection scenarios plus 60-day lagoon storage of disinfected effluent would be sufficient to reduce norovirus genogroups I and II (NoV GI and GII) risk of infection to tolerable levels for a golfer who incidentally ingests NoV after handling wet golf balls at a golf course irrigated with reclaimed water. We used our RT-qPCR NoV enumeration datasets from the four treatment-disinfection scenarios above and modeled detected and non-detected NoV by Bayesian inference in ‘R’. Monte Carlo simulation included pre-disinfection NoV GI and GII gene copy densities; Ontario WWTP-derived chlorine and UV log10 reductions; literature-derived effluent storage decay parameters and golfer ingested volumes, followed by six different NoV dose-response (DR) models. Quantitative Microbial Risk Assessment (QMRA) results suggest that there is an unacceptable NoV infection risk when using the conservative assumption that all detected NoV particles (RT-qPCR gene copies) are infectious, in both aggregated or disaggregated form. However, after adjusting for PCR target sequences and for infectiousness using data from recently published studies on cultivation of human NoV in human intestinal enteroids, we noted a significant reduction of infection risk. However, this less conservative (i.e., less protective) assumption for water reuse applications such as golf course irrigation may not be corroborated until human NoV are efficiently and routinely grown in cell cultures. In addition, further studies on drivers of NoV risk estimation by DR models are needed, e.g., the extent of NoV particle aggregation resulting from wastewater treatment, as well as the role of immunity. Meantime, regulatory agencies could consider more stringent treatment-disinfection requirements that target enteric viruses rather than E. coli and testing of actual reclaimed irrigation waters.