From a case-control survey to a diagnostic viral gastroenteritis panel for testing of general practitioners' patients.

OBJECTIVE: To evaluate the pathogenicity of a broad range of 11 possible gastroenteritis viruses, by means of statistical relationships with cases vs. controls, or Ct-values, in order to establish the most appropriate diagnostic panel for our general practitioner (GP) patients in the Netherlands (2010-2012). METHODS: Archived stool samples from 1340 cases and 1100 controls were retested using internally controlled multiplex real-time PCRs for putative pathogenic gastroenteritis viruses: adenovirus, astrovirus, bocavirus, enterovirus, norovirus GI and GII, human parechovirus, rotavirus, salivirus, sapovirus, and torovirus. RESULTS: The prevalence of any virus in symptomatic cases and asymptomatic controls was 16.6% (223/1340) and 10.2% (112/1100), respectively. Prevalence of astrovirus (adjusted odds ratio (aOR) 10.37; 95% confidence interval (CI) 1.34-80.06) and norovirus GII (aOR 3.10; CI 1.62-5.92) was significantly higher in cases versus controls. Rotavirus was encountered only in cases. We did not find torovirus and there was no statistically significant relationship with cases for salivirus (aOR 1,67; (CI) 0.43-6.54)), adenovirus non-group F (aOR 1.20; CI 0.75-1.91), bocavirus (aOR 0.85; CI 0.05-13.64), enterovirus (aOR 0.83; CI 0.50-1.37), human parechovirus (aOR 1.61; CI 0.54-4.77) and sapovirus (aOR 1.15; CI 0.67-1.98). Though adenovirus group F (aOR 6.37; CI 0.80-50.92) and norovirus GI (aOR 2.22, CI: 0.79-6.23) are known enteropathogenic viruses and were more prevalent in cases than in controls, this did not reach significance in this study. The Ct value did not discriminate between carriage and disease in PCR-positive subjects. CONCLUSIONS: In our population, diagnostic gastroenteritis tests should screen for adenovirus group F, astrovirus, noroviruses GI and GII, and rotavirus. Case-control studies as ours are lacking and should also be carried out in populations from other epidemiological backgrounds.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages.

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

Aetiology of acute diarrhoea in children in Shanghai, 2015-2018.

Diarrhoea remains a major cause of childhood morbidity and mortality worldwide. This study aimed to monitor the aetiology of acute diarrhoea in children in Shanghai. Paediatric outpatients with acute diarrhoea were enrolled in the study from Jan 2015 to Dec 2018. Faecal samples were collected for testing. Enteric bacteria were identified and typed by culture and serotyping, respectively. Enteric viruses were identified by real-time PCR. Enteric pathogens were identified in 1572 (58.4%) of the 2692 enrolled children with acute diarrhoea. Viruses were detected more frequently than bacteria (41.3% versus 25.0%). Nontyphoidal Salmonella spp. (NTS) was the most common (10.3%) bacteria isolated, followed by enteropathogenic Escherichia coli (EPEC) (6.5%), enteroaggregative Escherichia coli (EAEC) (6.2%), Campylobacter spp. (3.6%), enterotoxigenic Escherichia coli (ETEC) (1.1%), Shigella spp. (0.2%), and enterohemorrhagic Escherichia coli (EHEC) (0.1%). Rotavirus was the most common (16.0%) virus detected, followed by norovirus (15.5%), adenovirus (7.2%), sapovirus (3.0%) and astrovirus (2.7%). Rotavirus, norovirus and NTS were the major pathogens responsible for diarrhoea in Shanghainese children. Improving uptake of the rotavirus vaccine and strengthening foodborne-pathogen prevention will aid in reducing the burden of diarrhoeal disease in children in Shanghai.

Targeted Profiling of Immunological Genes during Norovirus Replication in Human Intestinal Enteroids.

Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were challenged with two norovirus pandemic GII.4 Sydney strains. Norovirus RNA replication in supernatants and cell lysates were quantified by RT-qPCR. RNA expression levels of immune-related genes were profiled using PCR arrays. The secreted protein levels of shortlisted upregulated genes were measured in supernatants using analyte-specific enzyme-linked immunosorbent assay (ELISA). Productive norovirus replications were achieved in three (75%) out of four inoculations. The two most upregulated immune-related genes were CXCL10 (93-folds) and IFI44L (580-folds). Gene expressions of CXCL10 and IFI44L were positively correlated with the level of norovirus RNA replication (CXCL10: Spearman's r = 0.779, p < 0.05; IFI44L: r = 0.881, p < 0.01). The higher level of secreted CXCL10 and IFI44L proteins confirmed their elevated gene expression. The two genes have been reported to be upregulated in norovirus volunteer challenges and natural human infections by other viruses. Our data suggested that HIE could mimic the innate immune response elicited in natural norovirus infection and, therefore, could serve as an experimental model for future virus-host interaction and antiviral studies.

Evaluation of the 50% Infectious Dose of Human Norovirus Cin-2 in Gnotobiotic Pigs: A Comparison of Classical and Contemporary Methods for Endpoint Estimation.

Human noroviruses (HuNoVs) are the leading causative agents of epidemic and sporadic acute gastroenteritis that affect people of all ages worldwide. However, very few dose-response studies have been carried out to determine the median infectious dose of HuNoVs. In this study, we evaluated the median infectious dose (ID50) and diarrhea dose (DD50) of the GII.4/2003 variant of HuNoV (Cin-2) in the gnotobiotic pig model of HuNoV infection and disease. Using various mathematical approaches (Reed-Muench, Dragstedt-Behrens, Spearman-Karber, exponential, approximate beta-Poisson dose-response models, and area under the curve methods), we estimated the ID50 and DD50 to be between 2400-3400 RNA copies, and 21,000-38,000 RNA copies, respectively. Contemporary dose-response models offer greater flexibility and accuracy in estimating ID50. In contrast to classical methods of endpoint estimation, dose-response modelling allows seamless analyses of data that may include inconsistent dilution factors between doses or numbers of subjects per dose group, or small numbers of subjects. Although this investigation is consistent with state-of-the-art ID50 determinations and offers an advancement in clinical data analysis, it is important to underscore that such analyses remain confounded by pathogen aggregation. Regardless, challenging virus strain ID50 determination is crucial for identifying the true infectiousness of HuNoVs and for the accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model.

Virus-Host Interactions Between Nonsecretors and Human Norovirus.

BACKGROUND & AIMS: Human norovirus infection is the leading cause of acute gastroenteritis. Genetic polymorphisms, mediated by the FUT2 gene (secretor enzyme), define strain susceptibility. Secretors express a diverse set of fucosylated histoblood group antigen carbohydrates (HBGA) on mucosal cells; nonsecretors (FUT2-/-) express a limited array of HBGAs. Thus, nonsecretors have less diverse norovirus strain infections, including resistance to the epidemiologically dominant GII.4 strains. Because future human norovirus vaccines will comprise GII.4 antigen and because secretor phenotype impacts GII.4 infection and immunity, nonsecretors may mimic young children immunologically in response to GII.4 vaccination, providing a needed model to study cross-protection in the context of limited pre-exposure. METHODS: By using specimens collected from the first characterized nonsecretor cohort naturally infected with GII.2 human norovirus, we evaluated the breadth of serologic immunity by surrogate neutralization assays, and cellular activation and cytokine production by flow cytometry. RESULTS: GII.2 infection resulted in broad antibody and cellular immunity activation that persisted for at least 30 days for T cells, monocytes, and dendritic cells, and for 180 days for blocking antibody. Multiple cellular lineages expressing interferon-γ and tumor necrosis factor-α dominated the response. Both T-cell and B-cell responses were cross-reactive with other GII strains, but not GI strains. To promote entry mechanisms, inclusion of bile acids was essential for GII.2 binding to nonsecretor HBGAs. CONCLUSIONS: These data support development of within-genogroup, cross-reactive antibody and T-cell immunity, key outcomes that may provide the foundation for eliciting broad immune responses after GII.4 vaccination in individuals with limited GII.4 immunity, including young children.

