Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

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.

Le virus Nipah (NiV) est un virus hautement pathogène responsable d’encéphalites et de syndromes respiratoires sévères chez l’humain. Les chauves-souris appartenant au genre Pteropus sont le réservoir naturel du NiV et ne développent pas de symptômes cliniques d’infection. Comprendre les relations entre l’hôte réservoir et le pathogène requiert la disponibilité de modèles pertinents pour l’étude des interactions. Les études portent à la fois sur le virus et son hôte. Ainsi, nous avons caractérisé phylogénétiquement la souche cambodgienne du NiV isolée de chauves-souris Pteropus et nous l’avons comparée avec les souches isolées chez l’homme. De plus, en absence du génome de référence pour l’espèce de chauve-souris Pteropus giganteus, nous avons séquencé et assemblé le génome de cette espèce, hôte réservoir de la souche NiV-Bangladesh, qui est en circulation actuellement. Enfin, afin d’obtenir des phénotypes cellulaires plus pertinents que des cellules immortalisées pour l’étude des interactions entre le NiV et les chauves-souris du genre Pteropus – les seules disponibles actuellement - nous avons utilisé la reprogrammation somatique sur des cellules primaires de chauve-souris Pteropus. Cette technique permet d’obtenir des cellules souches présentant la capacité d’autorenouvellement et de différenciation. En utilisant une combinaison originale de trois facteurs de transcription, nous avons généré les premières cellules reprogrammées de chauves-souris Pteropus exprimant des caractéristiques de cellules souches. Nous avons démontré que ces cellules sont très susceptibles à l’infection par le NiV mais incapables de produire de l’interféron et d’activer les cascades de signalisations antivirales en réponse à une stimulation avec de l’ARN double brin, contrairement aux cellules primaires. Le développement de ce modèle original ouvre de nouvelles perspectives pour l’étude des interactions entre l’hôte réservoir et le pathogène et pour l’identification de facteurs contrôlant la susceptibilité à l’infection par le NiV, et potentiellement par d’autres virus hébergés par des chauves-souris. / Nipah virus (NiV) is a highly pathogenic virus that causes encephalitis and severe respiratory syndromes in humans. Pteropus bats are the reservoir of NiV and do not show any clinical symptoms. In order to understand the host reservoir - pathogen interactions, the relevant models are needed. Such studies focus on both the virus and its host. A phylogenetically characterization of the NiV Cambodian strain obtained from Pteropus bats was performed and this virus was compared with human ones. In addition, we sequenced and assembled the genome of Pteropus giganteus bat, the natural host of the NiV-Bangladesh strain, which is currently circulating. Up to date, most studies have used immortalized primary cells that are not natural target of the virus. In order to get reprogrammed stem cells, a somatic reprogramming approach was applied to various Pteropus primary cells. The reprogrammed cells are capable of self-renew and differente in different cell lineages. Using an original mix of transcription factors, we derived reprogrammed cells exhibiting stem cells features. We demonstrated the high susceptibly of these cells to henipavirus infections compared with the very low level of infection of the initial primary cells. Generated bat reprogrammed cells do not induce interferon production and signalisation in response to dsRNA. The development of this original model opens new perspectives on virus-host interaction studies, especially that of cellular anti-viral response by identifying factors controlling either susceptibility or restriction to the NiV infection, and possibly other viruses hosted by bats.

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.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.

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.