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With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.

Die präsentierte Studie stellt die regionale Epidemiologie humaner Rhinoviren in einer erwachsen Patientenpopulation des Raums Leipzig dar. Hierfür wurden 284 Proben von ambulanten und stationären Patienten in 4 Saisons von 2013 bis 2017 aus respiratorischen Untersuchungsmaterialien gesammelt und mittels RT-PCR und anschließender Sanger Sequenzierung untersucht. Die resultierenden Ergebnisse wurden gemeinsam mit retrospektiv erhobenen klinischen Daten aus den jeweiligen Patientenakten statistisch untersucht. Die drei Spezies RV-A, RV-B und RV-C stellten sich in einer Verteilung von 60,9% zu 12,7% und 24,6% dar. Es zeigte sich eine große Vielfalt an verschiedenen Genotypen, deren Verteilung und Zirkulationsmuster untersucht wurden. Unter Einbeziehung der klinischen Patienteninformationen konnten Assoziationen zwischen RV-Genotypen, Ko-Infektionen und Klinik dargestellt werden. Abschließend sind die Daten und Resultate im Kontext europäischer und internationaler Studienergebnisse reflektiert und evaluiert worden.:Abkürzungsverzeichnis 1. Einführung 1.1 Rhinoviren 1.1.1 Genomorganisation und Virusstruktur 1.1.2 Oberflächenrezeptoren 1.1.3 Taxonomie 1.1.4 Virologische Diagnostik 1.1.5 Transmission, Infektion und Epidemiologie 1.1.6 Pathophysiologie und Klinik 1.1.7 Ableitung der Rationale für die publizierte Studie 2. Formatierte Publikation 3. Zusammenfassung der Arbeit Literaturverzeichnis Darstellung des eigenen Beitrags zur Arbeit Erklärung über die eigenständige Abfassung der Arbeit Lebenslauf Verzeichnis wissenschaftlicher Veröffentlichungen Danksagung

Les entérovirus du groupe B (EV-B) sont une cause majeure de myocardite aiguë, précurseur de la myocardite chronique et de la cardiomyopathie dilatée (CMD) chez l’homme. Les mécanismes moléculaires viraux impliqués dans la progression de la myocardite aiguë vers la phase chronique et la CMD restent inconnus. En utilisant une approche NGS, nous avons détecté des populations persistantes majoritaires d’EV-B tronquées en extrémité 5’, associées à des formes complètes mineures dans des cas de CMD. Afin évaluer leur impact sur la fonctionnalité des cardiomyocytes, nous avons transfecté dans des cardiomyocytes primaires (HCM) des ARN viraux clonés et identiques à ceux détectés dans les cas de CMD. Les formes EV-B majoritaires tronquées en extrémité 5’, seules ou associées à des populations complètes « auxiliaires » pourraient altérer les fonctions des HCM par des activités de la P2A virale. L'existence de mécanismes de recombinaison génomique entre les populations virales persistantes tronquées et complètes a été étudiée par un test de recombinaison d’ARN EV-B défectifs transfectés dans des HCM. Cette approche in vitro a produit majoritairement des recombinants non-homologues caractérisés par des échanges génétiques dans la région 5’NC (spacer1/2). Nos résultats indiquent l’existence d’événements de recombinaison génomique en région 5’ entre les populations d’EV-B tronquées et complètes qui pourraient contribuer au développement de la CMD. Une meilleure compréhension des mécanismes de persistance virale permettra le développement de nouvelles stratégies thérapeutiques pour lutter contre les infections chroniques par les EV-B. / Group-B Enteroviruses (EV-B) are a common cause of human acute myocarditis, a disease that is a precursor of chronic myocarditis and dilated cardiomyopathy (DCM). However, the viral molecular mechanisms involved in the progression of acute to chronic myocarditis and subsequently to DCM remain unknown. Using NGS approach, we detected persistent major EV-B populations characterized by 5’ terminal genomic deletions ranging from 17 to 50 nucleotides associated with minor complete viral forms in explanted hearts of DCM cases. To assess their impact on cardiomyocyte functions, we transfected viral RNA clones mimicking the viral genomes found in patients’ tissues into primary human cardiomyocytes (HCM). Our findings demonstrated that the major persistent 5’ deleted viral forms alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by viral 2Apro activities in EV-DCM cases. To assess the existence of genomic recombination mechanisms between persistent deleted and full-length viral helper populations, we used a recombination assay based on the rescue of non-replicative EV-B RNAs transfected in HCM. This in vitro approach produced major (75%) non-homologous recombinants that nucleotides sequencing characterized modular exchanges into the spacers 1 & 2 of the 5’NC region. Our findings indicate the existence of genomic recombination events through which, 5’ deleted and complete collaborative EV-B populations could significantly contribute to the pathogenesis of unexplained DCM cases. A better understanding of these viral persistence mechanisms will stimulate new therapeutic strategies research for chronic infections caused by EV-B.

Made available in DSpace on 2016-01-26T12:51:40Z (GMT). No. of bitstreams: 1 marcelacristinabygermini_dissert.pdf: 1767156 bytes, checksum: 3ac31a5282ff9f5d13908111e0e281cb (MD5) Previous issue date: 2012-07-31 / Introduction Childhood acute infectious diarrhea is one of the biggest health problems faced by developing countries and its incidence has been increased in children who attend daycare. Objective To evaluate the possible relation between bacterial and viral enteropathogens with diarrhea in a children population of a public daycare in São José do Rio Preto city São Paulo state. Material and methods The group of Microorganisms Investigation Center (CIM) of the Medicine College of São José do Rio Preto (FAMERP) collected and processed 100 fecal samples of 50 healthy children (control group) and other 50 children who presented fecal material with compatible aspect to diarrheic clinic. Stool samples were transported in Cary Blair transport media for bacterial analysis. All specimens were examined on the day of collection according to standard bacteriologic procedures. Briefly, suggestive bacterial colonies were isolated from McConkey, Salmonella Shigella, brilliant green (after enrichment in tetrathionate broth), and Columbia agar. Isolates identified by biochemical tests were serotyped by standard techniques (EPM-Milli and Oxidase stripes plus commercially available antisera; PROBAC, Brazil). For a viral analysis, an aliquot of the obtained fecal material was frozen under -70 degrees Celsius and, afterwards, conducted to the Virology Section of the Institute Evandro Chagas, Ananindeua, Pará state. The identification of the astrovirus and calicivirus was done by RT-PCR (Polymerase chain reaction, through reverse transcriptase). Polyacrylamide gel electrophoresis (PAGE) was carried out in Tris glycine buffer and rotavirus genome profile was defined following electrophoresis of extracted dsRNA through vertical 5% acrylamide bisacrylamide gels. Results and discussion There was no difference concerning the gender between the two groups, with a slight higher representation of female 52 (52,0%). The age group ranged from 6 months to 7 years old (an average of 1,6 years). The most frequent bacteria in the population was 38 strains of E.coli (38%), distributed like this: EPEC (12%), EIEC (3%), Pseudomonas spp. (2%) and E.coli O157 (1%). Fourteen children presented mixed colonization of Enterobacter and E.coli (14,1%). The circulating of enteric viruses in the children population are the Norovirus (2%) and Astrovirus (1%). The presence of Norovirus and Astrovirus is traditionally associated with the urban area inhabitants. The food intake out of the daycare and home indicated the presence of enteropathogens. The bacterial and viral agents detected are not associated with the diarrhea occurrence in the studied population. Conclusion: The results obtained in this study demonstrated that the children who attend daycare are asymptomatic carriers of potential pathologic agents, this fact deserves further investigation in this area, as well as in other country areas. This study will be useful for creating effective strategies of prevention, control and treatment, in order to improve the life condition of the group in this work. / Introdução: A diarreéia infecciosa aguda infantil é um dos maiores problemas de saúde enfrentado pelos países em desenvolvimento e tem sua incidência aumentada em crianças que frequentam creches. Objetivo: Avaliar a possível associação de enteropatógenos bacterianos e virais com a diarreéia em uma população infantil de uma creche pública do município São José do Rio Preto SP. , pela equipe do Centro de Investigação de Microrganismos (CIM) da Faculdade de Medicina de São José do Rio Preto (FAMERP). Material e Método: A equipe do Centro de Investigação de Microrganismos (CIM) da Faculdade de Medicina de São José do Rio Preto (FAMERP) efetuou a coleta e o processamento de Foram analisadas 100 amostras fecais, provenientes de sendo 50 crianças sadias no (grupo controle) e de outras 50 crianças que apresentaram material fecal com aspecto compatível à clínica diarreéica. Para análise bacteriológica, parte do material fecal foi utilizado meio detransportadoenviado em meio de transporte Cary Blair, com imediata semeadura as amostras foram semeadas em meio Ágar MacConkey (DIFCO), Ágar SS (DIFCO), em caldo Tetrationato, anterior à semeadura em em Ágar Verde Brilhante (DIFCO) e em Àgar Columbia (DIFCO) com carvão ativado. A técnica de aglutinação a partir de uma suspensão bacteriana foi utilizada para a identificação tipagem sorológica das enterobactérias. Para análise viral, uma alíquota do material fecal obtido foi congelada a -70 graus Ccelsius e, posteriormente, encaminhada ao Setor de Virologia do Instituto Evandro Chagas, Ananindeua, Estado do Pará. APara detecção dos Astrovírus e Calicivírus foram foi realizadas por RT-PCR (Reação em Cadeia da Polimerase via transcriptase reversa). Já Aa detecção dos rotavírus foi realizada efetuada por meio de eletroforese em gel de poliacrilamida (PAGE) em tampão Tris-glicina, e oseu perfil do genômico do rotavírus foi definido após eletroforese do RNA fita dupla (dsRNA) extraído em géis verticais de bisacrilamida-acrilamida a 5%. Resultados e Discussão?: Não houve diferença quanto ao gênero entre os dois grupos, com ligeira maior representação maior frequencia do sexo feminino 52 (52,0%). A faixa etária variou de seis meses a sete anos de idade (média de 1,6 anos). As bactérias mais frequentes na população são foram 38 cepascasos de E. coli (38%), assim distribuídas: sendo EPEC (- 12%), EIEC - (3%), Pseudomonas spp. - (2%) e E. coli O157 (- 1%). Houve tambémCatorze 14 crianças apresentaram casos de colonizaçãoção mista por mista de Enterobacter e E. coli (14,1%). Os vírus entéricos circulantes nessas população infantil crianças são o Norovírus (2%) e o Astrovírus (1%). A presença de Norovírus e Astrovírus está tradicionalmente associada com àa população residente em área urbana. O consumo de alimentos fora da creche e do domicílio foi indicativo dae presença de enteropatógenos. Os agentes bacterianos e virais detectados não estão associados aos casos de diarréeia na população estudada. Conclusão: Os dados obtidos neste estudo demostram que as crianças que frequentam creches são portadores assintomáticas de potenciais agentes patogênicos, fato este merece investigação adicional nesta região área, bem como em outras regiõesdo país. Este estudo contribuirá para a criação de estratégias efetivas de prevenção, controle e tratamento, melhorando assim a condição de vida do grupo em estudo.

Les entérovirus (EV) sont des Picornavirus (virus nus à génome ARN positif), caractérisés par une grande diversité génétique et antigénique (116 types classés en 4 espèces taxonomiques EV-A à D) et une évolution rapide. Les infections humaines sont très fréquentes, hautement contagieuses à partir des selles et épidémiques. La plupart des infections sont asymptomatiques ou bénignes ; elles peuvent être graves voire mortelles, en particulier chez les jeunes enfants. La poliomyélite, modèle d’infection à EV, est en voie d’éradication grâce aux programmes de vaccination et de surveillance sous l’égide de l’OMS. La détection de poliovirus sauvages dans des pays déclarés exempts de polio depuis plusieurs années et l’émergence récente de plusieurs EV non poliomyélitiques (EV-A71, EV-D68) associés à des manifestations cliniques sévères dans plusieurs régions du monde montrent l’importance de surveiller la circulation des EV dans la population humaine. Le but de la thèse était de rechercher et caractériser les EV dans les eaux usées de l’agglomération de Clermont-Ferrand et de comparer les données à celles de la surveillance clinique pour avoir une image plus complète de la circulation virale dans la population générale. Une méthode de concentration virale à partir des eaux usées prélevées en entrée (eaux usées brutes) et sortie (eaux usées traitées) de station d’épuration a été mise au point, permettant la détection moléculaire des EV et de 6 autres virus entériques humains. La présence de génomes viraux a été détectée dans tous les échantillons d’octobre 2014 à octobre 2015, avec une médiane de 6 virus différents en entrée de station et de 4 virus en sortie. L’analyse phylogénétique des séquences d’EV et des virus des hépatites A et E présents dans les eaux usées et les prélèvements cliniques des patients hospitalisés au CHU de Clermont-Ferrand pendant la même période, a validé l’approche mise en place pour surveiller la circulation communautaire d’un virus entérique. La diversité des EV présents dans les eaux usées brutes a été analysée par séquençage d’amplicons avec une technique haut débit Illumina (metabarcoding). Les résultats montrent la présence d’une grande diversité d’EV et la circulation silencieuse de 25 types (notamment 9 EV-C, dont des séquences de poliovirus 1 vaccinal) dans la population générale. L’analyse phylogénétique des variants intra-typiques a mis en évidence plusieurs profils épidémiques parmi les principaux types ayant circulé pendant la période d’étude. Les données obtenues montrent la faisabilité et la sensibilité de la stratégie développée pour détecter et caractériser les EV présents dans les eaux usées. Ils permettent de discuter la place de la surveillance environnementale dans la surveillance des infections à EV non polio (études épidémiologiques, prévention des épidémies, alertes sanitaires). Surveiller conjointement les virus entériques dans l’environnement et chez les patients permet une meilleure compréhension de leur prévalence. Cette approche globale de la circulation virale et de l’écologie de la santé représente un engagement important de la part des laboratoires et nécessitera une intégration dans des réseaux structurés de collaboration nationales et internationales dépassant la seule surveillance des EV. / Enterovirus (EV) are Picornaviruses (non-enveloped, positive-sense RNA viruses), characterized by a large genetic and antigenic diversity (116 types classified within 4 taxonomic species EV-A to D) and rapid evolution. Human infections are frequent, highly contagious from stools and occur as outbreaks. The infections are mainly asymptomatic or benign but severe or fatal cases can be reported in young children. Poliomyelitis is the model EV infection. Combined with clinical and virological surveillance, mass vaccination is closer than ever to achieve the WHO program of the Global Polio Eradication Initiative. However, the detection of wild type polioviruses in polio-free countries and the recent worldwide emergence of non-polio enteroviruses (EV-A71, EV-D68) associated with severe clinical manifestations underscore the importance of surveilling EV circulation in the general population. The aim of the PhD thesis was the detection and identification of EV strains in wastewater treated in the sewage treatment plant at Clermont-Ferrand (France). The viral data were compared with those reported through clinical surveillance to obtain a comprehensive picture of the viral circulation in the local population. A method was developed to concentrate viruses from raw and treated wastewater and molecular assays were used to detect EVs and 6 other human enteric viruses. The viral genomes were detected in all samples from October 2014 to October 2015, with a median of 6 and 4 different viruses in raw and treated wastewater respectively. Phylogenetic analysis of viral sequences (EV, hepatitis A and E viruses) determined in wastewater and reported in patients during the sampling period, showed the efficiency of the method for surveilling enteric viruses in the community. The EV diversity in raw wastewater was analyzed by sequencing of amplicons with the Illumina high throughput technology (metabarcoding). The analysis revealed a large viral diversity and the silent circulation of 25 types not detected from hospital data (in particular 9 EV-C, of which sequences of vaccine poliovirus 1). The phylogenetic analyses of intra-typic variants showed different epidemic patterns in the predominant EV types circulating over the study period. The data demonstrate the feasibility and sensitivity of the strategy developed for the detection and characterization of EV in wastewater and provide a future prospect for the implementation of environmental surveillance of non-polio EV infections in epidemiological studies, epidemic prevention, and for health alert. Combining the surveillance of enteric viruses in the environment and in the clinical setting allows a better understanding of their prevalence. This global approach of virus circulation and ecological health represents an important investment for laboratories, which will require integration in national and international collaboration networks beyond the scope of enterovirus surveillance.

<p>The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied <i>in vitro</i> in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound.</p><p>It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which <i>in vivo</i> may increase a virus’s ability to persist in islets.</p><p>Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect.</p><p>All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis <i>in vivo</i>.</p><p>Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study.</p><p>To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells <i>in vivo</i>, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction <i>in vivo</i>.</p>

The process of β-cell destruction that leads to type 1 diabetes (T1D) is incompletely understood and it is believed to be a result of both genetic and environmental factors. Enterovirus (EV) infections of the β-cells have been proposed to be involved, however, the effects of EV infections on human β-cells have been little investigated. This thesis summarises studies of three different Coxsackie B4 virus strains that have previously been shown to infect human islets. The effects of infections with these EV were studied in vitro in human islets and in a rat insulin-producing cell line. In addition, a pilot study was performed on isolated human islets to investigate the ability to treat such infections with an antiviral compound. It was found that one of the virus strains replicated in human β-cells without affecting their main function for at least seven days, which in vivo may increase a virus’s ability to persist in islets. Nitric oxide was induced by synthetic dsRNA, poly(IC), but not by viral dsRNA in rat insulinoma cells in the presence of IFN-γ, suggesting that this mediator is not induced by EV infection in β-cells and that poly(IC) does not mimic an EV infection in this respect. All three virus strains were able to induce production of the T-cell chemoattractant interferon-γ-inducible protein 10 (IP-10) during infection of human islets, suggesting that an EV infection of the islets might trigger insulitis in vivo. Antiviral treatment was feasible in human islets, but one strain was resistant to the antiviral compound used in this study. To conclude, a potential mechanism is suggested for the involvement of EV infections in T1D. If EV infections induce IP-10 production in human islet cells in vivo, they might recruit immune cells to the islets. Together with viral persistence and/or virus-induced β-cell damage, this might trigger further immune-mediated β-cell destruction in vivo.

To enhance the global control of encephalitis and hepatitis caused by rabies virus (RABV), Japanese encephalitis virus (JEV), enterovirus 71 (EV71) and hepatitis B virus (HBV), novel immunisation strategies are needed. All four diseases particularly affect low income countries with marginal health services – an affordable combined vaccine strategy could alleviate the large burden of disease. Therefore, we aimed to construct a multipathogen vaccine assessing the immunising activity of a recombinant modified vaccinia Ankara (MVA), expressing key antigens (RABV-glycoprotein, JEV pre-membrane & envelope protein, EV71-P1 protein and large hepatitis B surface antigen) from the various pathogens. Successful delivery of the pathogen sequences into non-essential sites (deletion site I, II, VI) of MVA via homologous recombination with a transfer plasmid, was demonstrated by transient color selection (LacZ-marker) in vitro. The stable insertion of the expression cassettes was validated over ten virus passages by PCR with specific primer sets, targeting the pathogen sequence. Two recombinants, one carrying the EV71 and JEV pathogen sequence and one carrying the RABV-HBV pathogen sequence were generated and validated by PCR.To ensure similar expression of the key antigens, a T7-promoter was linked to the expression cassettes of all pathogen sequences. Direct regulation of this promoter was achieved through co-infection with a second T7-polymerase expressing MVA under the control of a vaccinia p7.5 promoter. Protein expression from recombinant MVA using the co-infection model of expression in vitro, was further characterised by Western blot, dot blot and immunocytochemistry. All inserted transgenes were expressed using an avian (chicken embryo fibroblasts) or mammalian (human fetal lung fibroblasts) cell culture system. To investigate the co-infection model of antigen delivery in vivo, a pilot murine immunogenicity study was performed in six Balb/c mice using the MVA-RABV-HBV recombinant in a homologous prime-boost regimen two weeks apart. To detect antibodies against the expressed pathogen sequences in the mouse serum an antibody-capture assay was performed (Western blot, dot blot). The antigen (used to capture murine antibodies) was purified RABV-glycoprotein or large hepatitis B surface antigen expressed from a baculovirus. The murine antibodies were detected by a secondary anti-mouse antibody, conjugated with horseradish peroxidase for a chemiluminescent reporter assay. Although, serum antibodies against MVA were induced in all mice, no serum antibodies against RABV or HBV could be detected. In summary, we were able to demonstrate that two transgenes, when inserted into one or two different loci in the MVA genome, can be expressed in vitro when using the co-infection model of gene expression with a T7-expression system. This project has provided new insights into a novel group of vaccines, the multipathogen viral vector vaccines, employing MVA as a vector. Future studies will be needed to further explore this vaccine-group, as well as the co-infection model of expression.

Les entérovirus (EV) humains, membres de la famille des Picornaviridae, comprennent plus de 100 génotypes appartenant à 4 espèces : Enterovirus A, B, C et D. Ces virus sont à l’origine de pathologies très variées et occupent une place importante en santé publique. La méthode conventionnelle de typage des EV consiste en une réaction de séroneutralisation avec des antisérums spécifiques à partir de souches isolées en culture cellulaire ; cette technique est longue, coûteuse et limitée par sa capacité à identifier correctement les variants antigéniques et les nouveaux génotypes. De plus, elle est limitée aux génotypes cultivables. De nouvelles méthodologies de typage moléculaire par séquençage partiel du génome ont été récemment développées ; elles consistent à analyser une partie variable de la région codant une des protéines de capside (VP1 ou alternativement VP2 ou VP4). Cependant ces techniques sont le plus souvent réalisées à partir de souches isolées en culture cellulaire. Le but de ce travail a été de développer une technique de typage des EV directement sur des prélèvements cliniques en se basant sur le séquençage partiel de la région VP2 dont le laboratoire avait montré précédemment l’intérêt (Nasri et al., 2007). Pour le dessin des amorces, nous avons utilisé la stratégie CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) de manière à améliorer à la fois la spécificité et la sensibilité de la méthode d’amplification. Nous présentons ici un premier article décrivant la nouvelle technique de typage VP2 et rapportons son application au typage d’échantillons cliniques trouvés positifs par une PCR ciblant la région 5’ non codante du génome des EV sur une période de trois ans. Le deuxième article présente pour la première fois l’application d’une technique de typage direct à des échantillons environnementaux d’eaux usées. Le troisième article montre l’intérêt de coupler deux techniques de typage ciblant des régions différentes (VP1 et VP2) pour l’identification de souches d’EV isolées par culture cellulaire en Centre-Afrique. Malgré des problèmes de sensibilité, cette nouvelle technique de typage directement à partir d’échantillons peut rendre de grands services tant en clinique humaine que pour la surveillance environnementale / Human enteroviruses (EV), members of the Picornaviridae family, comprise more than 100 genotypes belonging to four species: Enterovirus A, B, C and D. These viruses are responsible for a wide range of pathologies and play an important role in Public Health. The classic method for typing EVs consists in a seroneutralisation assay with specific antisera using strains isolated by cell culture; this technique is cumbersome, expensive and unable to type currently antigenic variants and new serotypes. In addition, it is limitated to culturable serotypes. New methods of molecular typing by partial sequencing of the genome have been recently developed; they consist in analysing a variable part of the region coding for capsid protein (VP1 or alternatively VP2 or VP4). However, these techniques are usually performed on strains isolated by cell culture. The aim of this work was to develop a typing method able to work from clinical specimens by partial sequencing of the VP2 region, which had been shown to exhibit a good typing performance (Nasri et al., 2007). For the design of primers, we used the CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) strategy on order to improve the sensitivity and the specificity of the amplification assay. We present herein a first article that describes in details the new VP2 typing method and requests its use for typing clinical specimens found positive by a PCR assay targeting the 5’ non coding region of EVs over a period of three years. The second paper describes for the first time the direct use of a typing method on environtmental wastewater samples. The third article shows the interest of coupling 2 typing techniques targeting different regions (VP1 and VP2) of the EV genome for the identification of strains isolated by cell culture in Republic of Central Africa. Despite a loss of sensitivity, the new VP2 typing method used directly on specimens was found to be of great help both for human diagnosis and environmental surveillance