Epidemiological and molecular surveillance of norovirus in the Brazilian Amazon: description of recombinant genotypes and improvement of evolutionary analysis.

Noroviruses are highly infectious, genetically diverse viruses. Global outbreaks occur frequently, making molecular surveillance important for infection monitoring. This cross-sectional descriptive study aimed to monitor cases of norovirus gastroenteritis in the Brazilian Amazon. Fecal samples were tested by immunoenzymatic assay, RT-PCR and genetic sequencing for the ORF1/ORF2 and protease regions. Bayesian inference with a molecular clock was employed to construct the phylogeny. The norovirus prevalence was 25.8%, with a higher positivity rate among children aged 0-24 months. Genogroup GII accounted for 98.1% of the sequenced samples, while GI accounted for 1.9% of them. The GII.P16/GII.4 genotype was the most prevalent, with an evolution rate of 2.87x10-3 and TMRCA estimated in 2012. This study demonstrates that norovirus is a primary causative agent of gastroenteritis and provides data on viral genetic diversity that may facilitate infection surveillance and vaccine development.

Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays.

BACKGROUND: The causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank. RESULTS: Among the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays. CONCLUSIONS: The suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

Cost-of-illness of gastroenteritis caused by rotavirus in Chinese children less than 5 years.

Acute gastroenteritis (AGE) caused by rotavirus (RV) remains a public health issue in China. To accelerate the mass rotavirus vaccination, it is important to inform the policy maker, and the public of the economic burden caused by rotavirus infection. A meta-analysis was conducted applying standardized algorithms. Articles published before January 1, 2023, in English and Chinese were searched through PubMed, CNKI, and WanFang Data. Studies with cost analysis of RV AGE were included. A random-effects model was applied to synthesize the total cost of RV AGE from the societal perspective. A prospective survey aimed to measure the cost of RV AGE was conducted in 2021 and 2022 in Shaoxing city, Zhejiang province, that can represent the developed region. The cost data was applied as deviation indicator, in comparison with the pooled estimate generated from meta-analysis. Totally 286 articles were identified, and eventually 12 studies were included. The pooled total social cost of RV AGE was US$282.1 (95%CI: US$213.4-350.7). The pooled private cost of RV AGE was US$206.4 (95%CI: US$155.2-257.5). RV AGE hospitalized and RV AGE incurred in developed regions caused remarkable higher burden (US$631.2 [95%CI: US$512.6-749.8], and US$333.6 [95%CI: US$234.1-433.2] respectively), compared to RV AGE treated at outpatient, and incurred in less developed regions. Our study demonstrates that RV AGE causes a significant economic burden in China. Given the promising effectiveness and highly cost-effective, introduction of rotavirus vaccines in national immunization programs could substantially reduce the economic burden in China.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Molecular epidemiology of rotavirus among children in Western Canada: Dynamic changes in genotype prevalence in four consecutive seasons.

Rotavirus molecular surveillance remains important in the postvaccine era to monitor the changes in transmission patterns, identify vaccine-induced antigenic changes and discover potentially pathogenic vaccine-related strains. The Canadian province of Alberta introduced rotavirus vaccination into its provincial vaccination schedule in June 2015. To evaluate the impact of this program on stool rotavirus positivity rate, strain diversity, and seasonal trends, we analyzed a prospective cohort of children with acute gastroenteritis recruited between December 2014 and August 2018. We identified dynamic changes in rotavirus positivity and genotype trends during pre- and post-rotavirus vaccine introduction periods. Genotypes G9P[8], G1P[8], G2P[4], and G12P[8] predominated consecutively each season with overall lower rotavirus incidence rates in 2016 and 2017. The demographic and clinical features of rotavirus gastroenteritis were comparable among wild-type rotaviruses; however, children with G12P[8] infections were older (p < 0.001). Continued efforts to monitor changes in the molecular epidemiology of rotavirus using whole genome sequence characterization are needed to further understand the impact of the selection pressure of vaccination on rotavirus evolution.

Comparison of IgA Antibody Titer Induced by Human-Bovine Rotavirus Candidate Vaccine with Bovine Rotavirus and Rotarix.

