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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-1β maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1β and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1β, facilitating elevated Nlrp3-dependent release of mature IL-1β upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1β 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. [ABSTRACT FROM AUTHOR]

Noroviruses (NoVs) are the leading cause of acute viral gastroenteritis outbreaks. From June 2015 to March 2017, fifteen outbreaks of acute gastroenteritis (AGE) were reported to the Jinan Center for Disease Control and Prevention in China. To identify the circulating NoV genotypes associated with outbreaks in Jinan, China, 414 specimens from the 15 outbreaks were collected and analyzed for the causative viruses, and phylogenetic analysis was performed on the NoV-positive strains. The NoV detection rate was 57.5% (238/414), and a total of 14 outbreaks were caused by NoVs (eight by infection with genogroup II (GII), five by mixed infection with GI and GII, and one by mixed infection with GII and rotavirus (RoV)-A). A total of 75 NoV sequences were obtained from 13 NoV-positive outbreaks and classified into seven genotypes (38 GII.17, 13 GII.2, 4 GII.3, 4 GII.1, 10 GI.6, 5 GI.5 and 1 GI.3), while GII.4 was not identified. The most prevalent genotype changed yearly during the 2015–2017 period. Phylogenetic analysis demonstrated that these NoV genotypes had high homology with the strains circulating worldwide, especially strains from Asian countries and cities. Our study illustrated that multiple non-GII.4 NoV genotypes were prevalent in outbreaks of AGE in Jinan, China. Year-round surveillance of multiple NoV genotypes could help health authorities reduce the impact of NoV outbreaks on public health. [ABSTRACT FROM AUTHOR]

The article discusses the interactions between the commensal microbiota of the intestine and norovirus and their implications. Topics include dependance of all strains of norovirus on the commensal microbiota for infection; role of commensal bacteria in regulating enteric virus infections through immune skewing; and therapeutic role of microbiome for infectious diseases in humans.

Cyclooxygenases (COXs)/prostaglandin E2 (PGE2) signaling pathways are known to modulate a variety of homeostatic processes and are involved in various pathophysiological conditions. COXs/PGE2 signaling pathways have also been demonstrated to have proviral or antiviral effects, which appeared different even in the same virus family. A porcine sapovirus Cowden strain, a member of genus Sapovirus within the Caliciviridae family, induces strong COX-2/PGE2 but transient COX-1/PGE2 signaling to enhance virus replication. However, whether infections of other viruses in the different genera activate COXs/PGE2 signaling, and thus affect the replication of viruses, remains unknown. In the present study, infections of cells with the feline calicivirus (FCV) F9 strain in the genus Vesivirus and murine norovirus (MNV) CW-1 strain in the genus Norovirus only activated the COX-2/PGE2 signaling in a time-dependent manner. Treatment with pharmacological inhibitors or transfection of small interfering RNAs (siRNAs) against COX-2 enzyme significantly reduced the production of PGE2 as well as FCV and MNV replications. The inhibitory effects of these pharmacological inhibitors against COX-2 enzyme on the replication of both viruses were restored by the addition of PGE2. Silencing of COX-1 via siRNAs and inhibition of COX-1 via an inhibitor also decrease the production of PGE2 and replication of both viruses, which can be attributed to the inhibition COX-1/PGE2 signaling pathway. These data indicate that the COX-2/PGE2 signaling pathway has proviral effects for the replication of FCV and MNV, and pharmacological inhibitors against these enzymes serve as potential therapeutic candidates for treating FCV and MNV infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study was addressed to the relationship between norovirus and acute diarrhea in hospitalized children, including hospital-acquired infection (HAI) and community-acquired infection (CAI) in a children's hospital in Beijing. RT-PCR was used to detect norovirus in stool specimen, followed by sequence analysis for PCR products. From 2010 to 2013, a total of 1248 specimens, including 661 from the HAI group and 587 from the CAI group were tested for norovirus. Norovirus were detected in 380 of 1248 (30.4%) diarrheal specimens. The positive rate for norovirus detection was higher in children within HAI group than CAI group (35.3%, 232/661 vs. 25.6%, 148/587), and the difference was significant (X2 = 14.35, P<0.05). For age distribution, the highest positivity rates of norovirus were in age of 0–5 months for HAI group and 12–23 months for CAI group. In the study, 262 amplicons of the VP1 region from norovirus-positive specimens were sequenced, which showed GII.3 and GII.4 norovirus were the most common genotypes detected in 50.0% (n = 131) and 48.9% (n = 128) of the positive specimens, respectively. Regarding the wards distribution, GII.3 norovirus was mainly detected in ward for neonatal diseases (36/85 in HAI group; 19/46 in CAI group), GII.4 norovirus was mainly detected in ward for respiratory and digestive diseases (21/85 in HAI group; 15/33 in CAI group). Conclusion: The data elaborated the importance of norovirus in hospital associated infectious diarrhea. The prevalence of norovirus is higher from HAI group than CAI group, and the norovirus from the patients in CAI group could be the source of infection in HAI group. [ABSTRACT FROM AUTHOR]

