Development of Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of Human Norovirus Major Capsid Protein.

Human Norwalk viruses (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. Although nucleic acid detection methods, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are the gold standard for the diagnosis of norovirus infection, alternative methods are needed for the specific and sensitive viral protein detection for rapid diagnosis and surveillance. In this study, we developed a robust and high-throughput targeted proteomic assay workflow to directly detect the VP1 major capsid protein of HuNoVs. A parallel reaction monitoring (PRM) assay using a high-resolution mass spectrometer was used to detect representative peptides derived from VP1 in six different HuNoV genotypes. An optimized protocol using synthesized heavy isotope-labeled peptides as internal standards was also used to simultaneously genotype and quantify the VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.

Generation of Nucleic Acid Aptamer Candidates against a Novel Calicivirus Protein Target.

Human norovirus is the leading cause of foodborne illness globally. One of the challenges in detecting noroviruses is the identification of a completely broadly reactive ligand; however, all detection ligands generated to date target the viral capsid, the outermost of which is the most variable region of the genome. The VPg is a protein covalently linked to the viral genome that is necessary for replication but hitherto remains underexplored as a target for detection or therapeutics. The purpose of this work was to generate nucleic acid aptamers against human norovirus (Norwalk) and cultivable surrogate (Tulane) VPgs for future use in detection and therapeutics. Eight rounds of positive-SELEX and two rounds of counter-SELEX were performed. Five and eight unique aptamer sequences were identified for Norwalk and Tulane VPg, respectively, all of which were predicted to be stable (∆G < -5.0) and one of which occurred in both pools. All candidates displayed binding to both Tulane and Norwalk VPg (positive:negative > 5.0), and all but two of the candidates displayed very strong binding (positive:negative > 10.0), significantly higher than binding to the negative control protein (p < 0.05). Overall, this work reports a number of aptamer candidates found to be broadly reactive and specific for in vitro-expressed VPgs across genus that could be used for future application in detection or therapeutics. Future work characterizing binding of the aptamer candidates against native VPgs and in therapeutic applications is needed to further evaluate their application.

Replication in Human Intestinal Enteroids of Infectious Norovirus from Vomit Samples.

A typical clinical symptom of human norovirus infection is projectile vomiting. Although norovirus RNA and viral particles have been detected in vomitus, infectivity has not yet been reported. We detected replication-competent norovirus in 25% of vomit samples with a 13-fold to 714-fold increase in genomic equivalents, confirming infectious norovirus.

Less severe clinical symptoms of Norwalk virus 8fIIb inoculum compared to its precursor 8fIIa from human challenge studies.

Noroviruses (NoV) are a leading cause of epidemic gastroenteritis. Human challenge studies have been used to examine the infectivity, pathogenicity, and host immune response to NoV as well as vaccine efficacy. The goal of this study was to conduct a meta-analysis of data from five previously completed human challenge trials and compare the response to the secondary NV inoculum (8fIIb) to its precursor (8fIIa). We investigated a total of 158 subjects: 76 subjects were experimentally challenged with NV inoculum 8fIIa, and 82 subjects were challenged with 8fIIb. We compared demographic characteristics, infection, illness, mean severity score, blood types, and duration of viral shedding between the two groups of subjects. There were no statistically significant differences in overall infection and illness rates between subjects inoculated with 8fIIa and 8fIIb. However, individuals challenged with 8fIIa had significantly higher severity scores (5.05 vs. 3.22, p = .008) compared with those challenged with 8fIIb. We also observed that infection with 8fIIb was associated with significantly longer duration of viral shedding compared with 8fIIa (11.0 days vs. 5.0 days, p = .0005). These results have serious implications for the development of new NoV inocula for human challenge studies to test candidate vaccine efficacy-where illness severity and duration of viral shedding are important outcomes.

Norovirus Seroprevalence among Adults in the United States: Analysis of NHANES Serum Specimens from 1999-2000 and 2003-2004.

