Norovirus: Facts and Reflections from Past, Present, and Future.

Human Norovirus is currently the main viral cause of acute gastroenteritis (AGEs) in most countries worldwide. Nearly 50 years after the discovery of the "Norwalk virus" by Kapikian and colleagues, the scientific and medical community continue to generate new knowledge on the full biological and disease spectrum of Norovirus infection. Nevertheless, several areas remain incompletely understood due to the serious constraints to effectively replicate and propagate the virus. Here, we present a narrated historic perspective and summarize our current knowledge, including insights and reflections on current points of interest for a broad medical community, including clinical and molecular epidemiology, viral-host-microbiota interactions, antivirals, and vaccine prototypes. We also include a reflection on the present and future impacts of the COVID-19 pandemic on Norovirus infection and disease.

The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update.

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

Norovirus diarrhea is significantly associated with higher counts of fecal histo-blood group antigen expressing Enterobacter cloacae among black South African infants.

The study tested the hypothesis that harboring high levels of histo-blood group antigen-expressing Enerobactero cloacae is a risk factor for norovirus diarrhea. The fecal E. cloacae abundance in diarrheic norovirus positive (DNP), non-diarrheic norovirus negative (NDNN), diarrhea norovirus negative (DNN), and non-diarrhea norovirus positive (NDNP) infants was determined by qPCR, and the risk of norovirus diarrhea was assessed by logistical regression. DNP infants contained significantly higher counts of E. cloacae than NDNN and DNN infants, p = .0294, and 0.0001, respectively. The risk of norovirus diarrhea was significantly high in infants with higher counts of E. cloacae than those with lower counts, p = .009. Harboring higher counts of E. cloacae is a risk factor for norovirus diarrhea.

EHEC O111:H8 strain and norovirus GII.4 Sydney [P16] causing an outbreak in a daycare center, Brazil, 2019.

BACKGROUND: This study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized. RESULTS: Two isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing. CONCLUSION: In this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

Brote de gastroenteritis causado por norovirus en un hotel de Menorca, España / Norovirus outbreak causing gastroenteritis in a hotel in Menorca, Spain

OBJETIVO: Determinar el agente responsable de un brote de gastroenteritis ocurrido en un hotel de Menorca en septiembre de 2016. MÉTODOS: Se estudió la epidemiología de los casos y se investigaron muestras ambientales y clínicas para la presencia de microorganismos indicadores y patógenos. RESULTADOS: Se detectaron 151 casos: 123 afectaron a clientes y 28 a personal. Los análisis microbiológicos detectaron la presencia de norovirus genotipo II en heces de pacientes, así como en habitaciones y zonas comunes. El plan de control implementado permitió la erradicación del brote. CONCLUSIONES: Este estudio del brote causado por norovirus del genotipo II demuestra que una rápida actuación es crítica para controlar este tipo de brotes

OBJECTIVES: To establish the agent responsible for a gastroenteritis outbreak in a hotel in Menorca (Spain) in September 2016. METHODS: The study included epidemiological and laboratory analysis. Environmental and stool samples were examined for bacterial and viral pathogens. RESULTS: One hundred and fifty-one cases were detected, 123 among the tourists staying in the hotel and 28 affecting the staff. The presence of genotypeII norovirus was discovered in the microbiological studies of patient's faeces, as well as in the surface samples of rooms and common areas. The control plan implemented allowed for control of the outbreak. CONCLUSIONS: This study on a genotypeII norovirus outbreak reveals the importance of a rapid response for controlling these types of outbreaks

Attach Me If You Can: Murine Norovirus Binds to Commensal Bacteria and Fungi.

The presence of commensal bacteria enhances both acute and persistent infection of murine noroviruses. For several enteric viral pathogens, mechanisms by which these bacteria enhance infection involve direct interactions between the virus and bacteria. While it has been demonstrated that human noroviruses bind to a variety of commensal bacteria, it is not known if this is also true for murine noroviruses. The goal of this study was to characterize interactions between murine noroviruses and commensal bacteria and determine the impact of bacterial growth conditions, incubation temperature and time, on murine norovirus attachment to microbes that comprise the mammalian microbiome. We show that murine noroviruses bind directly to commensal bacteria and show similar patterns of attachment as human norovirus VLPs examined under the same conditions. Furthermore, while binding levels are not impacted by the growth phase of the bacteria, they do change with time and incubation temperature. We also found that murine norovirus can bind to a commensal fungal species, Candidaalbicans.

