Sporadic Acute Gastroenteritis Research Articles

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.

Enteric viruses other than rotavirus and norovirus in children under 5 years of age with gastroenteritis in Argentina, 2010-2021. A descriptive study.

Introduction. Data on the frequency of enteric adenoviruses, sapoviruses, and astroviruses in cases of sporadic acute gastroenteritis in Argentina are scarce. Methods. Descriptive design of a selection of fecal samples of children with diarrhea younger than 5 years referred between 2010 and 2021, with a previous negative result for rotavirus and norovirus. The presence of enteric adenovirus, sapovirus, and astrovirus was tested by molecular methods, with subsequent genotyping of positive samples. Results. At least 1 of the tested viruses was detected in 226 (39.4%) of the 574 selected samples. Specifically, adenovirus, sapovirus, and astrovirus were detected in 30.7%, 5.6%, and 3.1% of the samples, respectively. The most frequent viruses detected were adenovirus 41, sapoviruses GI.1 and GI.2, and astrovirus 1. Non-classic astroviruses were detected in 2 samples. Conclusions. Despite being less frequent, these enteropathogens are responsible for a large number of sporadic diarrhea events. Therefore, their study and surveillance contribute significantly to reduce the gap of undiagnosed cases.

Biological Specimen Banking as a Time Capsule to Explore the Temporal Dynamics of Norovirus Epidemiology.

Norovirus is recognised as a major cause of epidemic and sporadic acute gastroenteritis (AGE) in all age groups. Information on the genetic diversity of the noroviruses circulating in the 1980s and 1990s, before the development and adoption of dedicated molecular assays, is limited compared with the last decades. Between 1986 and 2020, uninterrupted viral surveillance was conducted in symptomatic children hospitalized with AGE in Palermo, Italy, providing a unique time capsule for exploring the epidemiological and evolutionary dynamics of enteric viruses. A total of 8433 stool samples were tested using real-time RT-PCR. All samples were stored at -20 or -80 °C until processing. In this 35-year long time span, noroviruses of genogroup II (GII) were detected in 15.6% of AGE requiring hospitalization, whilst GI noroviruses were detected in 1.4% of AGE. Overall, the predominant norovirus capsid (Cap) genotype was GII.4 (60.8%), followed by GII.3 (13.3%) and GII.2 (12.4%). Temporal replacement of the GII.4 Cap variants associated with different polymerase (Pol) types were observed over the study period. The chronology of emergence and circulation of the different GII.4 variants were consistent with data available in the literature. Also, for GII.3 and GII.2 NoVs, the circulation of different lineages/strains, differing in either the Cap or Pol genes or in both, was observed. This long-term study revealed the ability of noroviruses to continuously and rapidly modify their genomic makeup and highlights the importance of surveillance activities in vaccine design.

Genetic diversity and declining norovirus prevalence in infants and children during Japan's COVID-19 pandemic: a three-year molecular surveillance.

Noroviruses (NoVs) are a global concern, causing widespread outbreaks and sporadic acute gastroenteritis (AGE) cases across all age groups. Recent research has shed light on the emergence of novel recombinant strains of NoV in various countries. To delve deeper into this phenomenon, we extensively analyzed 1,175 stool samples collected from Japanese infants and children with AGE from six different prefectures in Japan over three years, from July 2018 to June 2021. Our investigation aimed to determine the prevalence and genetic characteristics of NoV associated with sporadic AGE while exploring the possibility of detecting NoV recombination events. Among the analyzed samples, we identified 355 cases positive for NoV, 11 cases attributed to GI genotypes, and 344 associated with GII genotypes. Notably, we discovered four distinct GI genotypes (GI.2, GI.3, GI.4, and GI.6) and seven diverse GII genotypes (GII.2, GII.3, GII.4, GII.6, GII.7, GII.14, and GII.17). The predominant genotypes were GII.4 (56.4%; 194 out of 344), followed by GII.2 and GII.3. Through dual genotyping based on sequencing of the ORF1/ORF2 junction region, we identified a total of 14 different RdRp/capsid genotypes. Of particular interest were the prevalent recombinant genotypes GII.4[P31] and GII.2[P16]. Notably, our study revealed a decrease in the number of children infected with NoV during and after the COVID-19 pandemic. These findings underscore the importance of continuous NoV surveillance efforts.