In Japan, the fraction of norovirus outbreaks attributable to human-to-human transmission has increased with time, and the timing of the increased fraction has coincided with the increase in the observed fraction of genogroup II genotype 4 (GII.4). The present study aimed to estimate the time-dependent changes in the transmissibility of noroviruses. The effective reproduction number (Ry), for year y, was estimated by analyzing the time series surveillance data for outbreak events from 2000 to 2016. Ry was estimated by using the fraction of outbreak events that were attributable to human-to-human transmission and by employing three different statistical models that are considered to mechanistically capture the possible data-generating process in different ways. The Ry estimates ranged from 0.14 to 4.15 in value, revealing an overall increasing trend (p<0.05 for all three models). The proportion of outbreaks caused by GII and GII.4 viruses among the total events also increased with time, and positive correlations were identified between transmissibility and these proportions. Parametric modeling of Ry indicated that the time-dependent patterns of Ry were better described by a step function plus linear trend rather than the step function alone that reflects the widespread use of reverse transcriptase PCR (RT-PCR) in and after 2007 for laboratory diagnosis. Accordingly, we conclude that norovirus transmissibility has increased over the past 16 years in Japan. The change is at least partially explained by the time-dependent domination of the contagious GII genogroup (e.g., GII.4), indicating that noroviruses better fitted to humans have selectively caused the human-to-human transmissions, thereby altering the epidemiology of this pathogen. [ABSTRACT FROM AUTHOR]

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining “static”) and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification (“static” versus “evolving”), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as “immunotypes”. We propose that “immunotypes” and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population. [ABSTRACT FROM AUTHOR]

Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics. [ABSTRACT FROM AUTHOR]

The extremely pathogenic Rabbit haemorrhagic disease virus (RHDV) and the completely benign Rabbit calicivirus (RCV) are closely related members of the genus Lagovirus (family Caliciviridae). The molecular mechanisms that determine the dramatic difference in virulence are unknown, but indirect evidence suggests that different properties of their RNA-dependent RNA polymerases (RdRps) may at least partially be responsible for the contrasting phenotypes. Here we report that the unusual ability of the RHDV RdRp to induce a striking rearrangement of the Golgi network is not specific to RHDV, but a common feature of virulent and benign rabbit caliciviruses alike. Expression of rabbit calicivirus RdRps induced a redistribution of both cis/medial and medial/trans Golgi membrane markers, but not that of an endoplasmic reticulum membrane marker. Inactivating mutations in the conserved GDD motif did not abolish the ability of RHDV RdRp to rearrange the Golgi network, suggesting that polymerase activity and metal co-factors are not required for this function. Finally, we discuss possible implications of RdRp-induced membrane rearrangements on virus replication and host immune responses. [ABSTRACT FROM AUTHOR]

The genus Sapovirus, in the family Caliciviridae, includes enteric viruses of humans and domestic animals. Information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in Africa. By screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the Serengeti ecosystem, East Africa, sapovirus RNA was detected in the spotted hyena (Crocuta crocuta, family Hyaenidae), African lion (Panthera leo, family Felidae), and bat-eared fox (Otocyon megalotis, family Canidae), but not in golden or silver-backed jackals (Canis aureus and C. mesomelas, respectively, family Canidae). A phylogenetic analysis based on partial RNA-dependent RNA polymerase (RdRp) gene sequences placed the sapovirus strains from African carnivores in a monophyletic group. Within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and African lion a second sub-group. The percentage nucleotide similarity between sapoviruses from African carnivores and those from other species was low (< 70.4%). Long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. The likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed. [ABSTRACT FROM AUTHOR]

Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-β production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-β release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response. [ABSTRACT FROM AUTHOR]

