Exploration of binary protein-protein interactions between tick-borne flaviviruses and Ixodes ricinus

International audienceBackground : Louping ill virus (LIV) and tick-borne encephalitis virus (TBEV) are tick-borne flaviviruses that are both transmitted by the major European tick, Ixodes ricinus . Despite the importance of I. ricinus as an arthropod vector, its capacity to acquire and subsequently transmit viruses, known as vector competence, is poorly understood. At the molecular scale, vector competence is governed in part by binary interactions established between viral and cellular proteins within infected tick cells. Methods : To investigate virus-vector protein–protein interactions (PPIs), the entire set of open reading frames for LIV and TBEV was screened against an I. ricinus cDNA library established from three embryonic tick cell lines using yeast two-hybrid methodology (Y2H). PPIs revealed for each viral bait were retested in yeast by applying a gap repair (GR) strategy, and notably against the cognate protein of both viruses, to determine whether the PPIs were specific for a single virus or common to both. The interacting tick proteins were identified by automatic BLASTX, and in silico analyses were performed to expose the biological processes targeted by LIV and TBEV. Results : For each virus, we identified 24 different PPIs involving six viral proteins and 22 unique tick proteins, with all PPIs being common to both viruses. According to our data, several viral proteins (pM, M, NS2A, NS4A, 2K and NS5) target multiple tick protein modules implicated in critical biological pathways. Of note, the NS5 and pM viral proteins establish PPI with several tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which are essential adaptor proteins at the nexus of multiple signal transduction pathways. Conclusion : We provide the first description of the TBEV/LIV- I. ricinus PPI network, and indeed of any PPI network involving a tick-borne virus and its tick vector. While further investigation will be needed to elucidate the role of each tick protein in the replication cycle of tick-borne flaviviruses, our study provides a foundation for understanding the vector competence of I. ricinus at the molecular level. Indeed, certain PPIs may represent molecular determinants of vector competence of I. ricinus for TBEV and LIV, and potentially for other tick-borne flaviviruses

High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

International audienceThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC B

: Caractérisation d’un système d’infection persistante par le virus de l’hépatite E dans les cellules hépatiques humaines HepaRG

International audienceHepatitis E virus (HEV) is considered as an emerging global health problem. The pathogen is responsible for more than 2 000 cases of acute hepatitis in France every year resulting in majority from zoonotic infections associated with the consumption of raw or undercooked pork. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. For decades, the lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin inhibited HEV replication in HepaRG cells. Using whole genome sequencing, 25 mutations including 8 non-synonymous mutations were detected in the genome of the virus recovered after 6 passages into HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to identify host–virus interactions and putative mutations within the viral genome than can occur in vitro in the context of prolonged hepatitis E infection and to test antiviral drugs against chronic HEV infection

: Caractérisation d’un système d’infection persistante par le virus de l’hépatite E dans les cellules hépatiques humaines HepaRG

International audienceHepatitis E virus (HEV) is considered as an emerging global health problem. The pathogen is responsible for more than 2 000 cases of acute hepatitis in France every year resulting in majority from zoonotic infections associated with the consumption of raw or undercooked pork. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. For decades, the lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin inhibited HEV replication in HepaRG cells. Using whole genome sequencing, 25 mutations including 8 non-synonymous mutations were detected in the genome of the virus recovered after 6 passages into HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to identify host–virus interactions and putative mutations within the viral genome than can occur in vitro in the context of prolonged hepatitis E infection and to test antiviral drugs against chronic HEV infection

Characterization of a cell culture system of persistent hepatitis E virus infection in the human HepaRG hepatic cell line

National audiencethan 2 000 cases of acute hepatitis in France every year resulting in majority from zoonotic infections associatedwith the consumption of raw or undercooked pork. In most cases, hepatitis E is a self-limiting disease and the virusis cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals candevelop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. For decades, thelack of efficient and relevant cell culture system and animal models has limited our understanding of the biologyof HEV and the development of effective drugs for chronic cases. In the present study, we developed a model ofpersistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture systemis based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering fromacute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after sevensuccessive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, the virus was still infectiousafter oral inoculation into pigs. We also showed that ribavirin inhibited HEV replication in HepaRG cells. Using wholegenome sequencing, 25 mutations including 8 non-synonymous mutations were detected in the genome of thevirus recovered after 6 passages into HepaRG. In conclusion, this system represents a relevant and efficient in vitromodel of HEV replication that could be useful to identify host–virus interactions and putative mutations withinthe viral genome than can occur in vitro in the context of prolonged hepatitis E infection and to test antiviral drugsagainst chronic HEV infection

Caractérisation d’un système d’infection persistante par le virus de l’hépatite E dans les cellules hépatiques humaines HepaRG