Norovirus is the most common cause of epidemic and endemic acute gastroenteritis. However, national estimates of the infection burden are challenging. This study used a nationally representative serum bank to estimate the seroprevalence to five norovirus genotypes including three GII variants: GI.1 Norwalk, GI.4, GII.3, GII.4 US95/96, GII.4 Farmington Hills, GII.4 New Orleans, and GIV.1 in the USA population (aged 16 to 49 years). Changes in seroprevalence to the three norovirus GII.4 variants between 1999 and 2000, as well as 2003 and 2004, were measured to examine the role of population immunity in the emergence of pandemic GII.4 noroviruses. The overall population-adjusted seroprevalence to any norovirus was 90.0% (1999 to 2000) and 95.9% (2003 to 2004). Seroprevalence was highest to GI.1 Norwalk, GII.3, and the three GII.4 noroviruses. Seroprevalence to GII.4 Farmington Hills increased significantly between the 1999 and 2000, as well as the 2003 and 2004, study cycles, consistent with the emergence of this pandemic strain. Seroprevalence to GII.4 New Orleans also increased over time, but to a lesser degree. Antibodies against the GIV.1 norovirus were consistently detected (population-adjusted seroprevalence 19.1% to 25.9%), with rates increasing with age. This study confirms the high burden of norovirus infection in US adults, with most adults having multiple norovirus infections over their lifetime.

Viral species richness and composition in young children with loose or watery stool in Ethiopia.

BACKGROUND: Stool consistency is an important diagnostic criterion in both research and clinical medicine and is often used to define diarrheal disease. METHODS: We examine the pediatric enteric virome across stool consistencies to evaluate differences in richness and community composition using fecal samples collected from children aged 0 to 5 years participating in a clinical trial in the Amhara region of Ethiopia. The consistency of each sample was graded according to the modified Bristol Stool Form Scale for children (mBSFS-C) before a portion of stool was preserved for viral metagenomic analysis. Stool samples were grouped into 29 pools according to stool consistency type. Differential abundance was determined using negative-binomial modeling. RESULTS: Of 446 censused children who were eligible to participate, 317 presented for the study visit examination and 269 provided stool samples. The median age of children with stool samples was 36 months. Species richness was highest in watery-consistency stool and decreased as stool consistency became firmer (Spearman's r = - 0.45, p = 0.013). The greatest differential abundance comparing loose or watery to formed stool was for norovirus GII (7.64, 95% CI 5.8, 9.5) followed by aichivirus A (5.93, 95% CI 4.0, 7.89) and adeno-associated virus 2 (5.81, 95%CI 3.9, 7.7). CONCLUSIONS: In conclusion, we documented a difference in pediatric enteric viromes according to mBSFS-C stool consistency category, both in species richness and composition.

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

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

Development of an infectious clone and replicon system of norovirus GII.4.

Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.

Estimating the Asymptomatic Ratio of Norovirus Infection During Foodborne Outbreaks With Laboratory Testing in Japan.

BACKGROUND: Foodborne norovirus outbreak data in Japan from 2005-2006, involving virological surveillance of all symptomatic and asymptomatic individuals, were reanalyzed to estimate the asymptomatic ratio of norovirus infection along with the risk of infection and the probability of virus shedding. METHODS: Employing a statistical model that is considered to capture the data-generating process of the outbreak and virus surveillance, maximum likelihood estimation of the asymptomatic ratio was implemented. RESULTS: Assuming that all norovirus outbreaks (n = 55) were the result of random sampling from an identical distribution and ignoring genogroup and genotype specificities, the asymptomatic ratio was estimated at 32.1% (95% confidence interval [CI], 27.7-36.7). Although not significant, separate estimation of the asymptomatic ratio of the GII.4 genotype appeared to be greater than other genotypes and was estimated at 40.7% (95% CI, 32.8-49.0). CONCLUSION: The present study offered the first explicit empirical estimates of the asymptomatic ratio of norovirus infection in natural infection settings. The estimate of about 30% was consistent with those derived from volunteer challenge studies. Practical difficulty in controlling GII.4 outbreaks was supported by the data, considering that a large estimate of the asymptomatic ratio was obtained for the GII.4 genotype.

Incorporating social contact data in spatio-temporal models for infectious disease spread.

