Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

Articles not assigned to an issueInternational audienceHuman enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3-18 RNA copies/µL for the targeted human pathogenic viruses and 20-40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination

Seasonal and spatial dynamics of enteric viruses in wastewater and in riverine and estuarine receiving waters

Enteric viruses represent a global public health threat and are implicated in numerous foodborne and waterborne disease outbreaks. Nonetheless, relatively little is known of their fate and stability in the environment. In this study we used carefully validated methods to monitor enteric viruses, namely adenovirus (AdV), JC polyomavirus (JCV), noroviruses (NoVs), sapovirus (SaV) and hepatitis A and E viruses (HAV and HEV) from wastewater source to beaches and shellfish beds. Wastewater influent and effluent, surface water, sediment and shellfish samples were collected in the Conwy catchment (North Wales, UK) once a month for one year. High concentrations of AdV and JCV were found in the majority of samples, and no seasonal patterns were observed. No HAV and HEV were detected and no related illnesses were reported in the area during the period of sampling. Noroviruses and SaV were also detected at high concentrations in wastewater and surface water, and their presence correlated with local gastroenteritis outbreaks during the spring and autumn seasons. Noroviruses were also found in estuarine sediment and in shellfish harvested for human consumption. As PCR-based methods were used for quantification, viral infectivity and degradation was estimated using a NoV capsid integrity assay. The assay revealed low-levels of viral decay in wastewater effluent compared to influent, and more significant decay in environmental waters and sediment. Results suggest that AdV and JCV may be suitable markers for the assessment of the spatial distribution of wastewater contamination in the environment; and pathogenic viruses can be directly monitored during and after reported outbreaks to prevent further environment-derived illnesses

Cosavirus, Salivirus and Bufavirus in Diarrheal Tunisian Infants

International audienceThree newly discovered viruses have been recently described in diarrheal patients: Cosa-virus (CosV) and Salivirus (SalV), two picornaviruses, and Bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroen-teritis (AGE) in diarrheal Tunisian infants. From October 2010 through March 2012, stool samples were collected from 203 children <5 years-old suffering from AGE and attending the Children's Hospital in Monastir, Tunisia. All samples were screened for CosV, SalV and BuV as well as for norovirus (NoV) and group A rotavirus (RVA) by molecular biology. Positive samples for the three screened viruses were also tested for astrovirus, sapovirus, ade-novirus, and Aichi virus, then genotyped when technically feasible. During the study period, 11 (5.4%) samples were positive for one of the three investigated viruses: 2 (1.0%) CosV-A10, 7 (3.5%) SalV-A1 and 2 (1.0%) BuV-1, whereas 71 (35.0%) children were infected with NoV and 50 (24.6%) with RVA. No mixed infections involving the three viruses were found, but multiple infections with up to 4 classic enteric viruses were found in all cases. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain since all infected children also presented infections with several enteric viruses, suggesting here potential water-borne transmission. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE

Evidence for human norovirus infection of dogs in the United kingdom.

Human noroviruses (HuNoVs) are a major cause of viral gastroenteritis, with an estimated 3 million cases per year in the United Kingdom. HuNoVs have recently been isolated from pet dogs in Europe (M. Summa, C.-H. von Bonsdorff, and L. Maunula, J Clin Virol 53:244-247, 2012, http://dx.doi.org/10.1016/j.jcv.2011.12.014), raising concerns about potential zoonotic infections. With 31% of United Kingdom households owning a dog, this could prove to be an important transmission route. To examine this risk, canine tissues were studied for their ability to bind to HuNoV in vitro. In addition, canine stool samples were analyzed for the presence of viral nucleic acid, and canine serum samples were tested for the presence of anti-HuNoV antibodies. The results showed that seven different genotypes of HuNoV virus-like particles (VLPs) can bind to canine gastrointestinal tissue, suggesting that infection is at least theoretically possible. Although HuNoV RNA was not identified in stool samples from 248 dogs, serological evidence of previous exposure to HuNoV was obtained in 43/325 canine serum samples. Remarkably, canine seroprevalence for different HuNoV genotypes mirrored the seroprevalence in the human population. Though entry and replication within cells have not been demonstrated, the canine serological data indicate that dogs produce an immune response to HuNoV, implying productive infection. In conclusion, this study reveals zoonotic implications for HuNoV, and to elucidate the significance of this finding, further epidemiological and molecular investigations will be essential.This collaborative project was facilitated by the Society of Microbiology's President's Fund awarded to S.L.C. and by the Region des Pays de la Loire ARMINA project. This work was supported by a Ph.D. studentship from the Medical Research Council to S.L.C. and a Wellcome Trust Senior Fellowship to I.G. (WT097997MA). I.G. is a Wellcome Senior Fellow.This is the final published version of the article. It was originally published in the Journal of Clinical Microbiology (Caddy S, et al., Journal of Clinical Microbiology, 2015, 53, 1873-1883, doi:10.1128/JCM.02778-14). The final version is available at http://dx.doi.org/10.1128/JCM.02778-1