Rotavirus (RV) is the most common cause of acute gastroenteritis in early childhood worldwide. Gastroenteritis is a preventable disease by the vaccine, and vigorous efforts were made to produce attenuated oral rotavirus vaccines. In recent years, despite the existence of three types of live attenuated rotavirus vaccines, several countries, such as China and Vietnam, have intended to produce indigenous vaccines based on rotavirus serotypes circulating among their population. In this study, the immunogenicity of homemade human-bovine reassortant RV candidate vaccine was tested in an animal model. Rabbits were randomly distributed into eight experimental groups with three animals per group. Afterward, three rabbits in each test group designated as P1, P2, and P3 were experimentally inoculated with the 106, 107, and 108 tissue culture infectious dose 50 (TCID50) of the reassortant virus, respectively. The N1 group received the reassortant rotavirus vaccine containing 107 TCID50+zinc. The N2, N3, and N4 groups received rotavirus vaccine strain, RV4 human rotavirus, and bovine rotavirus strain, respectively, and the control group received phosphate-buffered saline. It is noteworthy that three rabbits have been included in each group. The IgA total antibody titer was measured and evaluated by non-parametric Mann-Whitney and Kruskal-Wallis tests. The antibody titer produced in the studied groups did not significantly differ. The candidate vaccine showed immunogenicity, protectivity, stability, and safety. The findings of this study indicated a critical role of IgA production, which can induce immunity against a gastroenteritis viral pathogen. Regardless of purification, candidate reassortant vaccine and cell adapted animal strains could be used as a vaccine candidate for production.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Minimal Antigenic Evolution after a Decade of Norovirus GII.4 Sydney_2012 Circulation in Humans.

Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably, ~52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include >1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of cross-protective vaccines to human noroviruses.

N-Glycosylation of Rotavirus NSP4 Protein Affects Viral Replication and Pathogenesis.

Rotavirus (RV), the most common cause of gastroenteritis in children, carries a high economic and health burden worldwide. RV encodes six structural proteins and six nonstructural proteins (NSPs) that play different roles in viral replication. NSP4, a multifunctional protein involved in various viral replication processes, has two conserved N-glycosylation sites; however, the role of glycans remains elusive. Here, we used recombinant viruses generated by a reverse genetics system to determine the role of NSP4 N-glycosylation during viral replication and pathogenesis. The growth rate of recombinant viruses that lost one glycosylation site was as high as that of the wild-type virus. However, a recombinant virus that lost both glycosylation sites (glycosylation-defective virus) showed attenuated replication in cultured cell lines. Specifically, replications of glycosylation-defective virus in MA104 and HT29 cells were 10- and 100,000-fold lower, respectively, than that of the wild-type, suggesting that N-glycosylation of NSP4 plays a critical role in RV replication. The glycosylation-defective virus showed NSP4 mislocalization, delay of cytosolic Ca2+ elevation, and less viroplasm formation in MA104 cells; however, these impairments were not observed in HT29 cells. Further analysis revealed that assembly of glycosylation-defective virus was severely impaired in HT29 cells but not in MA104 cells, suggesting that RV replication mechanism is highly cell type dependent. In vivo mouse experiments also showed that the glycosylation-defective virus was less pathogenic than the wild-type virus. Taken together, the data suggest that N-glycosylation of NSP4 plays a vital role in viral replication and pathogenicity. IMPORTANCE Rotavirus is the main cause of gastroenteritis in young children and infants worldwide, contributing to 128,500 deaths each year. Here, we used a reverse genetics approach to examine the role of NSP4 N-glycosylation. An N-glycosylation-defective virus showed attenuated and cell-type-dependent replication in vitro. In addition, mice infected with the N-glycosylation-defective virus had less severe diarrhea than mice infected with the wild type. These results suggest that N-glycosylation affects viral replication and pathogenesis. Considering the reduced pathogenicity in vivo and the high propagation rate in MA104 cells, this glycosylation-defective virus could be an ideal live attenuated vaccine candidate.

Incidence and risk factors for norovirus-related diarrhea in Japanese geriatric intermediate care facilities: A prospective cohort study.