Human rotavirus strains circulating among children in the capital of China (2018–2022)_ predominance of G9P[8] and emergence ofG8P[8]

ObjectiveThis study aimed to update the genetic diversity of Rotavirus (RV) infections in children under five years old in Beijing, China. MethodsA 5-year active hospital-based surveillance for sporadic acute gastroenteritis (AGE) from January 2018 to December 2022 in the capital of China was performed. A total of 748 fecal samples from AGE patients were collected for followed by RV antigen detection by ELSIA, RNA detection by reverse transcription PCR, G/P genotyping and phylogenetic analyzing. ResultsRV antigen was detected in 11.0% of the collected samples, with 54 samples confirmed to be RV RNA positive. G9 and G8 genotypes were identified in 43 (79.6%) and 7 (13.0%) samples, respectively, all of which were allocated to P[8]. The predominant G/P combination was G9P[8] (79.6%), following by G8P[8] (13.0%), G4P[8] (5.6%) and G3P[8] (1.9%). A significant change in G/P-type distribution was observed, with the G9P[8] being predominant from 2018 to 2021, followed by the emergence of an uncommon G8P[8] genotype, which was first reported in 2021 and became predominant in 2022. Blast analysis showed that one G1 isolate had a high similarity of 99.66% on nucleotide acid with RotaTeq vaccine strain with only one amino acid difference L150V. Additionally, one P[8] isolate was clustered into a branch together with RotaTeq vaccine strain G6P[8]. ConclusionsThe study reveals that G8P[8] has become the predominant genotype in pediatric outpatients in China for the first time, indicating a significant change in the composition of RV genetic diversity. The importance of RVA genotyping in surveillance is emphasized, as it provides the basis for new vaccine application and future vaccine efficacy evaluation.

Incidence, Etiology, and Healthcare Utilization for Acute Gastroenteritis in the Community, United States.

Knowledge of the epidemiology of sporadic acute gastroenteritis (AGE) in the United States is limited. During September 2016-September 2017, we surveyed Kaiser Permanente Northwest members in Oregon and Washington, USA, to collect data on the 30-day prevalence of dually defined AGE and diarrhea disease and related health-seeking behavior; from a subset of participants, we obtained a stool specimen. Using the iterative proportional fitting algorithm with raked weights, we generated AGE prevalence and annualized rate estimates. We detected norovirus, rotavirus, astrovirus, and sapovirus from submitted stool specimens through real-time quantitative reverse transcription PCR (qRT-PCR). We estimated a 30-day prevalence of 10.4% for AGE and 7.6% for diarrhea only; annual rates were 1.27 cases/person/year for AGE and 0.92 cases/person/year for diarrhea only. Of those with AGE, 19% sought medical care. Almost one quarter (22.4%) of stool specimens from those reporting AGE tested positive for ≥1 viral pathogen, compared with 8.2% from those without AGE.

Genotypes and transmission routes of noroviruses causing sporadic acute gastroenteritis among adults and children, Japan, 2015-2019.

Noroviruses (NoVs) are major causes of acute viral gastroenteritis at all ages worldwide. The molecular epidemiology of sporadic cases remains poorly understood, especially in adults. Additionally, no studies have analyzed the transmission route in sporadic acute gastroenteritis. In this study, we investigated cases of very mild sporadic NoV acute gastroenteritis in adults (medical staff) who do not visit the outpatient clinic and child outpatients. We also evaluated genotype differences between adults and children and possible transmission routes in adults during 5years. The number of NoV positives were 58 in adults and 124 in children. In adults, the NoV positivity rate in this study was higher (64.4%) than that in previous reports of outpatients (10%) and inpatients (5%) in the United State. This finding suggested that the NoV positivity rate might be high in adults with very mild acute gastroenteritis. In adults, human-to-human transmission rates from children and food-borne transmission (raw oysters) were 21.6% (11/51) and 19.6% (10/51), respectively. Among adults, GII.2, GII.4, and GII.17 were the predominant genotypes, with rates of 32.7%, 30.9%, and 21.8%, respectively. Among children, GII.4 and GII.2 were the predominant genotypes, with rates of 45.5% and 40.6%, respectively. GII.17 was only detected in 0.8% (1/123) of children. Trends in NoV genotypes are expected to differ depending on the patient's age. Investigating sporadic cases including the patient's background (age and transmission route) may be helpful to monitor the trend of NoV strains, forecast prevalent NoV GII genotypes, and develop NoV vaccines.

Identification of compelling inhibitors of human norovirus 3CL protease to combat gastroenteritis: A structure-based virtual screening and molecular dynamics study.

