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International audience; Knowledge of rotavirus epidemiology is necessary to make informed decisions about vaccine introduction and to evaluate vaccine impact. During April 2010-March 2012, rotavirus surveillance was conducted among 9,745 children 80% of isolates during the second year. Hospitalization and death rates and severe dehydration among rotavirus case-patients did not differ during the 2 years. The emergence of G12P[8] warrants close attention to the characteristics of associated epidemics and possible prevention measures.

Background Diarrheal disease remains a leading cause of death in children in low-income countries. We investigated the etiology, risk factors and effects on nutritional status of severe diarrhea in children from two districts in Madagascar. Methods We performed a matched case-control study in 2011 to 2014, on children under the age of five years from Moramanga and Antananarivo. The cases were children hospitalized for severe diarrhea and the controls were children without diarrhea selected at random from the community. Stool samples were collected from both groups. Anthropometric measurements were made during follow-up visits about one and two months after enrolment. Results We enrolled 199 cases and 199 controls. Rotavirus infection was the most frequently detected cause of diarrhea. It was strongly associated with severe diarrhea (OR: 58.3; 95% CI: 7.7–439.9), accounting for 42.4% (95% CI: 37.6–43.1) of severe diarrhea cases. At the household level, possession of cattle (OR = 0.3; 95% CI: 0.1–0.6) and living in a house with electricity (OR = 0.4; 95% CI: 0.2–0.8) were protective factors. The presence of garbage around the house was a risk factor for severe diarrhea (OR = 3.2; 95% CI: 1.9–5.4). We found no significant association between severe diarrhea and the nutritional status of the children at follow-up visits, but evident wasting at enrolment was associated with a higher risk of severe diarrhea (OR = 9; 95% CI: 4.5–17.9). Conclusions Severe childhood diarrhea is mostly caused by rotavirus infection. An anti-rotavirus vaccine has already been introduced in Madagascar and should be promoted more widely. However, post-licensing surveillance is required. Interventions to improve the nutritional status of children, preventive measures focused on household and personal hygiene and nutritional rehabilitation during severe diarrheal disease should be reinforced.

International audience; The structure and function of the intestinal epithelium is briefly described, with the principal mechanisms involved in diarrhea. Human enteric viruses and probiotics are presented. We then review how probiotic bacteria could interfere with virus-induced pathology, we present our own view and describe specific interactions that would be valuable targets for future studies.

We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

International audience; The prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats harbor diverse viruses, some of which are of particular interest because they cause severe human diseases. However, little is known about the diversity of the global population of viruses found in bats (virome). We determined the viral diversity of five different French insectivorous bat species (nine specimens in total) in close contact with humans. Sequence-independent amplification, high-throughput sequencing with Illumina technology and a dedicated bioinformatics analysis pipeline were used on pooled tissues (brain, liver and lungs). Comparisons of the sequences of contigs and unassembled reads provided a global taxonomic distribution of virus-related sequences for each sample, highlighting differences both within and between bat species. Many viral families were present in these viromes, including viruses known to infect bacteria, plants/fungi, insects or vertebrates, the most relevant being those infecting mammals (Retroviridae, Herpesviridae, Bunyaviridae, Poxviridae, Flaviviridae, Reoviridae, Bornaviridae, Picobirnaviridae). In particular, we detected several new mammalian viruses, including rotaviruses, gammaretroviruses, bornaviruses and bunyaviruses with the identification of the first bat nairovirus. These observations demonstrate that bats naturally harbor viruses from many different families, most of which infect mammals. They may therefore constitute a major reservoir of viral diversity that should be analyzed carefully, to determine the role played by bats in the spread of zoonotic viral infections.

International audience; Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.

