Bats asymptomatically host a number of viruses that are the cause of recently emergent infectious diseases in humans. While the mechanisms underlying this asymptomatic infection are currently not known, studies of sequenced bat genomes help uncover genetic adaptations in bats that may have functional importance in the antiviral response of these animals. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of the Egyptian rousette bat (ERB; Rousettus aegyptiacus), a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We used this genome to understand the evolution of immune genes and gene families in bats, and describe several observations relevant to defense against viruses. We observed an unusual expansion of the NKG2/CD94 natural killer (NK) cell receptor gene families in Egyptian rousette bats relative to other species, and found genomic evidence of unique features and expression of these receptors that may result in a net inhibitory balance within bat NK cells. The expansion of NK cell receptors is matched by an expansion of potential major histocompatibility complex (MHC) class I ligands, which are distributed both within and, surprisingly, outside the canonical MHC loci. We also observed that the type I interferon (IFN) locus is considerably expanded and diversified in the ERB, and that the IFN-ω subfamily contributes most to this expansion. To understand the functional implications of this expansion, we synthesized multiple IFN-ω proteins and examined their antiviral effects. Members of this subfamily are not constitutively expressed but are induced after viral infection, and show antiviral activity in vitro, with different antiviral potencies observed for different IFN-ω proteins. Taken together, these results show that multiple bats, including the ERB, have expanded and diversified numerous antiviral loci, and potentially developed unique adaptations in NK cell receptor signaling, and type I IFN responses. The concerted evolution of so many key components of immunity in the ERB is strongly suggestive of novel modes of antiviral defense that may contribute to the ability of bats to asymptomatically host viruses that are pathogenic in humans.

Marburg virus (MARV)–along with Ebola Virus–comprises Filoviridae, a family of virus which causes the life-threatening hemorrhagic fever in human and non-human primates for which there is no clinically approved vaccine. For this reason, this virus can potentially lend itself to pandemic and weapons of bioterrorism. Strikingly, this virus yields asymptomatic responses in its recently discovered host Rousettus aegyptiacus. Understanding of the interaction between MARV and different animal hosts will enable the improved understanding of filovirus immunology and the development of effective therapeutic agents. Although cell lines and primary cells have been used to investigate gene expression analysis of this virus, the transcriptomic view of MARV infection on the tissue samples of animal hosts has been an uncharted territory. The comprehensive analysis of transcriptome in hosts and spillover hosts will shed light on the immune responses on a molecular level and potentially allow the comparative analysis to understand the phenotypical differences. However, there have been gaps in resources necessary to carry the transcriptome research for MARV. For example, MARV host Rousettus aegyptiacus genome and transcriptome had not been available. Furthermore, the statistical machinery necessary to analyze multi-tissue/multi-time data was not available. In this dissertation, I introduce the two items that fill these gaps and show the application of the tools I built for novel biological discovery. In particular, I have built 1) the comprehensive de novo transcriptome reference of Rousettus aegyptiacus and 2) the Multilevel Analysis of Gene Expression (MAGE) pipeline to analyze the RNA-seq data with the complex experimental design. I show the application of MAGE in multi-time, multi-tissue transcriptome data of Macaca mulata infected with MARV. In this study, 15 rhesus macaques were sequentially sacrificed via aerosol exposure to MARV Angola over the course of 9 days, and 3 types of lymph node tissues (tracheobronchial, mesenteric, and inguinal) were extracted from each sample and sequenced for gene expression analysis. With MAGE pipeline, I discovered that the posterior median log2FC of genes separates the samples based on day post infection and viral load. I discovered the set of genes such as CD40LG and TMEM197 with interesting trends over time and how similar and different pathways have been influenced in three lymph nodes. I also identified the biologically meaningful clusters of genes based on the topology-based clustering algorithm known as Mapper. Using the MAGE posterior samples, I also determined the genes that are preferentially expressed in tracheobronchial lymph nodes. In addition to new analysis tools and biological findings, I built the gene expression exploration tool for biologists to examine differential gene expression over time in various immune-related pathways and contributing members of the pathways. In conclusion, I have contributed to the two important components in the transcriptome analysis in MARV research and discovered novel biological insights. The MAGE pipeline is modular and extensible and will be useful for the transcriptome research with the complex experimental designs which are becoming increasingly prevalent with the decrease in the cost of sequencing.

Le nombre de virus détectés chez les chauves-souris est en augmentation, la plupart étant des virus à ARN. L'identification chez différentes espèces de chauves-souris, de virus ayant été responsables d'épidémies voire de pandémies chez l'homme (coronavirus agent du SRAS, virus Nipah et Hendra, filovirus Ebola et Marburg) a fait prendre conscience du risque que peuvent présenter ces animaux pour la santé humaine, ainsi que des possibilités réelles d'émergence de nouvelles pathologies dans les années futures. Ce travail avait donc pour objectifs: (i) d'identifier et caractériser les virus circulant au sein des populations de chauves-souris d'Afrique Centrale et (ii) d'explorer et d'identifier des déterminants bioécologiques, qui pourraient expliquer la richesse virale observée chez certaines espèces de chauves-souris rencontrées en Afrique tropicale forestière. A partir d'un total de 3472 individus testés, représentant 16 espèces provenant du Gabon, de la République du Congo et de la République Centrafricaine, nous avons confirmé la présence du virus Marburg chez les roussettes d'Egypte (Rousettus aegyptiacus) au Gabon, et mis en évidence des séquences virales de paramyxovirus très proches de virus zoonotiques émergents (les virus Nipah et Hendra) et réémergents (virus des oreillons) chez des chauves-souris frugivores. Des séquences de nouveaux coronavirus, flavivirus et paramyxovirus ont été également identifiées. Par ailleurs, la fragmentation de l'aire de distribution et le type de gîte ont été identifiés comme des déterminants de la richesse virale chez 15 espèces de chauves-souris d'Afrique Centrale. Les chauves-souris en Afrique Centrale seraient donc des réservoirs de virus apparentés à des virus pathogènes pour l'homme. Ces animaux pourraient donc être à l'origine de l'émergence des encéphalites à hénipavirus en Afrique et de la réémergence de certaines maladies humaines comme les oreillons, la rougeole. Des recherches futures s'orienteront vers la poursuite de la caracterisation génétique des virus détectés chez les chauves-souris d'Afrique Centrale et la détermination du risque zoonotique associé à ces virus. Des études écologiques seront également réalisées pour identifier les facteurs de risque d'émeregence des virus de chauves-souris potentiellement pathogènes pour l'homme. / The number of viruses détected in bats is growing, the most common are RNA viruses. The identification in different bat species of viruses that cause major epidemics or pandemics in human such as SARS coronavirus, Nipah and Henda viruses, the filoviruses Ebola and Marburg has raised awareness of potential risk that these animals may present to human health, as well as real possibilities of development of new diseases in future years. This work had two objectives: (i) to identify and characterize the viruses circulating in populations of bats in Central Africa and (ii) to explore and identify bioecological factors that could explain the viral richness observed in some bats species seen in tropical Africa forest. From 3472 individuals tested accounting for 16 species from Gabon, Congo and the Central African Republic, we established the presence of Marburg virus in Egyptian fruit bats (Rousettus aegyptiacus) in Gabon and identified viral sequences of paramyxoviruses close related to emerging and re-emerging zoonotic paramyxoviruses (Nipah virus, Hendra viruses and mumps virus) in fruit bats. Sequences of novel coronaviruses, paramyxoviruses and flaviviruses have also beenidentified. Moreover, the fragmentation of the range and roost type have been identified as determinants of viral richness in 15 bats species of Central Africa. Bats in Central Africa thus would be reservoirs of viruses related to viruses pathogenic for humans. These animals would lead to the emergence of encephalitis Henipavirus in Africa and the reemergence of certain human diseases such as mumps, measles. Further research will be conducted to continue the genetic characterization of viruses detected from bats in Central Africa and to determine the zoonotic risk associated with these viruses. Ecological studies will also be performed to identify the risk factors for the emergence of bats viruses potentially pathogenic for humans.