Molecular chaperone Hsp90 is a therapeutic target for noroviruses.

UNLABELLED: Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5' and 3' extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE: HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients

Role of the A, B, H and Lewis histo-blood group antigens in the evolution of GII.4 noroviruses

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d’acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu’une configuration stricte des aa directement impliqués dans l’accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d’attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s’attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s’accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d’attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l’accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002.Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants

Rôle des antigènes tissulaires de groupes sanguins humains A, B, H et Lewis dans l'évolution des Norovirus GII.4

Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants.Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d’acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu’une configuration stricte des aa directement impliqués dans l’accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d’attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s’attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s’accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d’attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l’accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002

Rôle des antigènes tissulaires de groupes sanguins humains A, B, H et Lewis dans l'évolution des Norovirus GII.4

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu une configuration stricte des aa directement impliqués dans l accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002.Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Spatial and temporal control of norovirus protease activity is determined by polyprotein processing and intermolecular interactions within the viral replication complex

Norovirus infections are a major cause of acute viral gastroenteritis and a significant burden to human health globally. A vital process for norovirus replication is the processing of the nonstructural polyprotein, by an internal protease, into the necessary viral components required to form the viral replication complex. This cleavage occurs at different rates resulting in the accumulation of stable precursor forms. In this report, we characterized how precursor forms of the norovirus protease accumulate during infection. Using stable forms of the protease precursors we demonstrated that these are all proteolytically active in vitro , but that when expressed in cells, activity is determined by both substrate and protease localization. Whilst all precursors could cleave a replication complex-associated substrate, only a subset of precursors lacking NS4 were capable of efficiently cleaving a cytoplasmic substrate. For the first time, the full range of protein-protein interactions between murine and human norovirus proteins were mapped by LUMIER assay, with conserved interactions between replication complex members, modifying the localization of a subset of precursors. Finally, we demonstrate that re-targeting of a poorly cleaved artificial cytoplasmic substrate to the replication complex is sufficient to permit efficient cleavage in the context of norovirus infection. This offers a model for how norovirus can regulate the timing of substrate cleavage throughout the replication cycle. The norovirus protease represents a key target in the search for effective antiviral treatments for norovirus infection. An improved understanding of protease function and regulation, as well as identification of interactions between the other non-structural proteins, offers new avenues for antiviral drug design

[Norovirus infections: an overview]

National audienceNoroviruses belong to the Caliciviridae family. They are a major cause of sporadic cases and outbreaks of gastroenteritis in all age groups, and are responsible for a considerable disease burden in industrialized countries. Noroviruses are single-stranded RNA viruses, and show great genetic diversity making their detection difficult. Noroviruses can be divided into 5 genogroups, which themselves are subdivided into genotypes. Besides chance mutations that occur during viral replication, the great heterogeneity observed among noroviruses is also due to intra and inter-genotypic recombination events between strains. Some of these new variants or new recombinants are frequently associated with new epidemic waves of gastroenteritis. Finally, it is worth pointing out that the discovery of mechanisms involved in NoV infections through blood antigen-related receptors and cultivation of the first norovirus, a murine norovirus, are milestones in research on this virus. These advances open new promising avenues of research that will help to the understanding of the -pathogenicity of this important pathogen