International audience; Continuous surveillance of acute diarrhea in France has been conducted by Inserm's sentinel network of general practitioners (GP) since 1991. Similar GP-based studies have been performed in the Netherlands, Austria and the UK. The causes of most cases of acute diarrhea are unclear. In case-controlstudies designed to identify viruses in stools, 35 to 40% of cases and virtually none of the controls were positive for one of 4 major viruses (rotavirus, calicivirus, astrovirus and adenovirus). Thus, no viral cause was identified in more than 60% of patients with acute diarrhea. The causative role of viruses such as torovirus, picobirnavirus, picornavirus and enterovirus 22 has rarely been investigated. Further investigations are needed to identify other viral, bacterial fungal or parasitic causes of acute diarrhea. In France, on average, more than 3 million people (predominantly children) visit a GP for acute diarrhea each year. Most of these patients recover spontaneously within a few days, but the medical, social and economic costs of acute diarrhea are sufficiently high to justify a more aggressive public policy to prevent and control epidemics in Europe.; COMMUNICATION SCIENTIFIQUESéance du 9 novembre 2010Malgré une surveillance permanente et continue des diarrhées aiguës sur le territoire français assurée par les médecins généralistes du réseau Sentinelles de l’Inserm depuis 1991, et la mise en place de systèmes de surveillance similaires ou des études en médecine générale dans d’autres pays d’Europe, notamment aux Pays-Bas, en Autriche et au Royaume-Uni, force est de constater que leur étiologie reste encore largement méconnue. Si des études cas-témoins conduites avec une recherche de virus dans les selles ont permis d’identifier un virus dans 35 à 40 % des cas et chez un faible nombre de témoins, en faveur d’un rôle causal probable de quatre virus principalement retrouvés lors des épidémies (rotavirus, calicivirus, astrovirus et adénovirus), dans plus de 60 % des cas la cause de ces diarrhées aigües hivernales ou estivales reste inconnue. Le rôle de virus actuellement peu ou pas recherchés comme les torovirus, picobirnavirus, picornavirus ou entérovirus 22 mériterait d’être précisé. Des recherches restent donc à conduire, notamment visant à l’identification plus étendue d’agents pathogènes potentiellement responsables, qu’ils soient viraux, bactériens, fongiques ou parasitaires, avant de pouvoir espérer prévenir et contrôler de manière efficace ces épidémies récurrentes en Europe, qui concernent de larges segments de la population. En France environ trois millions de personnes consultent en moyenne chaque année leur médecin généraliste pour un épisode de diarrhée aiguë (ce sont des patients de tous âges, bien que plus souvent des enfants). Si pour l’immense majorité des cas leur évolution favorable est spontanée en quelques jours, le coût sanitaire, social et économique de ces épidémies d’ampleur nationale est très élevé et justifierait une politique de prévention et de lutte plus ambitieuse à l’échelle de l’Europe,.

Continuous surveillance of acute diarrhea in France has been conducted by Inserm's sentinel network of general practitioners (GP) since 1991. Similar GP-based studies have been performed in the Netherlands, Austria and the UK. The causes of most cases of acute diarrhea are unclear. In case-controlstudies designed to identify viruses in stools, 35 to 40% of cases and virtually none of the controls were positive for one of 4 major viruses (rotavirus, calicivirus, astrovirus and adenovirus). Thus, no viral cause was identified in more than 60% of patients with acute diarrhea. The causative role of viruses such as torovirus, picobirnavirus, picornavirus and enterovirus 22 has rarely been investigated. Further investigations are needed to identify other viral, bacterial fungal or parasitic causes of acute diarrhea. In France, on average, more than 3 million people (predominantly children) visit a GP for acute diarrhea each year. Most of these patients recover spontaneously within a few days, but the medical, social and economic costs of acute diarrhea are sufficiently high to justify a more aggressive public policy to prevent and control epidemics in Europe., COMMUNICATION SCIENTIFIQUESéance du 9 novembre 2010Malgré une surveillance permanente et continue des diarrhées aiguës sur le territoire français assurée par les médecins généralistes du réseau Sentinelles de l’Inserm depuis 1991, et la mise en place de systèmes de surveillance similaires ou des études en médecine générale dans d’autres pays d’Europe, notamment aux Pays-Bas, en Autriche et au Royaume-Uni, force est de constater que leur étiologie reste encore largement méconnue. Si des études cas-témoins conduites avec une recherche de virus dans les selles ont permis d’identifier un virus dans 35 à 40 % des cas et chez un faible nombre de témoins, en faveur d’un rôle causal probable de quatre virus principalement retrouvés lors des épidémies (rotavirus, calicivirus, astrovirus et adénovirus), dans plus de 60 % des cas la cause de ces diarrhées aigües hivernales ou estivales reste inconnue. Le rôle de virus actuellement peu ou pas recherchés comme les torovirus, picobirnavirus, picornavirus ou entérovirus 22 mériterait d’être précisé. Des recherches restent donc à conduire, notamment visant à l’identification plus étendue d’agents pathogènes potentiellement responsables, qu’ils soient viraux, bactériens, fongiques ou parasitaires, avant de pouvoir espérer prévenir et contrôler de manière efficace ces épidémies récurrentes en Europe, qui concernent de larges segments de la population. En France environ trois millions de personnes consultent en moyenne chaque année leur médecin généraliste pour un épisode de diarrhée aiguë (ce sont des patients de tous âges, bien que plus souvent des enfants). Si pour l’immense majorité des cas leur évolution favorable est spontanée en quelques jours, le coût sanitaire, social et économique de ces épidémies d’ampleur nationale est très élevé et justifierait une politique de prévention et de lutte plus ambitieuse à l’échelle de l’Europe,.