Background: Noroviruses are an important cause of gastroenteritis but little is known about disease and re-infection rates in community settings in Asia. Methods: Disease, re-infection rates, strain prevalence and genetic susceptibility to noroviruses were investigated in a birth cohort of 373 Indian children followed up for three years. Stool samples from 1856 diarrheal episodes and 147 vomiting only episodes were screened for norovirus by RT-PCR. Norovirus positivity was correlated with clinical data, secretor status and ABO blood group. Results: Of 1856 diarrheal episodes, 207 (11.2%) were associated with norovirus, of which 49(2.6%) were norovirus GI, 150(8.1%) norovirus GII, and 8 (0.4%) were mixed infections with both norovirus GI and GII. Of the 147 vomiting only episodes, 30 (20.4%) were positive for norovirus in stool, of which 7 (4.8%) were norovirus GI and 23 (15.6%) GII. At least a third of the children developed norovirus associated diarrhea, with the first episode at a median age of 5 and 8 months for norovirus GI and GII, respectively. Norovirus GI.3 and GII.4 were the predominant genotypes (40.3% and 53.0%) with strain diversity and change in the predominant sub-cluster over time observed among GII viruses. A second episode of norovirus gastroenteritis was documented in 44/174 (25.3%) ever-infected children. Children with the G428A homozygous mutation for inactivation of the FUT2 enzyme (se428se428) were at a significantly lower risk (48/190) of infection with norovirus (p = 0.01). Conclusions: This is the first report of norovirus documenting disease, re-infection and genetic susceptibility in an Asian birth cohort. The high incidence and apparent lack of genogroupII specific immunity indicate the need for careful studies on further characterization of strains, asymptomatic infection and shedding and immune response to further our understanding of norovirus infection and disease. [ABSTRACT FROM AUTHOR]

Background: The epidemiology of cases of acute gastroenteritis (AGE) of viral etiology is a relevant public health issue. Due to underreporting, the study of outbreaks is an accepted approach to investigate their epidemiology. The objective of this study was to investigate the epidemiological characteristics of AGE outbreaks due to norovirus (NoV) and sapovirus (SV) in Catalonia. Material and Methods: Prospective study of AGE outbreaks of possible viral etiology notified during two years in Catalonia. NoV and SV were detected by real time reverse transcription polymerase (RT-PCR). Results: A total of 101 outbreaks were registered affecting a total of 2756 persons and 12 hospitalizations (hospitalization rate: 0.8x1,000,000 persons-year); 49.5% of outbreaks were foodborne, 45.5% person to person and 5% waterborne. The distribution of outbreaks according to the setting showed a predominance of catering services (39.6%), nursing homes and long term care facilities (26.8%) and schools (11.9%). The median number of cases per outbreak was 17 (range 2–191). The total Incidence rate (IR) was 18.3 per 100,000 persons-years (95%CI: 17.6–19.0). The highest IR was in persons aged ≥65 years (43.6x100,000 (95% CI: 41.0–46.2)) (p<0.001). A total of 1065 samples were analyzed with a positivity rate of 60.8%. 98% of positive samples were NoV (GII 56.3%; GI 4.2%; GII+GI 4.2%; non- typable 33.0%). SV was identified in two person-to-person transmission outbreaks in children. Conclusions: These results confirm the relevance of viral AGE outbreaks, both foodborne and person-to-person, especially in institutionalized persons. SV should be taken into account when investigating viral AGE outbreaks. [ABSTRACT FROM AUTHOR]

Background: Norovirus is a leading cause of gastroenteritis episodes and outbreaks in US military deployments, but estimates of endemic disease burden among military personnel in garrison are lacking. Methods: Diagnostic codes from gastroenteritis-associated medical encounters of active duty military personnel and their beneficiaries from July 1998–June 2011 were obtained from the Armed Forces Health Surveillance Center. Using time-series regression models, cause-unspecified encounters were modeled as a function of encounters for specific enteropathogens. Model residuals (representing unexplained encounters) were used to estimate norovirus-attributable medical encounters. Incidence rates were calculated using population data for both active duty and beneficiary populations. Results: The estimated annual mean rate of norovirus-associated medically-attended visits among active duty personnel and their beneficiaries was 292 (95% CI: 258 to 326) and 93 (95% CI: 80 to 105) encounters per 10,000 persons, respectively. Rates were highest among beneficiaries <5 years of age with a median annual rate of 435 (range: 318 to 646) encounters per 10,000 children. Norovirus was estimated to cause 31% and 27% of all-cause gastroenteritis encounters in the active duty and beneficiary populations, respectively, with over 60% occurring between November and April. There was no evidence of any lag effect where norovirus disease occurred in one population before the other, or in one beneficiary age group before the others. Conclusions: Norovirus is a major cause of medically-attended gastroenteritis among non-deployed US military active duty members as well as in their beneficiaries. [ABSTRACT FROM AUTHOR]