AIM: The risk of developing infectious diarrhea among elderly residents at Japanese geriatric intermediate care facilities is unclear. We investigated the incidence rate and risk factors of norovirus-related diarrhea at such facilities. METHODS: This prospective cohort study followed 1727 residents from November 2018 to April 2020 at 10 geriatric intermediate care facilities in Osaka, Japan regarding the occurrence of diarrhea. Resident data were collected from their medical records using structured forms at two to three of the following three time points: at recruitment, if they developed diarrhea, and when they left the facility. Residents who developed diarrhea were tested using rapid diagnostic tests for norovirus. Cox proportional hazard model was employed to hazard ratios (HRs) with 95% confidence intervals (CIs) to estimate the risk factors for norovirus-related diarrhea. RESULTS: During the study period, 74 residents developed diarrhea, 13 of whom were norovirus positive. The incidence rate of norovirus-related diarrhea was 10.11 per 1000 person-years (95% CI: 4.61-15.61). In terms of risk factors, people with care-needs level 3 were at a higher risk for developing norovirus-related diarrhea (adjusted HR [aHR] = 7.35, 95% CI: 1.45-37.30). Residents with hypertension (aHR = 3.41, 95% CI: 1.05-11.04) or stroke (aHR = 8.84, 95% CI: 2.46-31.83), and those who walked with canes (aHR = 16.68, 95% CI: 1.35-206.52) also had a significantly higher risk for norovirus-related diarrhea. CONCLUSIONS: Throughout the study period, the incidence of development of diarrhea was low. Care-needs level 3, stroke, hypertension and use of a cane were identified as risk factors for norovirus-related diarrhea in Japanese geriatric intermediate care facilities. Geriatr Gerontol Int 2023; 23: 179-187.

Advanced simulations and screening to repurposing a 3C protease inhibitor against the rupintrivir-resistant human norovirus-induced gastroenteritis.

Human norovirus (HuNoV) causes acute viral gastroenteritis in all age groups, and dehydration and severe diarrhea in the elderly. The World Health Organization reports ∼1.45 million deaths from acute gastroenteritis annually in the world. Rupintrivir, an inhibitory medicine against the human rhinovirus C3 protease, has been reported to inhibit HuNoV 3C protease. However, several HuNoV 3C protease mutations have been revealed to reduce the susceptibility of HuNoV to rupintrivir. The structural details behind rupintrivir-resistance of these single-point mutations (A105V and I109V) are not still clear. Hence, in this study, a combination of computational techniques were used to determine the rupintrivir-resistance mechanism and to propose an inhibitor against wild-type and mutant HuNoV 3C protease through structure-based virtual screening. Dynamic structural results indicated the unstable binding of rupintrivir at the cleft binding site of the wild-type and mutant 3C proteases, leading to its detachment. Our findings presented that the domain II of the HuNoV 3C protease had a critical role in binding of inhibitory molecules. Binding energy computations, steered molecular dynamics and umbrella sampling simulations confirmed that amentoflavone, the novel suggested inhibitor, strongly binds to the cleft site of all protease models and has a good structural stability in the complex system along the molecular dynamic simulations. Our in silico study proposed the selected compound as a potential inhibitor against the HuNoV 3C protease. However, additional experimental and clinical studies are required to corroborate the therapeutic efficacy of the compound.

Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins.

Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, is a primary cause of acute gastroenteritis in young children. In countries where rotavirus vaccines are widely used, norovirus (NoV) has emerged as the major cause of acute gastroenteritis. Towards the goal of creating a combined rotavirus-NoV vaccine, we explored the possibility of generating recombinant rotaviruses (rRVs) expressing all or portions of the NoV GII.4 VP1 capsid protein. This was accomplished by replacing the segment 7 NSP3 open reading frame with a cassette encoding, sequentially, NSP3, a 2A stop-restart translation element, and all or portions (P, P2) of NoV VP1. In addition to successfully recovering rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the segment 7 RNA of the RIX4414 vaccine strain. An immunoblot assay confirmed that rRVs expressed NoV capsid proteins as independent products. Moreover, VP1 expressed by rRVs underwent dimerization and was recognized by conformational-dependent anti-VP1 antibodies. Serially passaged rRVs that expressed the NoV P and P2 were genetically stable, retaining additional sequences of up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.6-kb VP1 sequence were less stable and gave rise to virus populations with segment 7 RNAs lacking VP1 coding sequences. Together, these studies suggest that it may be possible to develop combined rotavirus-NoV vaccines using modified segment 7 RNA to express NoV P or P2. In contrast, development of potential rotavirus-NoV vaccines expressing NoV VP1 will need additional efforts to improve genetic stability. IMPORTANCE Rotavirus (RV) and norovirus (NoV) are the two most important causes of acute viral gastroenteritis (AGE) in infants and young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of universal mass vaccination with live attenuated RV vaccines, similar highly effective NoV vaccines are not available. To pursue the development of a combined RV-NoV vaccine, we examined the potential of using RV as an expression vector of all or portions of the NoV capsid protein VP1. Our results showed that by replacing the NSP3 open reading frame in RV genome segment 7 RNA with a coding cassette for NSP3, a 2A stop-restart translation element, and VP1, recombinant RVs can be generated that express NoV capsid proteins. These findings raise the possibility of developing new generations of RV-based combination vaccines that provide protection against a second enteric pathogen, such as NoV.