Community burden and transmission of acute gastroenteritis caused by norovirus and rotavirus in the Netherlands (RotaFam): a prospective household-based cohort study.

BACKGROUND: Norovirus and rotavirus are the dominant pathogens causing acute gastroenteritis in children. To quantify their natural disease burden and transmission, we prospectively monitored households in an endemic setting in the Netherlands, a high-income country that does not have a rotavirus vaccination programme. METHODS: We did a prospective, household survey-based cohort study in the Netherlands. Randomly selected households from the Dutch Population Register were invited to participate if they had at least three household members, including a child younger than 2 years. A member of each household was asked to record the gastrointestinal symptoms of all household members every day for 10 consecutie weeks using an interactive smartphone application. Real-time detection of acute gastroenteritis onset on the basis of entered symptoms activated requests for the case and one other household member to complete disease questionnaires and provide stool samples. Stool samples were analysed by real-time PCR for norovirus, rotavirus, adenovirus 40/41, and astrovirus. We calculated the per-pathogen proportion of households with at least one secondary acute gastroenteritis episode (epidemiologically but not microbiologically linked), the probability of a secondary episode in household members at risk (secondary attack rate), and the microbiologically confirmed symptomatic and asymptomatic transmission rates. FINDINGS: During two seasons (January to March) in 2016 and 2017, 30 660 households were invited to participate, of which 604 households including 2298 individuals were enrolled. 697 acute gastroenteritis episodes were detected in 358 households, with samples obtained from 609 (87%) of 697 episodes. Norovirus (150 [25%] of 609 cases) and rotavirus (91 [15%] cases) were most frequently detected. Astrovirus was detected in 50 (8%) samples and adenovirus 40/41 in 24 (4%) samples. Overall disease severity was higher in patients with rotavirus-positive acute gastroenteritis than those with norovirus-positive acute gastroenteritis. Norovirus led to higher disease burden in adults than did rotavirus. Following an index case, a secondary acute gastroenteritis episode occurred in 34 (35%) of 96 households for norovirus and 26 (46%) of 56 households for rotavirus. Secondary attack rates were 15% (37 of 244 participants) for norovirus and 28% (33 of 120 participants) for rotavirus and asymptomatic transmission rates were 51% (52 of 102 household members) for norovirus and 22% (12 of 55 household members) for rotavirus. The microbiologically confirmed symptomatic transmission rate for norovirus was 10% (25 of 254 household members) and 18% for rotavirus (21 of 119 household members). INTERPRETATION: In households with young family members in a setting without rotavirus vaccination, norovirus is the dominant acute gastroenteritis pathogen, but rotavirus is associated with more severe disease. There was substantial within-household transmission, both symptomatic and asymptomatic. The study provides key quantities on transmission, which can inform vaccine policy decisions and act as a baseline for impact evaluations in high-income settings. FUNDING: The Netherlands Organisation for Health Research and Development (grant 91616158).

Viral uptake and stability in Crassostrea gigas oysters during depuration, storage and steaming.

More stable than bacteria in environmental samples, enteric viruses are generally related to outbreaks of gastroenteritis caused by the consumption of contaminated oysters. This study evaluated: i) the dynamic processes of enteric viral models bioaccumulation by Crassostrea gigas oysters artificially contaminated; ii) the stability of these viruses in oysters in controlled temperature conditions and iii) the effect of UV light in inactivating these viruses in depurated oysters. Plaque assay (PA) was used to assess the infectivity of both viral models. Cell culture coupled with RT-qPCR (ICC-RT-qPCR) was used to measure infectious adenovirus type 2 (HAdV-2) genomes and qPCR to measure genome copies of murine norovirus (MNV-1). The virus uptake through bioaccumulation behave differently: HAdV-2 reached its peak of uptake faster than MNV-1. Both viruses showed high stability in oysters when maintained under 4 °C, but were completely inactivated in steamed oysters. The HAdV-2 was completely inactivated after 12 h of depuration with UV light and after 24 h without UV light. After 72 h of depuration, MNV-1 was still detected in both tanks, probably due to the stronger interaction of this virus with the oyster's tissues. This study demonstrated the importance of a secure depuration time in ensuring a clean and safe product, and that the steaming process is the safest way to prepare oysters for consumption.

A robust human norovirus replication model in zebrafish larvae.

Human noroviruses (HuNoVs) are the most common cause of foodborne illness, with a societal cost of $60 billion and 219,000 deaths/year. The lack of robust small animal models has significantly hindered the understanding of norovirus biology and the development of effective therapeutics. Here we report that HuNoV GI and GII replicate to high titers in zebrafish (Danio rerio) larvae; replication peaks at day 2 post infection and is detectable for at least 6 days. The virus (HuNoV GII.4) could be passaged from larva to larva two consecutive times. HuNoV is detected in cells of the hematopoietic lineage and the intestine, supporting the notion of a dual tropism. Antiviral treatment reduces HuNoV replication by >2 log10, showing that this model is suited for antiviral studies. Zebrafish larvae constitute a simple and robust replication model that will largely facilitate studies of HuNoV biology and the development of antiviral strategies.

Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells.

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.

Viral Pathogen-Specific Clinical and Demographic Characteristics of Children with Moderate-to-Severe Diarrhea in Rural Bangladesh.

Diarrheal disease is a leading cause of childhood morbidity and mortality worldwide, but particularly in low-income countries in sub-Saharan Africa and South Asia. The Global Enteric Multicenter Study (GEMS) examined the infectious etiologies as well as associated demographics, socioeconomic markers, health-care-seeking behaviors, and handwashing practices of the households of children with diarrhea and their age- and gender-matched controls in seven countries over a 3-year period (December 2007-December 2010). Stool studies to determine diarrheal etiologies and anthropometry were performed at baseline and at 60-day follow-up visits, along with surveys to record demographics and living conditions of the children. We performed secondary analyses of the GEMS data derived from the Bangladesh portion of the study in children with diarrhea associated with viral enteropathogens and explored pathogen-specific features of disease burden. Rotavirus and norovirus were the most prevalent pathogens (39.3% and 35%, respectively). Disease due to rotavirus and adenovirus was more common in infants than in older children (P < 0.001 and P = 0.001, respectively). Height for age decreased from baseline to follow-up in children with diarrhea associated with rotavirus, norovirus, and adenovirus (P < 0.001). Based on these analyses, preventive measures targeted at rotavirus, norovirus, and adenovirus will be expected to have meaningful clinical impact. Cost of treatment was highest for rotavirus as well, making it an obvious target for intervention. Association of specific viruses with stunting is particularly notable, as stunting is an attributable risk factor for poor cognitive development and future productivity and economic potential.

Norovirus Attachment and Entry.

Human norovirus is a major human pathogen causing the majority of cases of viral gastroenteritis globally. Viral entry is the first step of the viral life cycle and is a significant determinant of cell tropism, host range, immune interactions, and pathogenesis. Bile salts and histo-blood group antigens are key mediators of norovirus entry; however, the molecular mechanisms by which these molecules promote infection and the identity of a potential human norovirus receptor remain unknown. Recently, there have been several important advances in norovirus entry biology including the identification of CD300lf as the receptor for murine norovirus and of the role of the minor capsid protein VP2 in viral genome release. Here, we will review the current understanding about norovirus attachment and entry and highlight important future directions.

Nlrp3 inflammasome activation and Gasdermin D-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection.

Norovirus infection is the leading cause of food-borne gastroenteritis worldwide, being responsible for over 200,000 deaths annually. Studies with murine norovirus (MNV) showed that protective STAT1 signaling controls viral replication and pathogenesis, but the immune mechanisms that noroviruses exploit to induce pathology are elusive. Here, we show that gastrointestinal MNV infection leads to widespread IL-1ß maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1ß and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1ß, facilitating elevated Nlrp3-dependent release of mature IL-1ß upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1ß as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment. While MNV-induced diarrheic responses were not affected, Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection. Together, these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death, thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice.

Development of a rapid and sensitive electrochemical biosensor for detection of human norovirus via novel specific binding peptides.

Human noroviruses cause acute foodborne gastroenteritis outbreaks worldwide. In this study, a highly sensitive and selective electrochemical biosensor was fabricated for the detection of human norovirus using novel peptides as recognition elements. The electrochemical biosensor was fabricated by assembling of eight novel peptides separately on the gold electrode and investigated their efficiencies for sensing human noroviruses. Among eight peptides, NoroBP peptide coated onto the gold electrode exhibited a high binding affinity towards human noroviruses, resulting a progressive decrease in current signals with increasing concentration of human norovirus (0-105 copies/mL). As a result, NoroBP-nonFoul(FlexL)2-coated gold electrode acts an efficient electrochemical biosensor for highly selective detection of human norovirus with a detection limit of 1.7 copies/mL, which is 3-fold lower than the reported methods. The developed electrochemical biosensor was successfully applied to detect human norovirus prepared by standard procedure from oyster, which suggests that the developed biosensor can be used as a very sensitive and selective point-of-care bioanalytical platform for the detection of human norovirus in various food samples.

Occurrence of viral gastroenteritis in children below 5 years: A hospital-based study from Assam, India.

Viral gastroenteritis is an important cause of mortality and morbidity in children under 5 years of age. Many a time, these cases go unnoticed causing immense scarcity of data on viral diarrhoea. The study aimed to determine the occurrence of viral gastroenteritis among children below 5 years and the aetiological viral agents. Stool samples were collected from patients suffering from acute gastroenteritis. Real-time polymerase chain reaction was done for detection of rotavirus, adenovirus, norovirus, astrovirus and sapovirus. Viruses were detected in 55% of children. Adenovirus was found to be the most common virus (33.7%), followed by rotavirus infection (28.7%).

Norovírus no município de São Paulo, 2010-2016: estudo transversal sobre a principal causa de gastroenterite infantil / Norovirus en el Municipio de São Paulo, Brasil, 2010-2016: estudio transversal sobre la principal causa de gastroenteritis infantil / Norovirus in São Paulo city, Brazil, 2010-2016: a cross-sectional study of the leading cause of gastroenteritis in children

Objetivo: descrever casos de doença diarreica aguda por norovírus em crianças menores de 5 anos do município de São Paulo, Brasil. Métodos: estudo transversal com dados provenientes da Vigilância Epidemiológica das Gastroenterites Causadas por Rotavírus; foi definido como caso o paciente internado em unidade sentinela por doença diarreica aguda e identificação laboratorial do norovírus como agente etiológico, entre os anos de 2010 e 2016. Resultados: durante o período estudado, a proporção de casos de norovírus em menores de 5 anos de idade ultrapassou a proporção de casos de rotavírus, agente considerado predominante na infância; o norovírus foi associado a 28,4% do total de casos notificados, ocorrendo o ano todo, principalmente nos meses mais quentes. Conclusão: norovírus foi o principal agente etiológico identificado em crianças menores de 5 anos com doença diarreica aguda no município de São Paulo.

Objetivo: describir casos de enfermedad diarreica aguda por Norovirus en niños menores de 5 años provenientes del Municipio de São Paulo, Brasil. Métodos: Estudio transversal con datos de la Vigilancia Epidemiológica de las Gastroenteritis causadas por Rotavirus. Se definió como caso el paciente internado en unidad centinela por enfermedad diarreica aguda e identificación de laboratorio del Norovirus como agente etiológico entre los años de 2010 y 2016. Resultados: Durante el período estudiado, la proporción de casos de Norovirus en menores de 5 años superó la proporción de casos de Rotavirus, agente considerado predominante en la infancia. El Norovirus fue asociado al 28,4% del total de los casos notificados, ocurriendo todo el año, principalmente en los meses más cálidos. Conclusión: el Norovirus fue el principal agente etiológico identificado en niños menores de 5 años con enfermedad diarreica aguda en el Municipio de São Paulo.

Objective: to describe cases of acute diarrheal disease caused by norovirus in children under 5 years old in São Paulo city, Brazil. Methods: this was a cross-sectional study using data from Epidemiological Surveillance of Gastroenteritis due to Rotavirus; cases were defined as patients hospitalized in a sentinel unit because of acute diarrheal disease and laboratory identification of norovirus as the etiological agent between 2010 and 2016. Results: during the study period, the proportion of norovirus cases in children under 5 years old exceeded the proportion of Rotavirus, an agent considered predominant in childhood; norovirus was associated with 28.4% of total reported cases, occurring all year round, especially in warmer months. Conclusion: norovirus was the leading etiological agent identified in children under 5 years old with acute diarrheal disease in São Paulo city.

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus.

Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.

HOIL1 Is Essential for the Induction of Type I and III Interferons by MDA5 and Regulates Persistent Murine Norovirus Infection.

The linear ubiquitin chain assembly complex (LUBAC), composed of heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1), HOIL1-interacting protein (HOIP), and SHANK-associated RH domain-interacting protein (SHARPIN), is a crucial regulator of multiple immune signaling pathways. In humans, HOIL1 or HOIP deficiency is associated with an immune disorder involving autoinflammation, immunodeficiency, and inflammatory bowel disease (IBD)-like symptoms. During viral infection, LUBAC is reported to inhibit the induction of interferon (IFN) by the cytosolic RNA sensor retinoic acid-inducible gene I (RIG-I). Surprisingly, we found that HOIL1 is essential for the induction of both type I and type III IFNs, as well as the phosphorylation of IFN regulatory factor 3 (IRF3), during murine norovirus (MNoV) infection in cultured dendritic cells. The RIG-I-like receptor, melanoma differentiation-associated protein 5 (MDA5), is also required for IFN induction and IRF3 phosphorylation during MNoV infection. Furthermore, HOIL1 and MDA5 were required for IFN induction after Theiler's murine encephalomyelitis virus infection and poly(I·C) transfection, but not Sendai virus or vesicular stomatitis virus infection, indicating that HOIL1 and LUBAC are required selectively for MDA5 signaling. Moreover, Hoil1-/- mice exhibited defective control of acute and persistent murine norovirus infection and defective regulation of MNoV persistence by the microbiome as also observed previously for mice deficient in interferon lambda (IFN-λ) receptor, signal transducer and activator of transcription factor 1 (STAT1), and IRF3. These data indicate that LUBAC plays a critical role in IFN induction to control RNA viruses sensed by MDA5.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis throughout the world but are challenging to study in vivo and in vitro Murine norovirus (MNoV) provides a tractable genetic and small-animal model to study norovirus biology and immune responses. Interferons are critical mediators of antiviral immunity, but excessive expression can dysregulate the immune system. IFN-λ plays an important role at mucosal surfaces, including the gastrointestinal tract, and both IFN-λ and commensal enteric bacteria are important modulators of persistent MNoV infection. LUBAC, of which HOIL1 is a component, is reported to inhibit type I IFN induction after RIG-I stimulation. We show, in contrast, that HOIL1 is critical for type I and III IFN induction during infection with MNoV, a virus that preferentially activates MDA5. Moreover, HOIL1 regulates MNoV infection in vivo These data reveal distinct functions for LUBAC in these closely related signaling pathways and in modulation of IFN expression.