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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.

RNA:protein interactions are central in many cellular processes, including activation of innate immune responses against microbial infection. Their study is essential to better understand the diverse biological events that occur within cells. However, isolation of RNA:protein complexes is often laborious and requires specialized techniques. This thesis is concerned with attempts to develop an improved purification protocol to isolate specific RNA:protein complexes. Taking advantage of the specific interaction of the Pseudomonas aeruginosa PP7 protein with its cognate RNA binding site, termed the PP7 recognition sequence (PRS), the aim was to identify cellular proteins involved in activating cell-signalling pathways, including the interferon-induction cascade, following viral infection with stocks of parainfluenza virus 5 (PIV5) rich in copyback defective interfering (DI) particles. Copyback DI genomes are powerful inducers of IFN and, here, I show they also activate the induction of IL-6, IL-8 and TNFα; cytokines that also have antiviral properties. Following the successful cloning of the PRS into a copyback DI genome, we investigated conditions for optimal in vitro capture of DI-PRS:protein complexes by PP7 on Dynabeads. When tested, the protocol led to the successful capture of ILF3 and PKR, two dsRNA binding proteins induced by IFN. We further developed a tap-tagging system to minimize the presence of non-specifically bound proteins to Dynabeads that may interfere with future mass spectrometry analysis. To isolate DI-PRS RNA:protein complexes from infected cells, attempts were made to rescue replicating DI-PRS genomes in the context of wild type PIV5. Similarly, efforts were made to isolate influenza A virus RNPs that contained the PRS in the neuraminidase (NA) gene from infected cells using the PP7-based protocol developed. However, for reasons discussed, unfortunately RNA:proteins complexes were not successfully purified from infected cells in either case.

La faune sauvage a été depuis longtemps incriminée dans la survenue de zoonoses et joue le rôle de réservoir d'agents pathogènes (virus Nipah, Hendra, Ebola, Hantaan etc.) pour l'homme. Les îles tropicales et subtropicales du Sud-Ouest de l'Océan Indien (SOOI) constituent l'une des 34 régions reconnues comme « hotspot » de biodiversité au niveau mondial. Elles sont caractérisées par un très fort endémisme de la faune sauvage surtout sur l'Ile de Madagascar. Le caractère multi-insulaire de la région du SOOI, la diversité de ses biotopes et ses disparités biogéographiques et humaines offrent un champ d'investigation unique pour explorer « in natura » la dynamique évolutive des agents infectieux et les relations hôtes-virus. Nos travaux de recherche ont porté sur deux modèles de virus à ARN de polarité négative, les paramyxovirus et les lyssavirus. Le premier modèle viral nous a permis d'aborder les questions relatives à la dynamique de transmission virale au sein de communauté d'hôtes, plus particulièrement, les chauves-souris et les petits mammifères terrestres de Madagascar et d'identifier les facteurs agissant sur cette dynamique de transmission et de diversification virale, en particulier les facteurs bio-écologiques associés à leurs hôtes. Le second modèle viral, les lyssavirus, nous a permis de décrire sur l'ensemble des îles du SOOI échantillonés, la circulation virale dans ce système multi-insulaire diversifié, au sein des chauves-souris dont la plupart des espèces sont endémiques à cette région. Dans l'ensemble, nos investigations ont permis de mettre en évidence des échanges viraux (« host-switch ») importants entre chauves-souris, petits mammifères terrestres endémiques de Madagascar et les rongeurs introduits, le rôle de ces mammifères en tant que réservoir viral majeur et souligner le rôle disséminateur de Rattus rattus. Par ailleurs, nous avons pu identifier ce phénomène de « host-switch » comme étant le mécanisme macro-évolutif prépondérant et l'importance des facteurs biotiques et abiotiques à l'origine de la dynamique de transmission et de la diversification virale observée chez les paramyxovirus de chauves-souris de Madagascar. / Since many decades, the wild fauna has been incriminated as an important reservoir of many zoonotic pathogens (Nipah, Hendra, Ebola, Hantaan viruses etc.) at risk for humans. Tropical and subtropical islands of the South West Indian Ocean (SWIO) are part of the 34 areas of the world recognized as "hotspot" of biodiversity. They are characterized by a strong wildlife endemism especially on Madagascar. The multi-island structure of the SWIO region, the diversity of its biotopes and its biogeographical human disparities, offer a unique opportunity to investigate "in natura" the evolutionary dynamics of infectious agents and the host-virus relationships. Our research has focused on two models of negative RNA viruses, paramyxoviruses and lyssaviruses. The first virus model allowed us to address issues related to the dynamics of viral transmission within a host community, in particular, bats and small terrestrial mammals of Madagascar and to identify the driving factors, especially bio-ecological factors associated with their hosts, affecting the dynamic of transmission and of viral diversification. The second model allowed us to describe on the islands of the SWIO, the intense circulation of bats lyssaviruses in this multi-island system which bats are endemic to this region. Overall, our investigations highlighted (i) intense viral exchanges ("host-switch") between bats, endemic terrestrial small mammals and introduced rodents from Madagascar, (ii) the role of these mammals as major viral reservoir and (iii) the key role played by Rattus rattus as viral spreader. Furthermore, we identified both the phenomenon of "host-switch" as the major macro-evolutionary mechanism among bat paramyxoviruses from Madagascar and the importance of biotic and abiotic factors in shaping the transmission dynamics and viral diversification.

Les virus Nipah (NiV) et Hendra (HeV) appartiennent au genre Henipavirus au sein de la famille des Paramyxoviridae. Cette famille comporte de nombreux pathogènes tel que le virus de la rougeole (MeV). Les paramyxovirus possèdent un génome de type ARN simple brin encapsidé par la nucléoprotéine (N) au sein d'une nucléocapside hélicoïdale. N interagit avec la phosphoprotéine (P) et cette dernière recrute la polymérase (L) qui assure la transcription et la réplication du génome viral. L'objectif de mon projet de thèse était de caractériser les protéines N et P ainsi que les interactions qui existent entre elles chez les trois virus, NiV, HeV et MeV. A la différence du MeV, qui a été intensivement étudié au cours des dernières années, les données moléculaires et structurales sur les Henipavirus étaient très limitées. A l'aide d'analyses computationnelles, nous avons pu déchiffrer l'organisation modulaire de N et de P, et nous avons montré que les régions, C-terminale de N (NTAIL) et N-terminale de P (PNT), sont prédites comme intrinsèquement désordonnées (RIDs). Les RIDs sont des régions fonctionnelles dépourvues de structures secondaires et tertiaires stables dans des conditions physiologiques. En utilisant des approches biochimiques et biophysiques, nous avons confirmé que NTAIL et PNT sont désordonnées. Elles conservent toutefois des structures secondaires transitoires qui pourraient correspondre à des éléments de reconnaissance moléculaire (ou MoREs) impliqués dans de transitions structurales en présence d'un partenaire. / The Paramyxoviridae family includes many important human and animal pathogens, such as measles virus (MeV), a morbillivirus, and the emerging Nipah (NiV) and Hendra (HeV) viruses, members of the Henipavirus genus. Paramyxoviruses possess a negative-strand RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. N interacts with the phosphoprotein (P), and this latter recruits the polymerase that ensures genome replication and transcription. My PhD project has mainly focused on the characterization of the N and P proteins and on the interactions between these two proteins from the three cognate viruses, namely NiV, HeV and MeV. While MeV has been extensively studied through the past years, structural and molecular information on Henipavirus N and P proteins were rather scarce. Using computational analyses, we deciphered the modular organization of Henipavirus N and P. Intrinsically disordered regions (IDRs) were predicted within these proteins, notably at the C-terminus of N (referred to as NTAIL), and at the N-terminus of P (referred to as PNT). IDRs are functional despite they lack of a well-defined 3-D structure under physiological conditions. Biochemical and biophysical approaches pointed out a mostly disordered state for both NTAIL and PNT, although they were shown to contain short-order prone segments (i.e. molecular recognition elements, MoREs). These latter are involved in partner recognition and in disorder-to-order transitions. The C-terminal domains of the P proteins (referred to as PXD) were found to bind to NTAIL and to induce an α-helical transition thereof.