Type III and Not Type I Interferons Efficiently Prevent the Spread of Rotavirus in Human Intestinal Epithelial Cells.

Rotavirus infects intestinal epithelial cells and is the leading cause of gastroenteritis in infants worldwide. Upon viral infection, intestinal cells produce type I and type III interferons (IFNs) to alert the tissue and promote an antiviral state. These two types of IFN bind to different receptors but induce similar pathways that stimulate the activation of interferon-stimulated genes (ISGs) to combat viral infection. In this work, we studied the spread of a fluorescent wild-type (WT) SA11 rotavirus in human colorectal cancer cells lacking specific interferon receptors and compared it to that of an NSP1 mutant rotavirus that cannot interfere with the host intrinsic innate immune response. We could show that the WT rotavirus efficiently blocks the production of type I IFNs but that type III IFNs are still produced, whereas the NSP1 mutant rotavirus allows the production of both. Interestingly, while both exogenously added type I and type III IFNs could efficiently protect cells against rotavirus infection, endogenous type III IFNs were found to be key to limit infection of human intestinal cells by rotavirus. By using a fluorescent reporter cell line to highlight the cells mounting an antiviral program, we could show that paracrine signaling driven by type III IFNs efficiently controls the spread of both WT and NSP1 mutant rotavirus. Our results strongly suggest that NSP1 efficiently blocks the type I IFN-mediated antiviral response; however, its restriction of the type III IFN-mediated one is not sufficient to prevent type III IFNs from partially inhibiting viral spread in intestinal epithelial cells. Additionally, our findings further highlight the importance of type III IFNs in controlling rotavirus infection, which could be exploited as antiviral therapeutic measures. IMPORTANCE Rotavirus is one of the most common causes of gastroenteritis worldwide. In developing countries, rotavirus infections lead to more than 200,000 deaths in infants and children. The intestinal epithelial cells lining the gastrointestinal tract combat rotavirus infection by two key antiviral compounds known as type I and III interferons. However, rotavirus has developed countermeasures to block the antiviral actions of the interferons. In this work, we evaluated the arms race between rotavirus and type I and III interferons. We determined that although rotavirus could block the induction of type I interferons, it was unable to block type III interferons. The ability of infected cells to produce and release type III interferons leads to the protection of the noninfected neighboring cells and the clearance of rotavirus infection from the epithelium. This suggests that type III interferons are key antiviral agents and could be used to help control rotavirus infections in children.

Profiling of humoral immune responses to norovirus in children across Europe.

Norovirus is a leading cause of epidemic acute gastroenteritis. More than 30 genotypes circulate in humans, some are common, and others are only sporadically detected. Here, we investigated whether serology can be used to determine which genotypes infect children. We established a multiplex protein microarray with structural and non-structural norovirus antigens that allowed simultaneous antibody testing against 30 human GI and GII genotypes. Antibody responses of sera obtained from 287 children aged < 1 month to 5.5 years were profiled. Most specific IgG and IgA responses were directed against the GII.2, GII.3, GII.4, and GII.6 capsid genotypes. While we detected antibody responses against rare genotypes, we found no evidence for wide circulation. We also detected genotype-specific antibodies against the non-structural proteins p48 and p22 in sera of older children. In this study, we show the age-dependent antibody responses to a broad range of norovirus capsid and polymerase genotypes, which will aid in the development of vaccines.