Contrasting Results from Two Commercial Kits Testing for the Presence of Clostridium perfringens Enterotoxin in Feces from Norovirus-Infected Human Patients.

BACKGROUND: Detection of Clostridium perfringens enterotoxin (CPE) is critical for disease surveillance; however, commercial testing kits produce contrasting results. METHODS: We examined the cause of the differing results from a reversed passive latex agglutination (RPLA) assay (PET-RPLA Toxin Detection Kit) and an enzyme-linked immunosorbent assay (C. perfringens Enterotoxin ELISA Kit) using 73 human norovirus-positive fecal samples from gastroenteritis patients across 22 episodes in Japan. RESULTS: CPE was detected in 39/73 samples using the RPLA method; however, ELISA-based examination of 10 RPLA-positive samples produced negative results. Moreover, cpe was not detected in any of the RPLA-positive (n = 32) or -negative (n = 5) samples, and C. perfringens was only isolated from one RPLA-positive sample. CONCLUSIONS: An ELISA-based testing approach may be more reliable than RPLA assays for CPE detection from human fecal samples. These findings may also be applicable to the detection of other foodborne diseases.

Comparing Mouse Health Monitoring Between Soiled-bedding Sentinel and Exhaust Air Dust Surveillance Programs.

To monitor rodent colony health in research facilities, soiled-bedding sentinel (SBS) animals have traditionally been used. SBS can be tested by various methods, which may include serology, PCR analysis, and necropsy. Several pathogens are unreliably detected by using SBS or transmitted poorly through soiled bedding, and collection and evaluation of SBS samples can be time-intensive. Recently, exhaust air dust (EAD) testing through PCR analysis has emerged as an adjunct or replacement method for rodent colony health monitoring. EAD monitoring may provide a more efficient, sensitive, and humane method for monitoring health status. Using both EAD and SBS health monitoring, we evaluated colony health over the course of 1 y in 3 research barrier rooms in which mice were housed exclusively on IVC racks. Three pathogens-Helicobacter spp., Rodentibacter spp. (previously Pasteurella pneumotropica), and murine norovirus (MNV)-were not excluded in 2 of the rooms, and we expected that these mice would test positive with some regularity. EAD monitoring was significantly more sensitive than SBS for detection of the bacterial agents. SBS failed to detect Helicobacter spp. at time points when EAD had 100% detection in the rooms that did not exclude the bacteria. The detection of MNV did not differ between health monitoring systems at any time point. The findings suggest that EAD is especially valuable in detecting bacteria poorly transmitted through soiled bedding. In addition, the corresponding results with MNV detection suggest that EAD surveillance can reliably be implemented as an alternative to SBS monitoring in a facility in which mice are housed exclusively on IVC racks.

The intestinal regionalization of acute norovirus infection is regulated by the microbiota via bile acid-mediated priming of type III interferon.

Evidence has accumulated to demonstrate that the intestinal microbiota enhances mammalian enteric virus infections1. For example, we and others previously reported that commensal bacteria stimulate acute and persistent murine norovirus infections2-4. However, in apparent contradiction of these results, the virulence of murine norovirus infection was unaffected by antibiotic treatment. This prompted us to perform a detailed investigation of murine norovirus infection in microbially deplete mice, revealing a more complex picture in which commensal bacteria inhibit viral infection of the proximal small intestine while simultaneously stimulating the infection of distal regions of the gut. Thus, commensal bacteria can regulate viral regionalization along the intestinal tract. We further show that the mechanism underlying bacteria-dependent inhibition of norovirus infection in the proximal gut involves bile acid priming of type III interferon. Finally, the regional effects of the microbiota on norovirus infection may result from distinct regional expression profiles of key bile acid receptors that regulate the type III interferon response. Overall, these findings reveal that the biotransformation of host metabolites by the intestinal microbiota directly and regionally impacts infection by a pathogenic enteric virus.

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.

Surveillance for norovirus and enteric bacterial pathogens as etiologies of acute gastroenteritis at U.S. military recruit training centers, 2011-2016.

