The emergence of viruses in the family Paramyxoviridae, especially those such as Hendra virus and Nipah virus (NiV) that are zoonotic, highlighted the severity of disease that could be caused by infection with viruses belonging to this family. In addition to causing disease outbreaks, several newly discovered paramyxoviruses were found to have unique genetic features, which provoked renewed interest in the study of previously unclassified or uncharacterised viruses in this family. J-virus (JPV) was isolated from wild mice, in Queensland, Australia, in 1972, and has been suggested to be a natural respiratory pathogen of mice. Beilong virus (BeiPV), another paramyxovirus, was first isolated from human mesangial cells in Beijing, China, in 2003, and was subsequently detected in rat mesangial cells. Following initial characterisation, the genomes of JPV and BeiPV were found to contain two genes, SH and TM, not common to other paramyxoviruses, as well as an extended attachment protein gene. BeiPV has the largest genome in the family Paramyxoviridae, which is, in fact, larger than that of any other virus within the order Mononegavirales. The genetic material of paramyxoviruses is not amenable to manipulation via classical genetics; a reverse genetics approach was therefore employed to study the evolution and classification of JPV and BeiPV. Minireplicon systems utilising green fluorescent protein as a reporter were established for JPV, BeiPV and NiV, and were used to better assess the taxonomic status of JPV and BeiPV, and to determine the relationship between these viruses and henipaviruses, which also have exceptionally large genomes. These studies indicate that JPV and BeiPV are closely related and should be classified in the same genus and their replication and transcription machinery is different from that of the henipaviruses. / To gain an understanding of the biology of JPV and BeiPV, viral surface proteins from JPV were expressed and evaluated. Chimeric JPV virions containing recombinant surface proteins were generated and electron microscopy was used to determine the localisation of the proteins encoded by those JPV genes which are uncommon in other paramyxoviruses. Analysis of the attachment protein gene of JPV indicated that the virus was able to assemble an exceptionally large protein (156 kDa) into the virion structure, providing evidence in support of the hypothesis that JPV and BeiPV may represent an ancient lineage of viruses within the family Paramyxoviridae. In order to determine tissue tropism of JPV during experimental infection and to aid future work with a full-length JPV infectious clone, a real-time PCR assay for JPV was developed and assessed on tissues collected from mice infected with JPV. A multiplex microsphere assay for JPV and BeiPV was developed and used to analyse the seroprevalence of these viruses in Australian and Malaysian rodents. Although there is currently no evidence for disease caused by JPV or BeiPV, this does not preclude the emergence of a zoonotic rodent paramyxovirus related to these viruses. If this were to occur, the tools for virus detection and serological monitoring are now established.

Les virus Nipah et Hendra sont deux Paramyxovirus émergents zoonotique apparu ces 15 dernières années en Asie du Sud-Est et en Australie. Ils sont responsables chez l'homme d'encéphalites dont le taux de mortalité peut dépasser les 90%. Il n'existe ni traitements, ni vaccins commercialisés. Ces virus sont donc classé P4. En étudiant la régulation négative de leur récepteur éphrineB2, j'ai pu mettre en évidence un mécanisme d'entrée endocytique pour le virus Nipah : la macropinocytose. Les Henipavirus sont les seuls Paramyxovirus connus dont on a pu démontré un tel mode d'entrée. En mimant le ligand naturel d'éphrineB2 (EphB4), les glycoprotéines virales G provoquent la rétractation des filopodes qui forment autour du virus des macropinosomes. De plus, l'entrée de ces virus peut-être bloquée in vitro par des inhibiteur de macropinocytose. Certain de ces inhibiteurs sont déjà utilisé en médecine humaine, ce qui ouvre la voie à un traitement peu onéreux contre ces dangereux pathogènes.

Public health professionals strive to understand how viruses are distributed in the environment, the factors that facilitate viral transmission, and the diversity of viral agents capable of infecting humans to characterize disease burdens and design effective disease intervention strategies. The public health discipline of conservation medicine supports this endeavor by encouraging researchers to identify previously unknown etiologic agents in wildlife and analyze the ecologic of basis of disease. Within this framework, this research reports the first examination of the prevalence in Southeast Asia of the orthoreovirus Nelson Bay virus in humans and in the Pteropus bat reservoir of the virus. Contact with Pteropus species bats places humans at risk for Nipah virus transmission, an important emerging infectious disease. This research furthermore explores the environmental determinants of Nelson Bay and Nipah viral prevalence in Pteropus bats and reports the characterization of two novel orthoreoviruses isolated from bat tissues collected in Bangladesh.

Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia, Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate the unique genomic characteristics of NiV and HeV, a new genus within the family Paramyxoviridae was created, named Henipavirus. NiV encodes two surface glycoproteins: the attachment glycoprotein (G) binds to the cellular receptor for the virus, while the fusion glycoprotein (F) mediates membrane fusion between the virus and cell membranes. Expression of F and G in the same cell results in cell-cell fusion in transfected cell monolayers, while expression of F and G on their own in cell monolayers does not result in fusion. Co-culture of singly-transfected F and G cells also does not result in fusion. Expression of NiV G in transgenic CRFK cells results in resistance to NiV- and HeV-induced cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3. Chimeric glycoproteins derived from NiV G and CDV H were constructed and characterized. None of the chimeric glycoproteins were able to fuse when coexpressed with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA). None of the chimeric glycoproteins altered cell surface expression levels of ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its release from infected cells. rCDV eGFPH NiVFG was poorly released from infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface down-regulation of the viral receptors ephrin-B2 and ephrin-B3. / October 2007

Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia, Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate the unique genomic characteristics of NiV and HeV, a new genus within the family Paramyxoviridae was created, named Henipavirus. NiV encodes two surface glycoproteins: the attachment glycoprotein (G) binds to the cellular receptor for the virus, while the fusion glycoprotein (F) mediates membrane fusion between the virus and cell membranes. Expression of F and G in the same cell results in cell-cell fusion in transfected cell monolayers, while expression of F and G on their own in cell monolayers does not result in fusion. Co-culture of singly-transfected F and G cells also does not result in fusion. Expression of NiV G in transgenic CRFK cells results in resistance to NiV- and HeV-induced cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3. Chimeric glycoproteins derived from NiV G and CDV H were constructed and characterized. None of the chimeric glycoproteins were able to fuse when coexpressed with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA). None of the chimeric glycoproteins altered cell surface expression levels of ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its release from infected cells. rCDV eGFPH NiVFG was poorly released from infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface down-regulation of the viral receptors ephrin-B2 and ephrin-B3.

Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia, Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate the unique genomic characteristics of NiV and HeV, a new genus within the family Paramyxoviridae was created, named Henipavirus. NiV encodes two surface glycoproteins: the attachment glycoprotein (G) binds to the cellular receptor for the virus, while the fusion glycoprotein (F) mediates membrane fusion between the virus and cell membranes. Expression of F and G in the same cell results in cell-cell fusion in transfected cell monolayers, while expression of F and G on their own in cell monolayers does not result in fusion. Co-culture of singly-transfected F and G cells also does not result in fusion. Expression of NiV G in transgenic CRFK cells results in resistance to NiV- and HeV-induced cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3. Chimeric glycoproteins derived from NiV G and CDV H were constructed and characterized. None of the chimeric glycoproteins were able to fuse when coexpressed with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA). None of the chimeric glycoproteins altered cell surface expression levels of ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its release from infected cells. rCDV eGFPH NiVFG was poorly released from infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface down-regulation of the viral receptors ephrin-B2 and ephrin-B3.

Les Henipavirus sont des paramyxovirus zoonotiques émergents hautement pathogènes. Ils sont capables d’infecter un large spectre d’hôtes incluant notamment la chauve-souris frugivore (réservoir naturel), le porc et l’homme. Etant donné leur très grande dangerosité et en l’absence de traitements curatifs ou prophylactiques efficaces, ces virus doivent être manipulés dans un laboratoire de classe P4. Dans une première partie, nous étudions l’effet de composés glyco-amino-glycanes sur l’infection par les Henipavirus ainsi que leur potentielle application en tant que traitement. Dans une seconde partie, nous nous attachons à comprendre les interactions entre le système immunitaire de l’hôte et le virus. Afin de mieux comprendre ces interactions, nous avons utilisé une approche basée sur l’utilisation de souris déficientes pour certaines voies de l’immunité. En effet, bien que les récepteurs cellulaires au virus (EFN B2 et B3) soient fonctionnels chez la souris, celle-ci est résistante à l’infection par voie intrapéritonéale. Nous avons analysé la susceptibilité au virus Nipah (NiV) de souris privées de différentes voies du système immunitaire inné et adaptatif. Les résultats obtenus permettent d’envisager certaines lignées de ces souris comme nouveaux modèles animaux pour l’étude de l’immunopathogénèse du NiV. Cette étude suggère aussi que le système interféron de type I joue un rôle crucial dans la limitation de la propagation virale vers le cerveau et que les lymphocytes T sont nécessaires à la complète élimination du virus. Les macrophages jouent, quant à eux, un rôle central et indispensable, à l’interface entre système inné et adaptatif. Enfin, nous abordons les prémices d’un projet visant à identifier les différences d’interactions au niveau moléculaire entre les protéines non-structurales du virus et les protéines du système immunitaire inné chez l’Homme et la souris afin de voir s’il se dégage des différences d’interactions pouvant expliquer les différences de pathogénie. Ces travaux ont donc permis d’identifier de nouveaux modèles animaux et de mieux caractériser les interactions entre le pathogène et le système immunitaire de l’hôte, de l’échelle moléculaire à l’échelle de l’organisme entier. Néanmoins, les mécanismes précis de ces interactions restent à élucider et permettront certainement de mieux comprendre la grande diversité de pathogénie des Henipavirus. / Henipaviruses are highly pathogenic emerging zoonotic paramyxoviruses. They can infect a broad spectrum of mammals including flying foxes (Pteropus fruit bats), its reservoir, pigs and humans. As there are neither therapeutic drugs nor efficient prophylactic treatment towards these highly lethal viruses, they have to be manipulated in biosafety level-4 laboratories. In the first part of this thesis, we study the role of glyco-amino-glycans on Henipavirus infection and their potential use as treatment. In the second part, we describe the interaction between the host immune system and the pathogen. To investigate these interactions, we took advantage of different transgenic mouse models deficient for some immune pathways. Indeed, although mice possess the viral entry receptor for Henipaviruses, they do not succumbed to intraperitoneal infection. We analyzed the susceptibility to Nipah virus (NiV) infection of mice deleted for different components of innate and adaptive immune systems. Obtained results showed that some of these mice can be used as new models for NiV immunopathogenesis study. This study also suggests that type I interferon system plays a major role in limitation of viral spreading to the brain and that T cells are necessary for full viral clearance. Macrophages act at the crossroad of immunity, between innate and adaptive system. Finally, we deal with the preliminary phases of a project which aims to identify the differences, at a molecular level, of interaction between non-structural viral proteins and innate immunity proteins in mice and human. Such differences could explain the different clinical patterns that are observed in these species. In conclusion, this thesis allowed to identify new animal models and to better characterize host-pathogen interactions, from molecular to whole organism level. However, the precise mecanisms of these interactions remain to be elucidated and would probably help to understand the great diversity of pathogeny of Henipaviruses.

