Thesis (Ph.D.)--UCLA, 2007. / Vita. Includes bibliographical references (leaves 105-108).

Thesis (P.h.D.)--UCLA, 2006. / Vita. Includes bibliographical references (leaves 144-166).

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.

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.

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

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.

Le virus Nipah (NiV) est un paramyxovirus zoonotique du genre Henipavirus, qui a émergé en 1998. NiV infecte l'homme et cause des troubles respiratoires et des encéphalites avec une forte létalité. A l’inverse, chez les hôtes naturels de NiV, les chauves-souris de la famille des Pteropodidae, l’infection est asymptomatique. Cependant, les mécanismes permettant aux Pteropodidae de contrôler l’infection sont inconnus à ce jour. NiV produit des protéines non structurales, V, W et C, qui sont des facteurs de virulence. V, W et C inhibent les voies de l’interféron de type 1. De plus, la protéine W inhibe la production de chimiokines in vitro et module la réponse inflammatoire in vivo, mais son mécanisme d’action reste inconnu. La voie NF-κB étant le principal régulateur de la réponse inflammatoire, nous avons émis l’hypothèse que W pourrait moduler la voie NF-κB. Nous avons démontré que la protéine W inhibe l'activation de la voie canonique de NF-κB induite par TNFα et IL-1β, effet pour lequel sa région C-terminale spécifique est nécessaire. Nous avons également identifié quels signaux d’import et d’export nucléaires de W sont nécessaires à son effet inhibiteur et ainsi mis en évidence l’importance du trafic nucléo-cytoplasmique de W pour l’inhibition de NF-κB. L’étude des interactions de W avec les protéines cellulaires nous a permis d’identifier un partenaire prometteur connu pour son rôle dans le rétrocontrôle négatif de NF-κB. Enfin, le rôle de W dans l'inhibition de la voie NF-κB a été démontré pendant l'infection par NiV. Les résultats obtenus ouvrent la voie à la compréhension du mécanisme par lequel W module la réponse inflammatoire. Finalement, afin de mieux comprendre le contrôle de l’infection de NiV par son hôte naturel, nous avons généré des lignées cellulaires primaires et immortalisées de chauve-souris Pteropus giganteus. Ces cellules devraient permettre de mieux comprendre les mécanismes par lesquels ces chauves-souris contrôlent l’infection virale. / Nipah virus (NiV), from Henipavirus genus, is a zoonotic paramyxovirus, which emerged in 1998. In humans, it causes acute respiratory distress and encephalitis with a high lethality. Conversely, the natural hosts of NiV, bats from the Pteropodidae family, are asymptomatic. The mechanisms by which the Pteropodidae control infection are unknown to date. NiV produces non-structural proteins, V, W and C, which are virulence factors. V, W and C inhibit the type 1 interferon pathways. Moreover, W inhibits the production of chemokines in vitro and modulates the inflammatory response in vivo, but its mechanism remains unknown. The NF-κB pathway being the main regulator of the inflammatory response, we hypothesized that W could modulate the NF-κB pathway. We demonstrated that protein W inhibits the activation of the NF-κB canonical pathway induced by TNFα and IL-1β. The specific C-terminal region of W is necessary for this effect. We have also identified which nuclear import and export signals of W are necessary for its inhibitory effect and thus highlight the importance of the nucleo-cytoplasmic trafficking of W for the inhibition of NF-κB. The study of the interactions of W with the cellular proteins allowed us to identify a promising partner known for its role in the negative feedback of NF-κB. Finally, the role of W in the inhibition of the NF-κB pathway was demonstrated during the infection with NiV. The results obtained open the way to understanding the mechanism by which W modulates the inflammatory response. Finally, to better understand the control of the infection of NiV by its natural host, we generated primary and immortalized cell lines of Pteropus giganteus bat. These cells should provide a better understanding of the mechanisms by which these bats control viral infection.