Human noroviruses (NV) are the most prevalent cause of sporadic and pandemic acute gastroenteritis. NV infections cause substantial morbidity and death globally, especially amongst the aged, immunocompromised individuals, and children. There are presently no authorized NV vaccines, small-molecule therapies, or prophylactics for humans. NV 3 C L protease (3CLP) has been identified as a promising therapeutic target for anti-NV drug development. Herein, we employed a structure-based virtual screening method to screen a library of 700 antiviral compounds against the active site residues of 3CLP. We report three compounds, Sorafenib, YM201636, and LDC4297, that were revealed to have a higher binding energy (BE) value with 3CLP than the control (Dipeptidyl inhibitor 7) following a sequential screening, in-depth molecular docking and visualization, physicochemical and pharmacological property analysis, and molecular dynamics (MD) study. Sorafenib, YM201636, and LDC4297 had BEs of -11.67, -10.34, and -9.78 kcal/mol with 3CLP, respectively, while control had a BE of -6.38 kcal/mol. Furthermore, MD simulations of the two best compounds and control were used to further optimize the interactions, and a 100 ns MD simulation revealed that they form stable complexes with 3CLP. The estimated physicochemical, drug-like, and ADMET properties of these hits suggest that they might be employed as 3CLP inhibitors in the management of gastroenteritis. However, wet lab tests are a prerequisite to optimize them as NV 3CLP inhibitors.

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand.

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n = 6), GII.4 (n = 1), GII.17 (n = 3), and GII.unclassified (n = 3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83-1.85 × 104 genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00 × 101 genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

Role of Noroviruses in Sporadic Acute Gastroenteritis Cases from Children Attending a Large Referral Children's Hospital in Buenos Aires City, Argentina.

Noroviruses (NVs) are recognized as the leading cause of sporadic and epidemic acute gastroenteritis worldwide, in all age groups. Although there is increasing knowledge that NVs are responsible for many acute gastroenteritis outbreaks in Argentina, studies to estimate prevalence in sporadic cases are scarce. A descriptive, observational and cross-sectional study was conducted with children under 5 years with acute gastroenteritis attending the outpatient department at the "Ricardo Gutiérrez" Children's Hospital (RGCH) in Buenos Aires City between June 2017 and June 2021. Sociodemographic, clinical and epidemiologic data were recorded. Stool samples were tested and genotyped for norovirus. Association between norovirus detection and demographic and clinical variables was assessed. A total of 350 children with acute diarrhea were included, of which stool samples were collected for 332 (94.9%). Norovirus was detected in 81 cases (24.4%). Vomiting and moderate/severe diarrhea were more frequent in norovirus-positive than norovirus-negative children. However, the presence of watery diarrhea and a history of rotavirus vaccination were significantly associated with norovirus etiology. GII and GII.4 were the most frequently detected genogroup and genotype, respectively. NVs were detected with high frequency, mostly in children between 6 months and 2 years old, reinforcing the hypothesis of a newly updated scenario of norovirus predominance over rotavirus. Watery diarrhea, complete vaccination against rotavirus and vomiting are 3 key parameters that should raise suspicion of possible norovirus gastroenteritis. Continuous and active norovirus surveillance in this age group is important because children represent a priority group for norovirus vaccine design and development.

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 therotavirus 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.

Immunization with Leishmania tarentolae-derived norovirus virus-like particles elicits high humoral response and stimulates the production of neutralizing antibodies

BackgroundNoroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome.ResultsThis study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 104) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT50) were observed for 1:5–1:320 serum dilutions.ConclusionsNorovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.

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

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

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new medium conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown the following: (i) a new HIE culture medium made with conditioned medium from a single cell line and commercial media promotes robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture medium made with conditioned media from 3 separate cell lines; (ii) GI.1, 11 GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14, and GII.17), and six GII.4 variants can be cultivated in HIEs; (iii) successful replication is more likely with virus in stools with higher virus titers; (iv) GII.4_Sydney_2012 virus replication was reproducible over 3 years; and (v) HuNoV infection is restricted to the small intestine, based on replication of two viral strains in duodenal and ileal HIEs, but not colonoids, from two susceptible donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system, and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control.IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse, and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies, and methods of virus inactivation.

Genetic analysis of sapoviruses detected in outbreaks and sporadic cases of acute gastroenteritis in Miyagi Prefecture, Japan.

