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.

Introduction This thesis concerns the mosquito-borne arbovirus Ross River (RR) virus. The main objectives were to determine the vector associations, the incidence, costs and natural history of disease, and behavioural and environmental risks for infection in tropical Queensland. 1. Literature review On the basis of the literature review there is strong evidence that Aedes vigilax, Ae. camptorhynchus and Culex annulirostris are important vectors in Australia. Aedes camptorhychus does not occur in Queensland. There is evidence that two peri-domestic container-breeding mosquitoes, Ae. notoscriptus and Ae. aegypti, may be vectors of the virus. The virus has been isolated from many other species but the role of most of these is unclear. It is unclear which vertebrate species are the major reservoirs for human infection. Studies are inconsistent with regard to the prevalence, duration and severity of symptoms and debility during RR virus disease. Nearly all epidemiological studies of RR virus have been descriptive. Therefore one can only surmise what the risks for human infection might be. Epidemiological studies to determine the associations between exposures and risk for disease do not exist. 2. Virus isolation from mosquitoes During 1996-1998 61,619 mosquitoes were processed for virus isolation. Thirty-three isolates of RR virus were made. The largest number (14) were from Ae. carmenti. The minimum infection rate (MIR) per 1,000 was 2.4. Isolates were also made from Ae. imprimens (1 isolate, MIR 10.3), Ae. kochi (2, 0.2), Ae. lineatus (1, 0.2), Ae. notoscriptus (1, 1.6), Ae. vigilax (1, 0.3), Cx. annulirostris (9, 0.3), Cx. vicinus (1, 4.0) and Mansonia septempunctata (3, 5.8). Ross River virus has been isolated from Cx. annulirostris, Ae. vigilax, Ae. notoscriputus and Ae. kochi but not from the other species. Ross River virus was not isolated from Ae. aegypti. Twenty-six isolates came from in or near a colony of 15,000 spectacled flying-fox, Pteropus conspicillatus. The proportion of RR virus positive pools from within 1 km. of this colony was significantly greater than elsewhere for all species combined and for Cx. annulirostris but not for Ae. carmenti. 3. The incidence and costs of Ross River virus disease Unpublished data on National, State and Territory notifications was collected. Crude incidence rates using census figures for denominator data were calculated. The same was done for the areas in which the other studies described in the thesis were carried out. An estimate of the cost of Ross River virus disease in Australia was made. During the period 1991-1998 of the States and Territories the Northern Territory (NT) had the highest and the Australian Capital Territory (ACT) the lowest notification incidences. These were 62-281 and 0-3 per 100,000 per annum, respectively. During this period the notification incidence for Queensland ranged from 70 to 149 per 100,000 per annum. For the local government areas of Cairns and Mareeba, where the majority of cases of RR virus disease for the studies described in this thesis were recruited, the notification incidences were between 74 and 267, and 28 and 200, respectively. On the basis of an average of 4,800 cases per annum in Australia the cost of serological testing and medical consultations were estimated at $443,520 and $105,600. Lost earnings were estimated at $1,798,560. The total cost for medical consultations, serological testing and lost earnings will therefore be over $2 million in an average year. 4. The natural history of Ross River virus disease In 1998 incident cases of RR virus disease were ascertained. Fifty-seven eligible cases were recruited but only 47 could be reviewed on 3 occasions and data on these were analysed. Cases were followed for up to 197 days. Review included history, examination and the administration of the Clinical Health Assessment Questionnaire (CLINHAQ) and Short Form-36 (SF-36) . On initial review the 3 most common symptoms were arthralgia, joint stiffness and myalgia affecting 97.9, 89.4 and 59.6% of cases, respectively. The joint types most commonly affected by pain at the initial review were the ankles, wrists, interphalangeal joints of the fingers, knees and metacarpophalangeal joints. Objective signs of joint inflammation were rare. The prevalence of signs of inflammation decreased and the prevalence of normal joints on examination increased through the reviews. The prevalence of use of NSAIDs decreased through the reviews. On the basis of CLINHAQ items regarding work performance functioning at work improved through the course of the reviews. Linear regression with days since symptom onset as the independent variable was performed for some variables. The 8 dimensions of the SF-36 were standardized to the Queensland population and analyzed longitudinally . Analyses of the CLINHAQ functional disability index (FDI) and the visual analogue scales (VASs) for pain, global severity, fatigue, gastrointestinal complaints and sleep, and the depression and anxiety scales were also performed. The slopes of all fitted regressions except the SF-36 general health dimension were significantly different from zero. All measures of disease severity returned to normal by 8 months from onset, many in a shorter period. 5. Behavioural and environmental risks for infection Fifty-five incident cases of RR virus disease were recruited and formed the basis of a case-control study of behavioural and environmental risks. They were matched to 85 controls. In the year prior to symptom onset the only leisure exposure that significantly altered risk was camping [Odds ratio (OR) = 2.15; 95% confidence interval (CI) = 1.07-4.35]. No peri-domestic activities in the year prior to onset significantly altered disease risk. Leisure exposures were also assessed in a 3-week exposure period ending 4 days prior to symptom onset. None significantly altered risk. No peri-domestic activity in this period significantly altered risk and nor did exposure to vertebrates or mosquitoes. Containers and vegetation around the subject's dwelling did not significantly increase risk. The presence of ice cream containers and buckets was significantly protective when assessed by questionnaire, however this was also assessed by inspection and was found to increase risk though not significantly so. It was concluded that the former finding was due to differential misclassification of exposure status. The premise condition index (PCI) was measured. A low PCI for the subject's house was associated with a significantly increased risk (3-4 relative to 7-9 as reference category: OR = 3.15, 95% CI = 1.07-9.25). Window screening did not alter disease risk and air-conditioning in the house or the bedroom decreased risk but not significantly so. Use of protective measures, except bed nets, in the year prior to onset was found to decrease disease risk. Personal repellents, mosquito coils and citronella candles significantly decreased risk. A dose response was shown for the number of protective measures from personal repellents, aerosol and surface sprays, mosquito coils, citronella candles and mosquito "zappers" used in the year prior to symptom onset. Pet ownership and proximity of dwelling to horses did not significantly alter risk. A preference for light coloured clothing was significantly protective (0.37, 0.15-0.89). Stratification by gender, date of symptom onset and geographical area was performed. Stratification by geographical area included a coastal and tablelands stratum. There were differences between the stratum-specific odds ratios for camping in the year prior to symptom onset, the presence of bromeliads in the subject's garden and a preference for light coloured clothing. Multivariate analysis demonstrated confounding by use of personal repellents, mosquito coils and citronella candles. When modeled together these were found to cause confounding among themselves. They also caused significant confounding of camping, outdoor work and the presence of banana trees in the subject's yard. Multivariate analysis of the association between PCI and disease risk failed to demonstrate confounding by use of protective measures or time between symptom onset and review. 6. Synthesis and conclusions The three research Chapters form a coherent body of public health research on the epidemiology (Chapters 5 and 6) and ecology (Chapters 4 and 7) of RR virus, and the natural history of RR virus disease (Chapter 6) in tropical Queensland. Conclusions are drawn from the research in the thesis. A set of priorities for future public health research on RR virus is suggested, and a pilot control program for Ross River virus disease in tropical Queensland is recommended.