International audience; The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

Tremendous progress has been made in recent years in the acquisition and treatment of electron micrographs of biological samples. These developments now allow retrieving much higher-resolution information from the scrutinized object than was possible until recently. In the case of highly symmetric particles, this approach is providing electron density maps comparable to those obtained by X-ray crystallography at least for large assemblies of equivalent sizes. Such accomplishments were made recently with icosahedral viruses, a bacteriophage (1) and an insect virus (2), and now, as reported in this issue of PNAS (3), this approach has revealed the details of the triple-layered architecture of the medically important rotavirus. This work follows from the recent 3D reconstruction of the double-layered rotavirus subparticles at the same level of resolution (4). There has indeed been steady methodological progress in this field. The first 3D fold of a structural protein of a human virus determined by EM, the hepatitis B virus (HBV) core protein, was reported ≈12 years ago (5, 6). The EM map of the HBV core particles obtained at the time was of sufficient quality to resolve the secondary structure elements of an α-helix-rich HBV core protein. From these pioneering studies, we have now moved to an era in which the quality of the EM maps permits the tracing of entire polypeptide chains for much larger proteins. Although the reported high-resolution 3D reconstructions are still special cases because of their high symmetry, the advances are not necessarily limited to symmetrical particles, but are likely to apply to any rigid macromolecular assembly, provided that enough high-quality images are available to reconstruct the object.

National audience; The performances of two diagnostic tests for rotavirus infection in stool samples were evaluated during a prospective study in children of less than 36 months in child-care centers of Lyon from November 2004 to May 2005. The VIKIA Rota-Adeno immuno-chromatographic test (bioMérieux) and the ELISA IDEIA Rotavirus kit (Dako) were compared with a referral method, the genotyping. Fifty-seven stool samples were collected and analyzed by RT-PCR. The virus genome was detected in 29 samples. The most frequent genotypic combinations were G9P[8] with a prevalence of 75.9%. Sensitivity and specificity of the VIKIA Rota-Adeno test and the ELISA IDEIA Rotavirus kit were strictly comparable and very good: 96.6% (83.0; 99.9) and 96.4% (81.6; 99.9), respectively. The immuno-chromatographic technique were in concordance with the ELISA tests in 93.6% of cases. Thus, the VIKIA Rota-Adeno test is a good alternative for the occasional analysis of stool samples in ambulatory practice.

Following a flooding event close to a shellfish production lagoon, 205 cases of gastroenteritis were linked to oyster consumption. Twelve stool samples from different individuals were collected. Analysis showed that eight samples were positive for multiple enteric viruses, and one stool sample had seven different enteric viruses. Analysis of shellfish implicated in the outbreak allowed detection of the same diversity of enteric viruses, with some viral genomic sequences being identical to those obtained from stool sample analysis. Shellfish were contaminated by as many as five different enteric viruses. For the first time in Europe, Aichi virus was identified in oyster samples. Shellfish samples collected over 3 weeks following the outbreak showed a progressive decline in the level of virus contamination as measured by the virus diversity detected and by quantitative reverse transcription-PCR.