Human sapovirus (SaV) causes sporadic and endemic acute gastroenteritis worldwide. However, little is known about the relationship between the mode of transmission and genetic characteristics of SaV. To investigate the molecular characteristics of SaV-associated acute gastroenteritis among sporadic cases, foodborne, and nonfoodborne outbreaks. We performed a systematic review of publications and genetic analysis of SaV in fecal specimens from 98 outpatients with acute gastroenteritis, 32 stool samples from 8 foodborne outbreaks, and 63 stool samples from 23 nonfoodborne outbreaks in Miyagi Prefecture, Japan from 1993 and between 2004 and 2020. Reverse transcription polymerase chain reaction (RT-PCR) was employed for the detection of SaV, and the partial capsid gene was sequenced for genotyping and phylogenetic analysis. The overall detection rate of SaV in sporadic cases, foodborne, and nonfoodborne outbreaks was 5.8, 1.7, and 4.3%, respectively. Genotypic analysis revealed GI.1 to be the predominant genotype in sporadic cases (31.5%) and nonfoodborne outbreaks (52.1%), whereas it was not detected in foodborne outbreaks. Some outbreaks occurred following sporadic cases with the same genotype. The distribution of SaV genotypes was different between foodborne outbreaks and other settings. The effective SaV infection control may differ depending on the genomic characteristics.

Human Intestinal Enteroids to Evaluate Human Norovirus GII.4 Inactivation by Aged-Green Tea.

Human noroviruses are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and the most common cause of foodborne illness in the United States. Several natural compounds, such as aged-green tea extract (aged-GTE), have been suggested as ingestible antiviral agents against human norovirus based on data using murine norovirus and feline calicivirus as surrogates. However, in vitro data showing their effectiveness against infectious human norovirus are lacking. We tested the activity of aged-GTE to inhibit human norovirus in a human intestinal enteroids (HIEs) model and Tulane virus in LLC-monkey kidney (LLC-MK2) cell culture. HIE monolayers pretreated with aged-GTE at different temperatures showed complete inhibition of human norovirus GII.4 replication at concentrations as low as 1.0 mg/ml for 37°C, 1.75 mg/ml for 21°C, and 2.5 mg/ml for 7°C. In contrast, a moderate decrease in Tulane virus infectivity of 0.85, 0.75, and 0.65 log TCID50/ml was observed for 2.5 mg/ml aged-GTE at 37, 21, and 7°C, respectively. Our findings demonstrate that GTE could be an effective natural compound against human norovirus GII.4, while only minimally effective against Tulane virus.

Molecular evolution of GII.P17-GII.17 norovirus associated with sporadic acute gastroenteritis cases during 2013-2018 in Zhoushan Islands, China.

In this study, we investigated the molecular characteristics and spatio-temporal dynamics of GII.P17-GII.17 norovirus in Zhoushan Islands during 2013-2018. We collected 1849 samples from sporadic acute gastroenteritis patients between January 2013 and August 2018 in Zhoushan Islands, China. Among the 1849 samples, 134 (7.24%) samples were positive for human norovirus (HuNoV). The complete sequence of GII.17 VP1 gene was amplified from 31 HuNoV-positive samples and sequenced. A phylogenetic tree was constructed based on the full-length sequence of the VP1 gene. Phylogenetic analysis revealed that the GII.17 genotype detected during 2014-2018 belongs to the new GII.17 Kawasaki variant. Divergence analysis revealed that the time of the most recent common ancestor (TMRCA) of GII.17 in Zhoushan Islands was estimated to be between 1997 and 1998. The evolutionary rate of the VP1 gene of the GII.17 genotype norovirus was 1.14 × 10-3 (95% HPD: 0.62-1.73 × 10-3) nucleotide substitutions/site/year. The spatio-temporal diffusion analysis of the GII.17 genotype identified Hong Kong as the epicenter for GII.17 dissemination. The VP1 gene sequence of Zhoushan Island isolates correlated with that of Hong Kong and Japan isolates.

Complete genome sequences of the first parechoviruses A associated with sporadic pediatric acute gastroenteritis in Russia

Parechovirus (the Picornaviridae family), recently classified as Parechovirus A and formerly known as Human parechovirus (HPeV), can cause a wide range of human diseases. Over the past decade, several studies have reported HPeV epidemiology in different regions; however, information from Russia is limited. A total of 632 stool samples collected in Novosibirsk, Russia during January–March 2012 were screened for HPeV by RT-PCR. The study cohort comprised 572 patients with acute gastroenteritis and 60 healthy children. Seven of 572 (1.2%) gastroenteritis cases were HPeV-positive, including one co-infection with rotavirus and astrovirus. All positive patients were ≤1 year old, and five of them were younger than 3 months. None of the healthy controls provided an HPeV-positive sample. Six HPeV isolates were classified as HPeV-1 and one as HPeV-5 using phylogenetic analysis. Two complete genome sequences of HPeV-1 and one of HPeV-5 were determined and analyzed. Phylogenetic analysis showed that the studied Russian strains are probably recombinants. P1 region sequences of two Russian HPeV-1 strains clustered with rare contemporary HPeV-1A strains, whereas their P3 regions were phylogenetically closer to the archival Harris strain. The Russian HPeV-5 strain formed a common cluster with other HPeV-5 strains only for the P1 region, while the P3 region grouped with the German HPeV-2 strain. In the Russian HPeV-5 strain, the lack of the arginine-glycine-aspartic acid (RGD) motif at the C-terminus of VP1 was observed. This is the first complete genome characterization of the Russian HPeV strains detected in sporadic cases of pediatric acute gastroenteritis.