See front file / Health Studies / DLITT ET PHIL (HEALTH ST)

See front file / Health Studies / DLITT ET PHIL (HEALTH ST)

Les chauve-souris représentant un réservoir important pour de nombreux virus pathogènes pour l’homme, un ensemble d’études en évolution moléculaire converge vers l’évidence d’une forte pression de sélection au niveau de gènes impliqués dans l’immunité dans l’ordre Chiroptera. En particulier, les chauves-souris du genre Pteropus hébergent des virus de la famille Henipavirus: Nipah et Hendra. Ces virus sont responsables d'épidémies en Asie du sud-est, et bien qu'ayant un taux d'incidence bas, les maladies résultantes de l'infection ont un taux de létalité allant de 40% à 90% chez l'homme. L’infection atteint aussi la plupart des mammifères avec des symptômes clinique graves, (e.g. porc ou cheval : espèces d’intérêt agronomique). La particularité du genre Pteropus est de ne pas développer ces symptômes cliniques graves d’infection. Afin d'en identifier les bases génétiques, nous avons utilisé l'analyse de sélection positive sur l’ensemble du génome codant sans restreindre notre analyse aux gènes de l’immunité. Nous avons mis en place les outils informatiques innovants et nécessaires au déploiement de cette démarche. Ces analyses, reposent sur des séquences de références pour les génomes de différentes espèces, et en absence du génome de référence pour P. giganteus, nous l’avons préalablement séquencé et assemblé. Or, tous les gènes sous sélection ne sont pas forcément liés à notre phénotype d’intérêt mais possiblement à d’autres (e.g. capacité de vol). Nous avons mis en place un algorithme afin d’établir un lien fonctionnel potentiel entre ces gènes identifiés sous sélection positive et un phénotype d’intérêt. / Bats represent a considerable reservoir for an extensive group of human pathogenic viruses. A number of molecular evolution studies points toward the evidence of a strong selection pressure in Chiroptera immune-related genes. Notably, Pteropus bats host viruses from Henipavirus genus: Nipah and Hendra. These viruses are responsible for epidemics in South-Est Asia, and, while the incidence is low, the resulting diseases are highly lethal, ranging between 40 to 90% in humans. Most of mammals are susceptible to the infection (including pigs and horses, animals valued in agronomy), and develop severe clinical symptoms. Specificity of Pteropus genus lies in the absence of clinical symptoms following the infection. In order to identify the genetic basis of this interesting phenomenon, we applied positive selection analysis to the entire coding genome, without bounding our analysis to immune-regulating genes. We have set breakthrough computational tools, without which our analysis would not have been possible. Reference sequences from genome of several species are the groundwork for our analysis. As P. giganteus reference genome has not yet been resolved, we sequenced and assembled it. However, not all genes under positive selection are necessarily linked to a phenotype of interest, but may be linked to other phenotypes (such as the flying ability). We have thus developed an algorithm to establish a possible functional link between the genes identified under positive selection and a phenotype of interest, which allows new perspectives in phylogenomic research.