Les paramyxovirus respiratoires humains sont des virus responsables d'infections chez les jeunes enfants, les personnes âgées et les patients immuno déprimés. Ces virus possèdent deux glycoprotéines à la surface de leur enveloppe, jouant un rôle dans l'entrée du virus dans la cellule cible. La glycoprotéine d’attachement (HN, G ou H) permet l’attachement du virus à son récepteur cellulaire et, dans le cas de HN, celle-ci est suspectée d’activer la seconde glycoprotéine, la protéinede fusion (F). Cette dernière réalise la fusion entre l'enveloppe du virus et la membrane cellulaire.Le mécanisme par lequel la protéine HN "active" la protéine F reste mal caractérisé, malgré la détermination récente de leurs structures en cristallographie. Plusieurs modèles sont actuellement proposés. Ce travail de thèse s’est focalisé principalement sur les glycoprotéines d’enveloppe des virus parainfluenza humain de type 2 (hPIV-2) et parainfluenza de type 5 (PIV-5), ainsi que sur la glycoprotéine de fusion du métapneumovirus humain (hMPV). La première partie de ce projet a consisté à caractériser une mutation retrouvée sur la protéine F de souches circulantes hPIV-2. Cette étude a notamment souligné l’importance de la sous-unité F2 dans la régulation de la fusion membranaire. Puis, dans un second temps, l’une des étapes du mécanisme d’entrée du métapneumovirus a été étudiée : la fusion membranaire induite par la glycoprotéine F. Il a été démontré qu’il était possible dans une certaine mesure, et par une approche de mutagenèse combinatoire, de dissocier les caractéristiques de F hMPV et ainsi de pouvoir mieux les étudier. Ce travail d’ingénierie de la glycoprotéine F hMPV s’est également inscrit dans un objectif de recherche appliquée afin de contribuer au développement de nouveaux outils prophylactiques et thérapeutiques. Cette perspective thérapeutique de F PIV-5 a été évaluée dans le cadre d’un vecteur oncolytique basé sur l’adénovirus de type 5 (AdV-5). L’expression de cette glycoprotéine hyperfusogène a ainsi contribué à un effet cytotoxique amplifié des vecteurs armés in vitro ainsiqu’en modèle animal immunocompétent. / Human respiratory paramyxoviruses are responsible for infectious diseases and hospitalisations among infants, children, elderly and the immunocompromised. These viruses harbour two glycoproteins implicated in virus entry into the cell. The attachment glycoprotein (HN,G or H) is implicated the virus attachment on a target cell receptor, and HN is also suspected to activate the second glycoprotein, the fusion protein (F). This latter glycoprotein can perform the fusion between the cellular membrane and the viral envelope. The mechanism of activation of the F protein, is not well-defined, even with the structural characterisation for some viruses studied inthis thesis. This thesis work is focussed on the viral glycoprotein of parainfluenza virus type 2 (hPIV-2), parainfluenza virus type 5 (PIV-5), and the fusion glycoprotein of human Metapneumovirus (hMPV).The first part of this project was the characterization of a mutation observed in the F protein natural variants of hPIV-2. This work highlights the importance of the F2 subunit of F in the fusion regulation. A second part of the project consisted of the study of the mechanism of F hMPV entry into the cell, induced by F glycoprotein. This work showed that it was possible to dissociate the characteristics of the F glycoprotein, in order to allow a better understanding of these characteristics. This engineering work on the F protein was used to understand the basic science but could also be used in the development of therapeutic tools.The therapeutic use of F PIV-5 was also evaluated in an oncolytic vector based on adenovirus type 5 (AdV-5). Its expression in tumours showed a highly cytotoxic activity for the target cells in vivo, but also in vitro on immunocompetent rodents.