An estimated 179 million cases of acute gastroenteritis (AGE) occur each year in the U.S. and AGE is commonly reported within both training and deployed U.S. military populations. Beginning in 2011, the Operational Infectious Diseases laboratory at Naval Health Research Center (NHRC) has undertaken routine surveillance of four U.S. military training facilities to systematically track the prevalence of AGE and to establish its etiologies among U.S. military recruits. Employing both molecular and standard microbiological techniques, NHRC routinely assays for pathogens of direct military relevance, including norovirus genogroups I and II, Salmonella, Shigella, and Campylobacter. During its initial surveillance efforts (2011-2016), NHRC identified norovirus as the primary etiology of both sporadic cases and outbreaks of AGE among trainees.

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013-2015.

BACKGROUND: Norovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana. METHODS: A total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real-time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0. RESULTS: The prevalence of norovirus was 9.3% (95% CI 6.7-11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March). CONCLUSION: The study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications.

Waterborne outbreak of gastroenteritis on the KwaZulu-Natal Coast, South Africa, December 2016/January 2017.

An unexpected increase in gastroenteritis cases was reported by healthcare workers on the KwaZulu-Natal Coast, South Africa, January 2017 with >600 cases seen over a 3-week period. A case-control study was conducted to identify the source and risk factors associated with the outbreak so as to recommend control and prevention measures. Record review identified cases and controls and structured-telephonic interviews were conducted to obtain exposure history. Stool specimens were collected from 20 cases along with environmental samples and both screened for enteric pathogens. A total of 126 cases and 62 controls were included in the analysis. The odds of developing gastroenteritis were 6.0 times greater among holiday makers than residents (95% confidence interval (CI) 2.0-17.7). Swimming in the lagoon increased the odds of developing gastroenteritis by 3.3 times (95% CI 1.06-10.38). Lagoon water samples tested positive for norovirus (NoV) GI.6, GII.3 and GII.6, astrovirus and rotavirus. Eleven (55%) stool specimens were positive for NoV with eight genotyped as GI.1 (n = 2), GI.5 (n = 3), GI.6 (n = 2), and GI.7 (n = 1). A reported sewage contamination event impacting the lagoon was the likely source with person-to-person spread perpetuating the outbreak. Restriction to swimming in the lagoon was apparently ineffective at preventing the outbreak, possibly due to inadequate enforcement, communication and signage strategies.

Asymptomatic norovirus infection associated with swimming at a tropical beach: A prospective cohort study.

BACKGROUND: Swimming in fecally-contaminated waterbodies can result in gastrointestinal infections. However, the pathogenic microorganisms responsible are not well understood because sporadic cases of illness are not reported completely, exposure information is often not collected, and epidemiology studies rely on self-reported symptoms. Noroviruses are considered a likely cause because they are found in high densities in sewage, resistant to wastewater treatment and survive in the environment. In this study, saliva samples were collected from subjects at a beach in Puerto Rico and tested for evidence of norovirus-specific IgG responses as an indicator of incident norovirus infection. METHODS: Saliva samples were collected from 1298 participants using an oral swab. Samples were collected on the day of the beach visit (S1); after 10-12 days (S2); and after three weeks (S3). Saliva was tested for IgG responses to GI.1 and GII.4 noroviruses using a microsphere based multiplex salivary immunoassay. Immunoconversion was defined as a four-fold increase in median fluorescence intensity (MFI) from S1 to S2 with the S3 sample at least three times above the S1 MFI. RESULTS: Thirty-four subjects (2.6%) immunoconverted to GI.1 or GII.4 norovirus. Swimmers who immersed their head in water had a higher rate of immunoconversion (3.4%), compared to either non-swimmers (0.0%, p = 0.003) or waders and non-swimmers combined (0.4%, Odds Ratio: 5.07, 95% Confidence Interval:1.48-17.00). Immunoconversion was not associated with gastrointestinal symptoms. CONCLUSIONS: This is the first study to demonstrate an association between swimming at a beach impacted by fecal contamination and asymptomatic norovirus infection. The findings implicate recreational water as potentially important transmission pathway for norovirus infection.

Therapeutic Opportunities in Intestinal Microbiota-Virus Interactions.

The host microbiota has emerged a third player in interactions between hosts and viral pathogens. This opens new possibilities to use different tools to modulate the intestinal microbial composition, aimed at reducing the risk of or treating viral enteric infections.

New perspectives regarding the antiviral effect of vitamin A on norovirus using modulation of gut microbiota.