International audienceLe virus de l'hépatite E (HEV) appartient à la famille des Hepeviridae. Quatre génotypes principaux circulent chez l'homme (HEV-1 à -4). Dans les pays en voie de développement, de sévères épidémies d’origine hydrique sont causées par HEV-1 et -2 retrouvés uniquement chez l’homme. Dans les pays industrialisés, HEV-3 et -4 sont responsables de cas sporadiques d’hépatite E résultant d’infections zoonotiques principalement associés à la consommation de viandes crues ou insuffisamment cuites. De nombreuses espèces animales domestiques et sauvages sont infectées par le HEV, mais le principal réservoir animal est le porc. En France, le HEV représente un problème majeur de santé publique et de sécurité alimentaire avec environ 60 000 cas annuels incluant plus de 500 hospitalisations et 20 décès. La gravité de l'infection par le HEV va d’une forme asymptomatique, à des hépatites aiguës résolutives mais peut aussi aller jusqu’à des formes très sévères avec des taux de mortalité élevés chez les femmes enceintes. De plus, les personnes immunodéprimées, notamment sous traitement immunosuppresseur comme les receveurs d’organes, sont susceptibles de développer des infections chroniques et des fibroses hépatiques qui peuvent progresser rapidement vers des cirrhoses et des insuffisances hépatiques. Aucun traitement spécifique contre le HEV n’a encore été validé même si plusieurs études ont rapporté un effet bénéfique de la ribavirine pour traiter les cas d’infection chronique. Des modèles de culture cellulaire du HEV ont été décrits mais ne permettent qu'une réplication virale modérée, ce qui limite les études sur la biologie du virus et le développement d’un traitement antiviral efficace contre les hépatites E chroniques. Dans cette étude, nous caractérisons un modèle d’infection persistante par le HEV d’hépatocytes humaines dans lesquelles le virus se réplique efficacement. Ce système d’infection repose sur l’utilisation de cellules HepaRG différentiées infectées avec un isolat d’HEV-3 provenant d’un patient souffrant d’hépatite E aigüe. Une réplication efficace a été maintenue pendant plusieurs semaines à plusieurs mois et après plusieurs passages sur cellules HepaRG avec des titres atteignant 1x109 copies d’ARN / ml de surnageant. De plus, la ribavirine a bien un effet inhibiteur sur la réplication du HEV dans ces cellules. Nous avons également trouvé, après inoculation orale chez le porc, que le virus était toujours infectieux après 6 passages sur HepaRG différentiées. Le séquençage complet du génome a permis d’identifier une duplication de 29 acides aminés dans la région Hypervariable de l’ORF-1 et de déterminer qu’après 7 passages du virus, 25 mutations ont été sélectionnées sur la totalité du génome, parmi lesquelles 8 sont non-synonymes. En conclusion, ce système d’infection représente un modèle in vitro pertinent et efficace de réplication du HEV qui pourra contribuer à l’étude de la biologie, des déterminants de virulence du HEV et l’identification de molécules antivirales contre les infections chroniques. Il pourra également être utile à l’évaluation de la présence de particules infectieuses dans des échantillons cliniques ou de terrain (provenant de l’alimentation ou de l’environnement) et la production de stocks de virus pour des infections in vitro et in vivo

Characterization of a cell culture system of persistent hepatitis E virus infection in the human HepaRG hepatic cell line

National audiencethan 2 000 cases of acute hepatitis in France every year resulting in majority from zoonotic infections associatedwith the consumption of raw or undercooked pork. In most cases, hepatitis E is a self-limiting disease and the virusis cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals candevelop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. For decades, thelack of efficient and relevant cell culture system and animal models has limited our understanding of the biologyof HEV and the development of effective drugs for chronic cases. In the present study, we developed a model ofpersistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture systemis based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering fromacute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after sevensuccessive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, the virus was still infectiousafter oral inoculation into pigs. We also showed that ribavirin inhibited HEV replication in HepaRG cells. Using wholegenome sequencing, 25 mutations including 8 non-synonymous mutations were detected in the genome of thevirus recovered after 6 passages into HepaRG. In conclusion, this system represents a relevant and efficient in vitromodel of HEV replication that could be useful to identify host–virus interactions and putative mutations withinthe viral genome than can occur in vitro in the context of prolonged hepatitis E infection and to test antiviral drugsagainst chronic HEV infection

First Complete Genome Sequence of a Salmonella enterica subsp. enterica Serovar Derby Strain Associated with Pork in France

In France, Salmonella enterica subsp. enterica serovar Derby is one of the most often isolated serovars in pigs. Here, we describe the draft genome sequence of a strain isolated from a pig. This strain had the most frequent pulsed-field gel electrophoresis (PFGE) and antimicrobial patterns (S, SSU, T) usually observed in pig production in France. Those patterns have been also highlighted in human isolates

Identification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology

Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis affecting humans in the European Union. Human cases are mainly due to Campylobacter jejuni or Campylobacter coli, and contamination is associated with the handling and/or consumption of poultry meat. In fact, poultry constitutes the bacteria’s main reservoir. A promising way of decreasing the incidence of campylobacteriosis in humans would be to decrease avian colonization. Poultry vaccination is of potential for this purpose. However, despite many studies, there is currently no vaccine available on the market to reduce the intestinal Campylobacter load in chickens. It is essential to identify and characterize new vaccine antigens. This study applied the reverse vaccinology approach to detect new vaccine candidates. The main criteria used to select immune proteins were localization, antigenicity, and number of B-epitopes. Fourteen proteins were identified as potential vaccine antigens. In vitro and in vivo experiments now need to be performed to validate the immune and protective power of these newly identified antigens