Background: In the US, noroviruses are estimated to cause 21 million cases annually with economic losses reaching $2 billion. Outbreak investigations frequently implicate vomiting as a major transmission risk. However, little is known about the characteristics of vomiting as a symptom or the amount of virus present in emesis. Methodology and Principal Findings: Emesis samples and symptomology data were obtained from previous norovirus human challenge studies with GI.1 Norwalk virus, GII.2 Snow Mountain virus, and a pilot study with GII.1 Hawaii virus. Viral titers in emesis were determined using strain-specific quantitative RT-PCR. In all four studies, vomiting was common with 40–100% of infected subjects vomiting at least once. However, only 45% of subjects with vomiting also had diarrhea. Most of the emesis samples had detectable virus and the mean viral titers were 8.0 x 105 and 3.9 x 104 genomic equivalent copies (GEC)/ml for GI and GII viruses, respectively (p = 0.02). Sample pH was correlated with GII.2 Snow Mountain virus detection. Conclusions and Significance: Half of all subjects with symptomatic infection experienced vomiting and the average subject shed 1.7 x 108 GEC in emesis. Unlike shedding through stool, vomiting is more likely to result in significant environmental contamination, leading to transmission through fomites and airborne droplets. This quantitative data will be critical for risk assessment studies to further understand norovirus transmission and develop effective control measures. The correlation between sample pH and virus detection is consistent with a single site of virus replication in the small intestine and stomach contents becoming contaminated by intestinal reflux. Additionally, the frequency of vomiting without concurrent diarrhea suggests that epidemiology studies that enroll subjects based on the presence of diarrhea may be significantly underestimating the true burden of norovirus disease. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines the resistance and susceptibility of humans to norovirus infection, which is recognized as the leading cause of gastroenteritis in children and adults. Topics include the intermediary factors that affect susceptibility including secretor status affecting gut microbiota composition, the interaction of noroviruses with secretor status and the diverse secretor and non-secretor human histo-blood group antigens that increased susceptibility.

Background: Infectious disease burden estimates provided by a composite health measure give a balanced view of the true impact of a disease on a population, allowing the relative impact of diseases that differ in severity and mortality to be monitored over time. This article presents the first national disease burden estimates for a comprehensive set of 32 infectious diseases in the Netherlands. Methods and Findings: The average annual disease burden was computed for the period 2007–2011 for selected infectious diseases in the Netherlands using the disability-adjusted life years (DALY) measure. The pathogen- and incidence-based approach was adopted to quantify the burden due to both morbidity and premature mortality associated with all short and long-term consequences of infection. Natural history models, disease progression probabilities, disability weights, and other parameters were adapted from previous research. Annual incidence was obtained from statutory notification and other surveillance systems, which was corrected for under-ascertainment and under-reporting. The highest average annual disease burden was estimated for invasive pneumococcal disease (9444 DALYs/year; 95% uncertainty interval [UI]: 8911–9961) and influenza (8670 DALYs/year; 95% UI: 8468–8874), which represents 16% and 15% of the total burden of all 32 diseases, respectively. The remaining 30 diseases ranked by number of DALYs/year from high to low were: HIV infection, legionellosis, toxoplasmosis, chlamydia, campylobacteriosis, pertussis, tuberculosis, hepatitis C infection, Q fever, norovirus infection, salmonellosis, gonorrhoea, invasive meningococcal disease, hepatitis B infection, invasive Haemophilus influenzae infection, shigellosis, listeriosis, giardiasis, hepatitis A infection, infection with STEC O157, measles, cryptosporidiosis, syphilis, rabies, variant Creutzfeldt-Jakob disease, tetanus, mumps, rubella, diphtheria, and poliomyelitis. The very low burden for the latter five diseases can be attributed to the National Immunisation Programme. The average disease burden per individual varied from 0.2 (95% UI: 0.1–0.4) DALYs per 100 infections for giardiasis, to 5081 and 3581 (95% UI: 3540–3611) DALYs per 100 infections for rabies and variant Creutzfeldt-Jakob disease, respectively. Conclusions: For guiding and supporting public health policy decisions regarding the prioritisation of interventions and preventive measures, estimates of disease burden and the comparison of burden between diseases can be informative. Although the collection of disease-specific parameters and estimation of incidence is a process subject to continuous improvement, the current study established a baseline for assessing the impact of future public health initiatives. [ABSTRACT FROM AUTHOR]

We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis. [ABSTRACT FROM AUTHOR]

Blood group-related glycans determining ABO and Lewis blood groups are known to function as attachment factors for most of the norovirus (NoV) strains. To identify binding specificity of each NoV, recombinant norovirus-like particles (VLPs) and human saliva samples with different ABO, Lewis phenotypes and secretor status have been commonly applied. When binding specificities of VLPs prepared from 16 different genotypes of NoVs in GI and GII genogroups were characterized in samples of human gastric mucosa compared to human saliva based on blood group phenotypes, considerable differences were observed for several strains. Novel binding specificities determined by an ELISA using preparations from human gastric mucosa were also ascertained by immunohistochemical analyses using human jejunal mucosa, widely believed to be susceptible to NoV infection. Further, A, B and O(H) blood group substances prepared from porcine and squid tissues were found to be effective for preventing ABO blood group-specific binding of VLPs to both saliva and mucosa samples. Therefore, these blood group substances might have potential for the prevention and treatment of NoV infection. [ABSTRACT FROM AUTHOR]

Norovirus is the commonest cause of acute gastrointestinal disease and is the main aetiological agent of outbreaks of gastroenteritis, particularly in semi-closed environments. Norovirus infections in England typically peak between December and March each year. The most commonly detected norovirus strains belong to the genetically diverse genogroup-II genotype-4 (GII-4) genocluster and in the previous two norovirus winter seasons the majority of GII-4 strains in circulation worldwide have been genetically similar to the GII-4 strain New Orleans 1805/2009/USA. At the beginning of the 2012/13 season a genetically distinct GII-4 strain (Sydney 2012/NSW0514/2012/AU) was described which emerged worldwide during the winter of 2012/13. Here we describe the emergence of norovirus strains genetically related to Sydney2012 in England during the 2012/13 season to replace NewOrleans2009 strains as the most commonly detected variant of GII-4 norovirus in England. Furthermore, we demonstrate that whilst the emergence of Sydney2012 coincided with an early peak in the number of norovirus outbreaks, there was not an overall increase in norovirus activity compared to the previous season. Finally, we show that the Sydney2012 strain is associated with distinct genetic changes compared to the NewOrleans2009 strain, and these changes may have contributed to the emergence of the Sydney2012 strain. [ABSTRACT FROM AUTHOR]

Background: Limited information is available on the molecular epidemiology of GII.4 Sydney-associated diarrhea in China in the winter of 2012–13 during the global epidemic associated with the emergence of GII.4 Sydney. Methods: Fecal specimens collected from 171 diarrhea outpatients (one from each) between late October 2012 and the middle of March 2013 were examined for NoV by reverse transcription-polymerase chain reaction and sequences corresponding to both the NoV partial polymerase and partial capsid regions were analyzed phylogenetically. Clinical characteristics of GII.4 Sydney cases versus other NoV-positive cases detected in a previous study were compared statistically. Results: Twenty-six (15.2%, 26/171) outpatients with diarrhea were infected with NoV. Twenty-two of the 26 (84.6%) identified NoV strains clustered into GII.4 Sydney. There was a significant difference in symptoms of fever (χ2, P<0.05 ), abdominal pain (χ2, P<0.05 ) and diarrhea frequency (Mann-Whitney U test, P<0.05) between the GII.4 Sydney case group and other NoV-positive case group. Conclusions: The new NoV variant, GII.4 Sydney, has been circulating in Beijing, China and became the predominant strain in the winter of 2012–13. GII.4 Sydney causes severe fever, abdominal pain and higher diarrhea frequency clinically compared to other NoV infections. [ABSTRACT FROM AUTHOR]

