"Study of Virological and Clinical Features and Inflammatory Response during Respiratory Tract Infection by Human Enterovirus"

Le genre Entérovirus (EV) (famille des picornaviridae) est composé de petits virus à ARN non enveloppées classés en 12 espèces dont 7 sont pathogènes pour l'homme : 4 espèces (A-D) d'enterovirus humains (HEV) et trois espèces (A-C) de rhinovirus humains (HRV). Dans le genre enterovirus, les HRV et HEV sont reconnus comme des pathogènes respiratoires fréquemment responsables d'infections des voies aériennes supérieures et inférieures chez l'enfant et l'adulte. Entre 2009 et 2012, de nouveaux génotypes d'HEV à tropisme respiratoire (HEV-68, 104, 109, et le CVA-21) ont été décrits dans des cas isolés ou épidémiques démontrant la capacité des espèces A à D à induire des infections respiratoires basses humaines.La première phase de ce travail de thèse a eu pour objectifs de préciser le rôle étiologique des infections à EVs; d'identifier les génotypes potentiellement responsables des pathologies respiratoires pédiatriques nécessitant une hospitalisation, mais aussi d'analyser et de comparer les caractéristiques cliniques et épidémiologiques entre les différents groupes de génotypes identifiés. Nous avons réalisé une étude rétrospective sur une cohorte de 309 enfants hospitalisés au CHU de Reims entre septembre 2009 et juin 2010 pour une infection respiratoire aiguë non documentée microbiologiquement par la réalisation des tests virologiques et bactériologiques conventionnels. Nos résultats montrent que le génome des EVs (HEV et HRV) est retrouvé dans 60,5% (187/309) des aspirations naso-pharyngées des enfants hospitalisés, distinguant 15 infections à HEV (dont 10 souches HEV-68) et 172 à HRV. Les cas de bronchiolite et d'exacerbation de l'asthme (133/187) positifs pour la détection des souches HEV (12/133) étaient plus âgés (P=0,003) et plus fréquemment associés avec une détresse respiratoire (P=0,01) et un besoin en oxygénothérapie au moment de leur hospitalisation (P=0,01) que les cas infectés par un HRV. De plus, nous avons mis en évidence pour la première fois en France la circulation épidémique de souches d'HEV-68 (10/15 des souches d'HEV détectées) isolées au cours de l'automne 2009 chez des enfants hospitalisés pour une infection respiratoire aiguë. Nos résultats fournissent de nouvelles informations sur ce génotype ré-émergent qui semble présenter un tropisme respiratoire spécifique des voies respiratoires inférieures.La seconde phase de ce travail de thèse s'est intéressée à étudier les mécanismes liés au développent des processus inflammatoires de la muqueuse au cours de l'infection des voies respiratoires basses par HEV. A l'aide d'un modèle in vitro de cellules respiratoires humaines (A549) infectées par HEV-B (CVB5, Mitchell), nous avons observé que l'infection réplicative des HEV dans les cellules A549 induisait une augmentation dose et temps-dépendante des ARNm, et des protéines IL-8, MCP-1 et RANTES.En conclusion, nos résultats obtenus à partir de prélèvements respiratoires dans le cadre de notre étude de cohorte suggèrent que les EVs représentent une cause étiologique fréquente d'infections respiratoires basses chez l'enfant avec une pathogénicité supérieure des HEVs (principalement dans notre étude HEV-68) par rapport aux souches HRVs. De plus, nos résultats obtenus à partir d'expérimentation in vitro démontrent que les HEVs du groupe B sont capables d'induire au cours de l'infection des cellules épithéliales alvéolaires humaines (A459) une sécrétion spécifique d'IL-8, MCP-1 et RANTES. La production de ces chimiokines correspond à une réponse innée de la cellule épithéliale humaine infectée par les HEVs: nous avons montré pour la première fois que ce mécanisme était en partie régulé par l'activation de la voie non canonique de NF-kB via la protéine NIK dans la cellule épithéliale respiratoire humaine.The Enterovirus (EV) genus (picornaviridae family) consists of small non-enveloped positive RNA viruses classified in 12 species of which 7 are pathogenic for humans: four species (A-D) of human enterovirus (HEV) and three species (A-C) of human rhinovirus (HRV). Among the EV genus, HEV and HRV are recognized as leading causes of acute respiratory tract infections (ARTIs) in human. Between 2009 and 2012, new HEV respiratory genotypes (e. g. HEV-68, 104, 109, 117 and CVA-21) have been described in isolated cases or outbreaks supporting the ability HEV species A to D to induce lower respiratory tract infections. This supports the hypothesis of an underestimation of the prevalence and etiological role of EVs in pediatric acute respiratory tract infections (ARTIs) (more specifically bronchitis, bronchiolitis and asthma exacerbation).To assess the etiological role and the clinical characteristics of HRV and HEV infections in pediatric patients hospitalized for ARTIs, we conducted a retrospective study of 309 hospitalized pediatric patients in University Hospital Centre of Reims with microbiologically unexplained ARTIs from September 2009 to June 2010. Among the 309 ARTIs, 15 HEV and 172 HRV strains were identified. Among bronchiolitis and asthma exacerbation cases (n=133), HEV infected cases were older (P=0.003) and were more frequently associated with a respiratory distress (P=0.01) and a need for oxygen therapy at the time of admission (P=0.01) than cases infected by HRV strains. Interestingly, during this retrospective study, we provided evidence that during the fall 2009 in France, HEV-D68 strains were responsible for a low proportion of pediatric cases hospitalized for acute airway diseases including bronchiolitis and asthma exacerbation.To identify the mechanisms that can regulate the development of airway mucosa inflammation during HEV respiratory lower tract infections, we investigated the production of chemokines by HEV infected human alveolar epithelial cells (A549). Using in vitro model A549 cells infected by HEV-B (CVB5, Mitchell), we demonstrated that HEV-B strains isolated from upper respiratory tract of child with bronchiolitis could actively replicate in various human airway epithelial cells, and that this replicative infection induced specific dose and time-dependent increases in mRNA and protein secretion of IL-8, MCP-1 and RANTES, but not of all other CC and CXC human chemokines tested. The protein secretion of these chemokines appeared to be significantly increased at 48 and 72 hours post-infection in culture treated by low-doses of IFN-γ in comparison with mock-infected cells (P <0.001), and was correlated to the viral replication activity. In second time, we explore the pathogenic mechanisms that can regulate inflammatory responses to HEV in lower respiratory airways. We show that HEV infection induced a time-dependent increase of NIK protein accumulation that peaks at 16 hours post-infection (H P-I). NIK protein accumulation mediated the processing of p100 in p52, which association with Rel B was evidenced in nuclear compartment between 16 and 48 H P-I.In conclusion, our findings indicate that EVs are a common cause of lower respiratory tract infections in pediatric patients with a potential higher pathogenicity of HEV strains (mainly HEV-68) by comparison to HRV strains. Moreover, our in vitro results demonstrated that HEV are capable to induce the release of specific chemokines (IL -8, MCP-1 and RANTES) by alveolar epithelial cells during a replicative infection. Finally, we demonstrated for the first time that this innate airway epithelial cell response against HEV infection was partly regulated by the activation of the non-canonical NF-kB via NIK protein

SARS-CoV-2 Vaccination: What Can We Expect Now?

International audienceAt the beginning of summer 2022, my colleagues and I wanted to share some thoughts about a vaccination success story [...

Entérovirus non poliomyélitiques et pathologies respiratoires

Les entérovirus (Picornaviridae) sont des agents infectieux communs divisés en 4 espèces (entérovirus humains, espèces A à D) qui regroupent actuellement 108 sérotypes. Ces virus non enveloppés à ARN simple brin de polarité positive, très résistants dans le milieu extérieur, se transmettent principalement par voie fécale-orale mais également par voie aérienne. Même si la grande majorité des infections à entérovirus est asymptomatique, ces pathogènes ubiquitaires sont responsables de syndromes infectieux incluant des infections des voies respiratoires hautes (sinusites, pharyngites, otites) ou basses (pneumonies, bronchiolites ou exacerbation de l’asthme infantile). Des travaux récents indiquent que les entérovirus seraient la troisième cause virale de bronchiolite chez les enfants âgés de 1 à 12 mois. Par ailleurs, des cas sporadiques et parfois mortels de pneumonies dues au virus coxsackie A16 (CV-A16), à l’entérovirus 71 (EV-71) et à un nouveau génotype d’entérovirus (EV-104) ont été décrits et montrent la capacité des entérovirus humains des espèces A à C à induire des infections respiratoires basses sévères. Les données épidémiologiques actuelles ainsi que la capacité d’évolution génétique rapide des souches d’entérovirus montrent que ces virus ont un fort potentiel d’émergence en tant que pathogènes humains. Elles soulignent la nécessité de développer de nouvelles stratégies préventives, diagnostiques et thérapeutiques pour lutter contre les infections pédiatriques respiratoires par le

Analysis of transcription and translation viral activities of wild type (WT) and 5’ terminally deleted enteroviruses in a model of cultured primary human cardiomyocytes

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Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection

International audienceThe early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (OAF). There is, however, accumulating evidence suggesting that E6 interaction with OAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential OAF co-receptor during E6 infection. Using stably transfected Chinese Hamster Ovary (CHO) cells expressing CAR and DAF receptors, we found that DAF expression allowed attachment of both haemagglutinating and non-haemagglutinating E6 strains but was not sufficient for promoting E6 cell entry. Interestingly, the co-expression of DAF and CAR rendered 0.1-0.2% of cells permissive to some E6 strains' infection. Although our results did not show a major role of the CAR/DAF cooperation for E6 infection, it nevertheless indicated the use of CAR in the cell entry step of some minor E6 quasispecies. Moreover, the present report validates the use of recombinant CHO cells as valuable cellular model for the further characterisation of E6 receptors. (C) 2011 Elsevier B.V. All rights reserved

Evaluation of Four Commercial Multiplex Molecular Tests for the Diagnosis of Acute Respiratory Infections

International audienceAcute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden's index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86-1.00; PPV:78.95-100.00; Sp:97.32-100.00] & B [YI:0.44-1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50-0.99; PPV:85.71-100.00; Sp:99.38-100.00], hMPV [YI:0.71-1.00; PPV:83.33-100.00; Sp:99.37-100.00], EV/hRV [YI:0.62-0.82; PPV:93.33-100.00; Sp:94.48-100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20-0.80; PPV:57.14-100.00; Sp:98.14-100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections

First demonstration of the circulation of a pneumovirus in French pigs by detection of anti-swine orthopneumovirus nucleoprotein antibodies

Abstract The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms

Major Persistent 5′ Terminally Deleted Coxsackievirus B3 Populations in Human Endomyocardial Tissues

We performed deep sequencing analysis of the enterovirus 5′ noncoding region in cardiac biopsies from a patient with dilated cardiomyopathy. Results displayed a mix of deleted and full-length coxsackievirus B3, characterized by a low viral RNA load (8.102 copies/μg of nucleic acids) and a low viral RNA positive-sense to RNA negative-sense ratio of 4.8

Rapid Detection of Respiratory Tract Viral Infections and Coinfections in Patients with Influenza-Like Illnesses by Use of Reverse Transcription-PCR DNA Microarray Systems ▿

We prospectively tested 95 nasal swabs or nasopharyngeal aspirates taken from 56 adults and 39 children visiting the Reims University Medical Centre (northern France) for influenza-like illnesses (ILI) during the early stage of the French influenza A/H1N1v pandemic (October 2009). Respiratory samples were tested using a combination of two commercially available reverse transcription-PCR (RT-PCR) DNA microarray systems allowing rapid detection of influenza A virus strains, including the new A/H1N1v strain as well as 20 other common or newly discovered respiratory viruses. Concomitantly, a generic and classical real-time RT-PCR assay was performed to detect all circulating influenza A virus strains in the same samples. Of the 95 respiratory samples tested, 30 (31%) were positive for the detection of influenza A/H1N1v virus infection by both RT-PCR DNA microarray and classical real-time RT-PCR detection assays. Among the infections, 25 (83%) were monoinfections, whereas 5 (17%) were multiple infections associating influenza A/H1N1v virus with coronavirus (CoV), human bocavirus (HBoV), respiratory syncytial virus (RSV), or human rhinoviruses (HRVs). Of the 95 respiratory samples tested, 35 (37%) were positive for respiratory viruses other than influenza A/H1N1v virus. Among these infections, we observed 30 monoinfections (HRVs [63%], parainfluenza viruses [PIVs] [20%]), influenza A/H3N2 virus [6%], coronavirus [4%], and HBoV [4%]) and 5 multiple infections, in which HRVs and PIVs were the most frequently detected viruses. No specific single or mixed viral infections appeared to be associated significantly with secondary hospitalization in infectious disease or intensive care departments during the study period (P > 0.5). The use of RT-PCR DNA microarray systems in clinical virology practice allows the rapid and accurate detection of conventional and newly discovered viral respiratory pathogens in patients suffering from ILI and therefore could be of major interest for development of new epidemiological survey systems for respiratory viral infections

Prevalence of Rotavirus, Adenovirus, Norovirus, and Astrovirus Infections and Coinfections among Hospitalized Children in Northern France▿

From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (P = 0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments