Viral Plasticity Underlying Tropism And Pathogenesis/ Innate Immune Evasion Of Emerging Viruses

During the past years, numerous novel viruses of zoonotic origin, including flavi-, corona-, and arenaviruses have emerged as causative agents of severe incurable human diseases posing important public health problems. A hallmark of emerging pathogenic human RNA viruses is their low-fidelity mechanism of replication, resulting in high mutation rates and consequent high genetic diversity. Actual RNA virus populations are therefore comprised of swarms of genetic variants with high information content referred to as quasispecies. The quasispecies nature of RNA viruses allows for rapid adaptation to dynamic environments, including changing hosts and tissues during zoonotic transmission and evading host immune responses. In our present project, we will apply cutting edge next generation sequencing (NGS) technologies for a comprehensive real-time analysis of quasispecies dynamics underlying viral plasticity of major emerging viruses in relevant experimental paradigms in vitro and in vivo. We will combine our complementary expertise on highly pathogenic coronaviruses (Thiel), neurotropic flaviviruses (Leib, Gäumann), and hemorrhagic arenaviruses (Kunz) to study the genetic basis of viral plasticity underlying the tropism and innate immune evasion. Importantly, our studies will be performed in natural target cells, such as primary human airway epithelium (HAE, Thiel), organotypic culture slices of brain tissue (OCS, Leib, Gäumann), and primary human endothelial cell cultures (Kunz). These systems recapitulate major features of living tissue in vivo, are biologically highly relevant, and provide well-controlled infection paradigms that, combined with NGS, allow us to track temporal dynamics of genetic changes in viral quasispecies as a function of virus-host cell interaction and adaptation to a changed environment. Upon spill-over into the human population, viruses are subject to strong selective pressure leading to positive selection of viral attachment proteins able to use human receptors and selection of viral proteins involved in cell interactions at later steps of the viral life cycle. Importantly, the subject of selective pressure is not the individual viral variant but the entire quasispecies population, involving complex interactions between variants. The selection of specific viral variants within a population is thus impossible to predict by biocomputational models and an experimental approach is needed. In our first aim, we will use an unbiased approach based on NGS analysis of the entire quasispecies population during passage in organotypic tissue cultures. We hypothesize that alterations in the viral quasispecies are important indicators and requirements for viral adaptation including host and target cell tropism.Innate anti-viral immunity, in particular the interferon (IFN)-I system, represents a powerful first line of defense against RNA viruses. In our second aim, we will treat organotypic tissue cultures with recombinant IFN-I in order to modulate the innate immune status of cells . We hypothesize that selection by innate immune pressure will induce changes in the viral quasispecies that will pinpoint general mechanisms of innate immune evasion. Finally, in our third aim, we will use of a wide panel of functional assays, reverse genetic systems, in vitro and in vivo systems to phenotypically analyze the most interesting virus mutants identified in aims 1 and 2, and we hypothesize that the use of primary organotypic culture systems and in vivo disease models will validate biologically relevant phenotypes present in the viral quasispecies.Using a comparative approach at the level of viral population genetics, streamlined experimental approaches and technologies (aim 4), our studies on coronaviruses, flaviviruses, and arenaviruses will reveal important commonalities and differences, allowing the definition of general principles of viral plasticity in the context of tropism and innate immune evasion. The results of our study could help to predict the risk of zoonotic transmission, adding to our preparedness. By combining cutting-edge technology with an important scientific question our project will address an unmatched problem of high relevance for the infectious disease community at large. Keywords type I interferon; Coronavirus; adaptation; quasispecies; viral plasticity; Flavivirus; innate immunity; RNA virus; Arenavirus; zoonosis Hauptdisziplin Molekularbiologie