Genomic and biological characterization of a pandemic norovirus variant GII.4 Sydney 2012.

Genogroup II, genotype 4 noroviruses (GII.4 NoVs) are a leading cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. In this study, we isolated a GII.4 NoV strain (designated 2015HN08) from a kid presenting with acute gastroenteritis and determined its near-complete genome sequence. We then performed sequence analysis by comparing this strain with the prototypical GII.4 strain. Virus-like particles (VLPs) derived from the major capsid protein (VP1) were expressed by using a recombinant-baculovirus expression system, and monoclonal antibodies (mAbs) were produced to compare changes in antigenic or histo-blood group antigens (HBGAs) binding sites with the previously characterized GII.4 NoV strain (JZ403). The genome of 2015HN08 was 7559 nucleotides (nt) long, excluding the poly(A) tail. Genotyping analysis indicated that this strain was a Sydney 2012 variant. In comparison with the prototype Sydney 2012 strain, there were 74, 35, and 16 differences in nucleotide sequences in ORF1, OFR2, and OFR3, causing 7, 10, and 6 amino acid (aa) changes, respectively. Expression of VP1 led to successful assembly of VLPs, as demonstrated by electron microscopy. Screening of hybridoma cell supernatants with an in vitro VLP-HBGAs binding blockade assay led to the identification of a cell clone 3G10 that exhibited HBGA-blocking effects. This mAb also exhibited blocking effects against JZ403 strain, suggesting maintenance of the antigenic site and/or HBGAs binding sites between the two strains. In summary, we determined the near-complete genome sequence of a GII.4 Sydney 2012 variant and produced an mAb with blocking effects that might be useful in evaluating the evolution of current Sydney 2012 NoV strains.

Genetic diversity and epidemiology of Genogroup II noroviruses in children with acute sporadic gastroenteritis in Shanghai, China, 2012\u20132017

BackgroundNoroviruses (NoVs) are considered an important cause of acute gastroenteritis (AGE) across all age groups, especially in children under 5 years of age. We investigated the epidemiology of noroviruses in outpatient children from the Children’s Hospital of Fudan University in Shanghai, China.MethodsStool specimens were collected between January 2012 and December 2017 from 1433 children under 5 years of age with acute gastroenteritis. All samples were analysed by conventional reverse transcription-polymerase chain reaction (RT-PCR) for genogroup II NoVs amplifying both the RNA-dependent RNA polymerase (RdRp) and partial capsid genes. The Norovirus Genotyping Tool v.2.0 (https://www.rivm.nl/mpf/typingtool/norovirus/) was used for genotyping the strains, and phylogenetic analyses were conducted by MEGA 6.0.ResultsFrom 2012 to 2017, GII NoVs were detected in 15.4% (220/1433) of the samples, with the highest detection rate in children aged 7–12 months (19.2%, 143/746). The seasons with the highest prevalence of GII NoVs infection were autumn and winter. Based on genetic analysis of RdRp, GII.Pe (74.5%%, 137/184) was the most predominant RdRp genotype from 2013 to 2017, while GII.P4 played a dominant role in 2012 (55.6%, 21/36). Among the capsid genotypes, the most prevalent NoV genotype from 2012 to 2017 was GII.4 (74.1%, 163/220). On the basis of genetic analysis of RdRp and capsid sequences, the strains were clustered into − 19 RdRp/capsid genotypes, and 12 of them were discordant, such as GII.Pe/GII.4-Sydney_2012, GII.P12/GII.3, GII.P7/GII.6, GII.Pe/GII.3, and GII.P16/GII.2. Starting with 2013, GII.Pe/GII.4-Sydney_2012 had completely replaced the pandemic GII.P4-2006b/GII.4-2006b subtype and was detected in children across all age groups.ConclusionsThe present study shows high detection rates and the genetic diversity of circulating NoV GII genotypes in paediatric AGE samples from Shanghai. The findings emphasize the importance of continuous molecular surveillance of emerging NoV strains.