Norovirus (NoV) constitutes the second most common viral pathogen causing pediatric diarrhea after rotavirus. In Africa, diarrhea is a major health problem in children, and yet few studies have been performed regarding NoV. The association of histo-blood group antigens (HBGA) and susceptibility to NoV infection is well established in Caucasian populations with non-secretors being resistant to many common NoV strains. No study regarding HBGA and NoV susceptibility has yet been performed in Africa. We collected 309 stool and 208 saliva samples from diarrheal children in Ouagadougou, Burkina Faso; May 2009 to March 2010. NoV was detected using real-time PCR, and genotyped by sequencing. Saliva samples were ABO, Lewis and secretor phenotyped using in house ELISA assays. NoV was detected in 12% (n = 37) of the samples. The genotype diversity was unusually large; overall the 37 positive samples belonged to 14 genotypes. Only children <2 years of age were NoV positive and the GII.4 NoVs were more frequent in the late dry season (Jan-May). NoV infections were observed less in children with the secretor-negative phenotype or blood group A (OR 0.18; p = 0.012 and OR 0.31; p = 0.054; respectively), with two non-secretors infected with genotypes GII.7 and GII.4 respectively. Lewis-negative (Lea−b−) children, representing 32% of the study population, were susceptible to GII, but were not infected with any NoV GI. GII.4 strains preferentially infected children with blood group B whereas secretor-positive children with blood group O were infected with the largest variety of genotypes. This is the first study identifying host genetic factors associated with susceptibility to NoV in an African population, and suggests that while the non-secretor phenotype provides protection; the Lewis b antigen is not necessary for GII infection. [ABSTRACT FROM AUTHOR]

Background: We studied the transmission of rotavirus infection in households in peri-urban Ecuador in the vaccination era. Methods: Stool samples were collected from household contacts of child rotavirus cases, diarrhea controls and healthy controls following presentation of the index child to health facilities. Rotavirus infection status of contacts was determined by RT-qPCR. We examined factors associated with transmissibility (index-case characteristics) and susceptibility (household-contact characteristics). Results: Amongst cases, diarrhea controls and healthy control household contacts, infection attack rates (iAR) were 55%, 8% and 2%, (n = 137, 130, 137) respectively. iARs were higher from index cases with vomiting, and amongst siblings. Disease ARs were higher when the index child was <18 months and had vomiting, with household contact <10 years and those sharing a room with the index case being more susceptible. We found no evidence of asymptomatic infections leading to disease transmission. Conclusion: Transmission rates of rotavirus are high in households with an infected child, while background infections are rare. We have identified factors associated with transmission (vomiting/young age of index case) and susceptibility (young age/sharing a room/being a sibling of the index case). Vaccination may lead to indirect benefits by averting episodes or reducing symptoms in vaccinees. [ABSTRACT FROM AUTHOR]

Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods. Here, we report the characterization of a broadly cross-reactive monoclonal antibody (MAb) raised against the capsid protein of a GII.3 norovirus strain. The MAb reacted with VLPs and denatured VP1 protein from GI, GII, GIV and GV noroviruses, and mapped to a linear epitope located in the inner shell domain. An alignment of all available VP1 sequences showed that the putative epitope (residues 52–56) is highly conserved across the genus Norovirus. This broadly cross-reactive MAb thus constitutes a valuable reagent for the diagnosis and study of these diverse viruses. [ABSTRACT FROM AUTHOR]

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity. [ABSTRACT FROM AUTHOR]

Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus. [ABSTRACT FROM AUTHOR]

Noroviruses (NoV) have been recognized as an important pathogen associated with acute gastroenteritis worldwide during the past three decades. In the spring of 2012, a series of foodborne outbreaks in tourist groups were reported to Xiamen Center for Disease Control and Prevention, Xiamen, Fujian province, China. Among a total of 268 tourists in 7 groups, the prevalence rate of acute gastroenteritis was 16.0% (43/268). Twenty-three feces or anal swabs were collected for laboratory tests of causative agents, no bacterial pathogen was identified, while 22 of them were positive for NoV RNA. In addition, thirteen NoV fragments were recovered from positive specimens and sequenced, belonging to five genotypes such as GI.3, GI.4, GII.4, GII.6, and GII.14, respectively. However, NoV fragments obtained from locally infected patients showed distinct genotypes. Therefore, epidemiological investigation and laboratory analyses demonstrated that the serial foodborne NoV outbreaks in tourists were co-infection of multiple genotypes induced acute gastroenteritis linked to a restaurant. [ABSTRACT FROM AUTHOR]