Routine public health surveillance of notifiable infectious diseases gives rise to weekly counts of reported cases-possibly stratified by region and/or age group. We investigate how an age-structured social contact matrix can be incorporated into a spatio-temporal endemic-epidemic model for infectious disease counts. To illustrate the approach, we analyze the spread of norovirus gastroenteritis over six age groups within the 12 districts of Berlin, 2011-2015, using contact data from the POLYMOD study. The proposed age-structured model outperforms alternative scenarios with homogeneous or no mixing between age groups. An extended contact model suggests a power transformation of the survey-based contact matrix toward more within-group transmission.

Natural Norovirus Infections in Rhesus Macaques.

Using a recently developed real-time reverse transcription PCR, I retested 500 fecal samples from rhesus macaques collected in 2008. Previous conventional reverse transcription PCR testing identified 1 isolate of GII norovirus; retesting found GI, GII, and possible GIV noroviruses in the samples, indicating the natural circulation of noroviruses in nonhuman primate colonies.

An examination of co-infection in acute gastroenteritis and histo-blood group antigens leading to viral infection susceptibility.

The aim of the present study was to evaluate co-infection in the gastrointestinal tract in terms of viruses, bacteria and the ABO blood group. We hypothesized that a combination of norovirus (NV) and bacteria in the gastrointestinal tract could affect the likelihood of an individual to contracting NV. Histo-blood group antigens (HBGAs) are considered to act as receptors that can lead to NV susceptibility. In addition to genetics, co-infection in the gastrointestinal tract may be associated with this mechanism. A total of 370 patients with acute gastroenteritis presenting with diarrhea (14-89 years) were recruited. The male/female ratio was 20/17. Single infection (bacteria or virus), co-infection with two viruses, and co-infection with one virus and one bacterium were statistically analyzed. In total, 88 of the 376 subjects (23.4%) were positive for one virus, and 50 (13.3%) were positive for one bacterium. Co-transfection with bacteria and a virus were detected in 46 (47.9%) of the 96 bacterial gastroenteritis cases. Statistical analysis revealed that co-infection of bacteria and NV was not significant in all viral infections (P=0.768). In terms of the ABO histo-blood group type and NV infection, the frequency in the O type was not significantly increased (P=0.052). Co-infection of bacteria and a virus occurred frequently in the gastrointestinal tract. The ABO blood phenotype expression was not a significant factor in NV infection in the present case series and the results did not suggest an affinity of NV for specific bacteria.

Etiology of Diarrheal Disease Years in Shenzhen from 2013 to 2014 / 现代检验医学杂志

Objective Through the analysis of the pathogens distribution of out-patient department and emergency department patients with diarrhea from Shenzhen Hospital of Peking University in 2013~2014,so as to provide reference for the clinical diagnosis and treatment of diarrhea disease.While understanding the improved Salmonella detection results.Methods Var-ied pathogenic bacteria were isolated and identified from 1 719 diarhea stool samples of native Shenzhen Hospital of Peking University from 2013 to 2014 through enrich culture,separate culture,biochemistry,serology etc.Pathogenic virus were test-ed for 451 watery stool specimens by fluorescence PCR.Analyzed statistical differences between the direct inoculation and selenite cystine broth enrichment for Salmonella.Results Picked out 143 disease germs from 1 719 examples diarrhea pa-tient’s stool samples,among which there were 25 strains of ETEC,12 strains of EPEC,8 strains of EAEC,1 strains of EIEC,19 strains ofVibrio parahemolyticus,76 strains Salmonella,2 strains Shigella and 0 strain ofVibrio cholera.There were 10 samples with two disease germs timely.Picked out 189 disease viruses from 451 examples diarrhea patient’s stool without disease germs,among which there were 79 Rotaviruspositive,91 Norwalkvirus positive,9 Adenoviruspositive,10 Astrovirus positive.There were 4 samples with Rotavirus and Norwalkvirus timely.After Salmonella ways to improve the positive rate of 0.6% (17/2 627)increased to 4.4% (76/1 719),χ2=67.2,P<0.01,the difference was statistically signifi-cant.Conclusion The detectable rate of Salmonella and Norwalk virus was the majority,and the clinic enhance the test of the diarrhea pathogenic microorganism,including the improvement of detection method,to reduce the missing rate of them,to provide the scientific basis for the diagnostical therapectic measures.

Production of Brazilian human norovirus VLPs and comparison of purification methods

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Serum Immunoglobulin A Cross-Strain Blockade of Human Noroviruses.