First Episodes of Norovirus and Sapovirus Gastroenteritis Protect Against Subsequent Episodes in a Nicaraguan Birth Cohort.

BACKGROUND: Norovirus and sapovirus cause a large burden of acute gastroenteritis (AGE) in young children. We assessed protection conferred by norovirus and sapovirus AGE episodes against future episodes. METHODS: Between June 2017 and July 2018, we recruited 444 newborns in León, Nicaragua. Weekly household surveys identified AGE episodes over 36 months, and AGE stools were tested by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) for norovirus genogroup (G)I/GII and sapovirus. We used recurrent-event Cox models and negative control methods to estimate protection conferred by first episodes, controlling for observed and unobserved risk factors, respectively. RESULTS: Sapovirus episodes conferred a 69% reduced hazard of subsequent episodes using the negative control method. Norovirus GI (hazard ratio [HR] = 0.67; 95% confidence interval [CI] = 0.31, 1.3) and GII (HR = 0.20; 95% CI = 0.04, 0.44) episodes also appeared highly protective. Protection against norovirus GII was enhanced following two episodes. CONCLUSIONS: Evidence of natural immunity in early childhood provides optimism for the future success of pediatric norovirus and sapovirus vaccines.

The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

A shift in circulating rotaviral genotypes among hospitalized neonates.

In neonates, rotavirus (RV) infection is generally nosocomial. The control of rotaviral infection within hospital settings is challenging due to prolonged shedding of the virus and contamination of the surrounding environment. There are few studies that have reported asymptomatic infection within neonates. In this study, neonates were screened for RV infection and possible clinical manifestations that may play a role in RV acquisition were analysed. Stool samples were collected from 523 hospitalized neonates admitted for > 48 h in a low-cost and higher-cost tertiary centre. RV antigen was screened using ELISA and the samples which tested positive were confirmed by semi-nested RT-PCR. RV was detected in 34% of participants and genotypes identified included G12P[11] (44.4%), G10 P[11] (42.6%), G10G12P[11] (10.1%) and G3P[8] (2.9%). ICU admissions were associated with higher viral shedding (p < 0.05). Hospitalization in the low-cost facility ICU was associated with higher RV acquisition risk (p < 0.05). RV was detected in higher rates (36.9%) among neonates with gastrointestinal manifestations. G10P[11] was the predominant genotype for several years (1988-2016) among neonates within India. The preponderance of an emerging G12P[11] genotype and heterotypic distribution was documented. RV surveillance is important to identify emerging strains and establish the road ahead in managing RV infection.

Molecular Epidemiology of Norovirus (NoV) Infection in Mie Prefecture: The Kinetics of Norovirus Antigenemia in Pediatric Patients.

Few studies have shown the presence of norovirus (NoV) RNA in blood circulation but there is no data on norovirus antigenemia. We examined both antigenemia and RNAemia from the sera of children with NoV infections and studied whether norovirus antigenemia is correlated with the levels of norovirus-specific antibodies and clinical severity of gastroenteritis. Both stool and serum samples were collected from 63 children admitted to Mie National Hospital with acute NoV gastroenteritis. Norovirus antigen and RNA were detected in sera by ELISA and real-time RT-PCR, respectively. NoV antigenemia was found in 54.8% (34/62) and RNAemia in 14.3% (9/63) of sera samples. Antigenemia was more common in the younger age group (0-2 years) than in the older age groups, and most patients were male. There was no correlation between stool viral load and norovirus antigen (NoV-Ag) levels (rs = -0.063; Cl -0.3150 to 0.1967; p = 0.6251). Higher levels of acute norovirus-specific IgG serum antibodies resulted in a lower antigenemia OD value (n = 61; r = -0.4258; CI -0.62 to -0.19; p = 0.0006). Norovirus antigenemia occurred more commonly in children under 2 years of age with NoV-associated acute gastroenteritis. The occurrence of antigenemia was not correlated with stool viral load or disease severity.

Interaction with mammalian enteric viruses alters outer membrane vesicle production and content by commensal bacteria.

Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome-wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus-infected mice compared to mock-infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection.