Les entérovirus (EV) sont des agents étiologiques de nombreuses pathologies chez les adultes et les enfants, y compris la poliomyélite qui a été éradiquée en France. Aujourd’hui, la surveillance d’EV se déroule dans le cadre de la vigilance post-éradication, et fournit des données épidémiologiques importants sur des entérovirus non polio.A Marseille, la surveillance a amené à l’analyse de 654 souches isolées entre 1985 et 2005. Les EV de l'espèce B étaient prédominants, parmi lesquels l’echovirus 30 (E30) a été le plus fréquemment isolé et l’E13 a émergé lors de l’épidémie en 2000. Notre analyse des souches cliniques sur 20 ans renforce la stratégie de sérotypage par la VP1. L'analyse phylogénétique a identifié des souches de différents sérotypes, avec des régions nonstructurales génétiquement proches associées aux VP1 divergents. Ceci contredit le modèle actuel de la recombinaison et pourrait être à l’origine de l'émergence d’E13 épidémique.Deux épidémies majeures d’E30 ont été décrites en 2000 et 2005. Entre elles, le protocole de diagnostic d’EV a été modifié: en 2000, la détection s’est fait par la culture cellulaire et la RT-PCR classique; en 2005, la technique de la RT-PCR en temps réel a été utilisée. Ainsi, l’épidémie de 2005 a été caractérisée par une réduction significative du délai nécessaire de livrer les résultats du diagnostic et de la durée du séjour hospitalier.Nous avons également adapté un système moléculaire pour la détection d'EV71. EV71 est associé à des épidémies du syndrome pied, main, bouche en Asie, mais peut aussi engendrer des complications neurologiques fatales. Nous avons testé 365 échantillons positifs pour l’EV. 3 cas d'EV71 du génogroupe C2 ont été détectés entre 2009 et 2011 chez les enfants sans histoires de voyage récent, ce qui confirme la circulation actuelle de ce génogroupe en France.Les entérovirus ont le potentiel de provoquer des épidémies fréquentes, à cause de leur diversité génétique importante et de leur capacité de réémergence. Il est nécessaire de maintenir la surveillance d'EV par la détection et l'analyse des virus en circulation, et de développer d’autres techniques rapides de détection et d’identification. / Enteroviruses are single-stranded RNA viruses associated with a myriad of pathologies in adult and paediatric populations, the most notable of which, poliomyelitis, has been eradicated in France. Today, enterovirus surveillance is carried out in the context of post-eradication monitoring, and provides important epidemiological data for nonpolio enteroviruses.In Marseille, surveillance efforts culminated in the compilation and analysis of 654 strains isolated between 1985 and 2005. Predominant serotypes belonged to the B species: Echovirus 30 (E30) was the most frequently isolated serotype and E13 emerged during the 2000 epidemic. Our analysis of clinical strains over 20 years lends credence to the VP1 serotyping strategy. Phylogenetic analysis identified strains of different serotypes which were genetically similar in the nonstructural regions despite distinct VP1 regions. This observation contradicts the current model of recombination and could explain the emergence of epidemic E13.Two large outbreaks of E30 were described in 2000 and 2005, in between which EV diagnostic protocol was changed: in 2000, detection was performed using cell culture and classic RT-PCR techniques; in 2005, this was done via real-time RT-PCR. As a result, the 2005 outbreak was characterised by a significant decrease in the time needed to deliver diagnostic results, as well as in the length of hospital stay.We also adapted a real-time molecular assay for the detection of EV71. EV71 is associated with major outbreaks of hand, foot and mouth disease in the Asia-Pacific region, but can also cause fatal neurological complications. We screened 356 EV-positive samples and detected three cases of genogroup C2 EV71 infection between 2009 and 2011 in young children with no history of travel, confirming the current circulation of EV71 in France.Enteroviruses have the potential to cause frequent epidemics, due to their great genetic diversity and their propensity for re-emergence. This underscores the need to maintain EV surveillance by analysing past and present circulating viruses, as well as to develop more rapid detection and identification techniques.

Le cycle cellulaire des entérovirus est connu grâce aux nombreux travaux publiés portant sur les<br />poliovirus. Il reste néanmoins de nombreuses interrogations concernant les premières étapes de<br />l'infection correspondant à l'attachement et à l'entrée du virus dans la cellule. Elles constituent pourtant des cibles potentielles pour de futurs antiviraux. Nous nous sommes donc intéressés aux étapes précoces de l'infection par echovirus 6, un des nombreux sérotypes du genre Enterovirus. A partir d'une souche isolée chez un patient, deux populations virales se distinguant par leur capacité à hémagglutiner (HAE6 et NHAE6) ont été sélectionnées par passages successifs sur cellules PLC et HeLa. L'utilisation de composés chimiques et de formes mutées de protéines impliquées dans l'endocytose ont permis de caractériser leurs voies d'entrée. HAE6 semble pénétrer dans la cellule par un sous-type de radeaux lipidiques, les caveolae, alors que NHAE6 a recours aux puits tapissés de clathrine. La comparaison des séquences codant pour les 4 protéines de capside a ensuite révélé 4 substitutions d'acides aminés localisées dans des régions très exposées de la capside virale impliquées dans la liaison du virus à son récepteur cellulaire, suggérant l'utilisation de récepteurs différents. Ainsi, une affinité 4 fois supérieure pour DAF, une protéine présente dans les radeaux lipidiques, orienterait l'endocytose de HAE6 vers les caveolae. Parmi les autres candidats potentiels évalués, ni les acides sialiques, ni l'intégrine VLA-2, ni CAR, le récepteur des coxsackie et adénovirus, interviennent dans l'infection par echovirus 6 tandis que les sulfates d'héparanes joueraient simplement le rôle de récepteurs d'attachement. Finalement, seule la co-expression de DAF et de CAR a permis l'infection productive par HAE6 et NHAE6 de cellules CHO démontrant pour la première fois le rôle de CAR comme récepteur pour un echovirus. Ces données montrent qu'une souche virale isolée d'un patient est en fait constituée d'un mélange de quasi-espèces susceptibles d'entrer dans la cellule par différentes voies d'endocytose conférant au virus une capacité d'adaptation majeure aux multiples environnements rencontrés au cours de l'infection.

The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health.

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.                   In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.                   As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

Title from PDF of title page. / Document formatted into pages; contains 86 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. / ABSTRACT: The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. / ABSTRACT: The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.