Gut microbiota has been revealed to play an important role in various health conditions, and recent studies have suggested the modulation of gut microbiota as a therapeutic strategy. There is no effective treatment of norovirus infection, though vitamin A has been suggested to have an antiviral effect in an epidemiological study. We demonstrated that vitamin A significantly inhibited murine norovirus replication. Vitamin A supplementation significantly increased the abundance of Lactobacillus sp. during norovirus infection, which played a crucial role in antiviral efficacy, inhibiting murine norovirus. Therefore, we elaborated the antiviral effect of vitamin A via modulation of gut microbiota. Furthermore, we suggest a novel strategy, using potential probiotics, as having a protective and therapeutic effect on noroviral infection.

Intestinal microbiome in children with severe and complicated acute viral gastroenteritis.

The aim of the present study was to evaluate the microbiota of children with severe or complicated acute viral gastroenteritis (AGE). To that end, next-generation sequencing (NGS) technology was used to sequence the 16S ribosomal RNA (16S rRNA) gene in 20 hospitalized pediatric patients with severe or complicated AGE and a further 20 otherwise healthy children; the fecal microbiome was then assessed. Comparative metagenomics data were analyzed by a Wilcoxon rank-sum test and hierarchical clustering analysis of bacterial reads. The statistical analyses showed a significantly decreased Shannon diversity index (entropy score) of the intestinal microbiota in patients with severe AGE compared with normal controls (P = 0.017) and patients with mild-to-moderate AGE (P = 0.011). The intestinal microbiota score of the 5 patients with rotavirus AGE was significantly lower than that of those with norovirus infection (P = 0.048). Greater richness in Campylobacteraceae (P = 0.0003), Neisseriaceae (P = 0.0115), Methylobacteriaceae (P = 0.0004), Sphingomonadaceae (P = 0.0221), and Enterobacteriaceae (P = 0.0451) was found in patients with complicated AGE compared with normal controls. The data suggest a significant reduction in intestinal microbial diversity in patients with severe AGE, particularly those with rotavirus infection.

Relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans.

Host genetic factors, such as histo-blood group antigens (HBGAs), are associated with susceptibility to norovirus (NoV) and rotavirus (RV) infections. Recent advances point to the gut microbiome as a key player necessary for a viral pathogen to cause infection. In vitro NoV attachment to host cells and resulting infections have been linked to interactions with certain bacterial types in the gut microbiota. We investigated the relationship between host genotype, gut microbiota, and viral infections. Saliva and fecal samples from 35 adult volunteers were analysed for secretor status genotype, the gut microbiota composition by 16S rRNA gene sequencing, and salivary IgA titers to NoV and RV. Higher levels of IgA against NoV and RV were related to secretor-positive status. No significant differences were found between the FUT2 genotype groups, although the multivariate analysis showed a significant impact of host genotype on specific viral susceptibilities in the microbiome composition. A specific link was found between the abundance of certain bacterial groups, such as Faecalibacterium and Ruminococcus spp., and lower IgA titers against NoV and RV. As a conclusion, we can state that there is a link between host genetics, gut microbiota, and susceptibility to viral infections in humans.

[Norovirus infection surveillance in early warning of outbreaks in population in oyster farming areas].

OBJECTIVE: To have an insight into the prevalence tendency caused by Noroviruses( NoVs) in population of oyster farming areas, and evaluate the early warning signification of acute gastroenteritis( AGE) monitoring. METHODS: Between January and December in 2014, a retrospective household survey of AGE during past four weeks was conducted to residents using multi-stage sampling in oyster farming areas. Norovirus capsid genome was detected using real-time RT-PCR and semi-nested PCR, sequenced and phylogenetic analyzed. RESULTS: A total of 75 cases of AGE were reported. The incidence rate was 0. 10 per person-year, the detection rate of norovirus was 20. 0%( 15 /75). The dominant epidemic No Vs strain in oyster farming was still GII. 4 Sydney 2012. Furthermore, a newly GII. 17 variant was detected and early in March 2014. CONCLUSION: The GII. 4 Sydney 2012 genotype is still prevalent strain that caused AGE currently in the community residents of oyster farming areas. NoVs GII. 17 variant was found in sporadic patients of sentinel hospital AGE monitoring in March 2014 that have an early warning effect to the outbreaks of winter in 2014.