International audience; Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 A resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus lambda3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence. Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.

Rotavirus nonstructural protein NSP3 interacts specifically with the 3′ end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.

International audience; Rotavirus is the leading cause of severe dehydrating diarrhoea in infants and young children worldwide. This virus infects mature enterocytes in the small intestine, and induces structural and functional damage. In the present study, we have identified a new mechanism by which rotavirus impairs a brush border-associated intestinal protein. We show that infection of enterocyte-like Caco-2/TC7 cells by rhesus monkey rotavirus (RRV) impairs the biosynthesis of dipeptidyl peptidase IV (DPP IV), an important hydrolase in the digestion of dietary proline-rich proteins. We show that the enzyme activity of DPP IV was reduced, and that rearrangements of the protein occurred at the apical domain of the RRV-infected cells. Using pulse-chase experiments and cell surface immunoprecipitation, we have demonstrated that RRV infection did not affect the stability or apical targeting of DPP IV, but did induce a dramatic decrease in its biosynthesis. Using quantitative RT-PCR, we showed that RRV had no effect on the level of expression of DPP IV mRNA, suggesting that the observed decrease in the biosynthesis of the protein is related to an effect of the virus at the translational level.

International audience; A large waterborne outbreak of infection that occurred during August 2000 in a local community in France was investigated initially via a rapid survey of visits to local physicians. A retrospective cohort study was then conducted on a random cluster sample of residents. Of 709 residents interviewed, 202 (28.5%) were definite cases (at least three liquid stools/day or vomiting) and 62 (8.7%) were probable cases (less than three liquid stools/day or abdominal pain). Those who had drunk tap water had a three-fold increased risk for illness (95% CI 2.4-4.0). The risk increased with the amount of water consumed (chi-square trend: p < 0.0001). Bacteriological analyses of stools were performed for 35 patients and virological analyses for 24 patients. Campylobacter coli, group A rotavirus and norovirus were detected in 31.5%, 71.0% and 21% of samples, respectively. An extensive environmental investigation concluded that a groundwater source to this community had probably been contaminated by agricultural run-off, and a failure in the chlorination system was identified. This is the first documented waterborne outbreak of infection involving human C. coli infections. A better understanding of the factors influencing campylobacter transmission between hosts is required.

International audience; Seven commercially available immunochromatographic assays were tested for the rapid detection of group A rotaviruses in fecal samples compared to a enzyme immunoassay (Argene). Detection of rotaviruses in 80 ELISA positive frozen stool samples showed rates superior to 90% for three reagents (Rota Strip (Cypress Diagnostics), 98.8%; Rotascreen (Microgen), 95.0%; VIKIA Rota/Adeno (bioMérieux), 92.5%); from 82.5% to 88.8% for three others (Diarlex with centrifugation (Orion Diagnostica), 88.8%; Combo Rota/Adeno (All Diag), 87.5%; Rota/Adeno Combi Stick (bmd), 82.5%) and only 70.0% for Diarlex with filtration vial (Orion Diagnostica). The evaluation of the specificity, performed on one hundred fresh rotavirus negative stools, did not show any false positives with any assay. Analysis of the different technical features of these tests showed that they are quick and suitable for a clinical laboratory and do not require expensive equipment.

The direct effect of the rotavirus NSP4114-135 and Norovirus NV464-483 peptides on 36Cl uptake was studied by using villus cell brush border membrane (BBM) isolated from young rabbits. Both peptides inhibited the Cl-/H+ symport activity about equally and partially. The interaction involved one peptide-binding site per carrier unit. Whereas in vitro NSP4114-135 caused nonspecific inhibition of the Cl-/H+ symporter, the situation in vivo is different. Because rotavirus infection in young rabbits accelerated both Cl- influx and Cl- efflux rates across villi BBM without stimulating Cl- transport in crypt BBM, we conclude that the NSP4114-135 peptide, which causes diarrhea in young rodents, did not have any direct, specific effect on either intestinal absorption or secretion of chloride. The lack of direct effect of NSP4 on chloride transport strengthens the hypothesis that NSP4 would trigger signal transduction pathways to enhance net chloride secretion at the onset of rotavirus diarrhea.

35 ref.; International audience; The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.