Background. Human noroviruses are the leading cause of acute viral gastroenteritis, justifying vaccine development despite a limited understanding of strain immunity. After genogroup I (GI).1 norovirus infection and immunization, blockade antibody titers to multiple virus-like particles (VLPs) increase, suggesting that GI cross-protection may occur. Methods. Immunoglobulin (Ig)A was purified from sera collected from GI.1-infected participants, and potential neutralization activity was measured using a surrogate neutralization assay based on antibody blockade of ligand binding. Human and mouse monoclonal antibodies (mAbs) were produced to multiple GI VLPs to characterize GI epitopes. Results. Immunoglobulin A purified from day 14 post-GI.1 challenge sera blocked binding of GI.1, GI.3, and GI.4 to carbohydrate ligands. In some subjects, purified IgA preferentially blocked binding of other GI VLPs compared with GI.1, supporting observations that the immune response to GI.1 infection may be influenced by pre-exposure history. For other subjects, IgA equivalently blocked multiple GI VLPs. Only strain-specific mAbs recognized blockade epitopes, whereas strain cross-reactive mAbs recognized nonblockade epitopes. Conclusions. These studies are the first to describe a functional role for serum IgA in norovirus immunity and the first to characterize human monoclonal antibodies to GI strains, expanding our understanding of norovirus immunobiology.

Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans.

Mucosal and Cellular Immune Responses to Norwalk Virus.

BACKGROUND: Noroviruses are a leading cause of acute gastroenteritis worldwide. Mucosal and cellular immune responses remain poorly understood, with most studies of noroviruses having focused on serological responses to infection. METHODS: We used saliva, feces, and peripheral blood mononuclear cells collected from persons who were administered Norwalk virus (NV) to characterize mucosal (salivary and fecal immunoglobulin A [IgA]) and cellular (NV-specific IgA and immunoglobulin G [IgG] antibody-secreting cells and total and NV-specific IgA and IgG memory B cells) immune responses following infection. RESULTS: Prechallenge levels of NV-specific salivary IgA and NV-specific memory IgG cells correlated with protection from gastroenteritis, whereas prechallenge levels of NV-specific fecal IgA correlated with a reduced viral load. Antibody-secreting cell responses were biased toward IgA, while memory B-cell responses were biased toward IgG. NV-specific memory B cells but not antibody-secreting cells persisted 180 days after infection. CONCLUSIONS: NV-specific salivary IgA and NV-specific memory IgG cells were identified as new correlates of protection against NV gastroenteritis. Understanding the relative importance of mucosal, cellular, and humoral immunity is important in developing vaccine strategies for norovirus disease prevention.

Epidemiology of norovirus gastroenteritis outbreaks in two primary schools in a city in eastern China.

We describe the epidemiology of 2 outbreaks of norovirus (GII) gastroenteritis in elementary schools in a city in eastern China using data from field investigations, pathogen testing, and face-to-face interviews. The transmission shows a point source type. In a case-control study, we identified airborne and person-to-person transmission as the source of the outbreaks.

Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.

In silico 3D structure analysis accelerates the solution of a real viral structure and antibodies docking mechanism.

Norwalk virus (NoV) is responsible for most outbreaks of non-bacterial gastroenteritis. NoV is genetically diverse and show antigenically variable. Recently, we produced a monoclonal antibody called 5B-18 that reacts broadly with NoV genogroup II (GII). We suspected the 5B-18 binds to a conformational epitope on 3D structure of virion. X-ray crystallography showed us that 5B-18 binds to NoV at the P domain, which protrudes from the capsid surface of the virion. However, there seems to be no space that would allow the IgG to approach the virion. To solve this problem, we used cryo-electron microscopy to examine NoV GII virus-like particles (VLPs). The P domain rises up higher in NoV GII than in NoV GI, and it seems to form an outer layer around the virion. Finally, using in silico modeling we found the 5B-18 Fab arms and NoV P region are quite flexible, so that 5B-18 can bind the NoV virion from bottom of P domain. This study demonstrates the shortcomings of studying biological phenomenon by only one technique. Each method has limitations. Multiple methods and modeling in silico are the keys to solving structural problems.