Analysis of the viral evolution of zoonotic RNA viruses in their natural host

Abstract

Lassa virus (LASV) can cause Lassa fever (LF), a severe febrile illness which is responsible for 900,000 infections and 18,000 deaths annually. LASV is a the negative-sense RNA virus, which belongs to the family of Arenaviridae, and is a zoonotic pathogen. Transmission of LASV to humans occurs through the body fluids and excretions of its primary rodent reservoir, Mastomys natalensis. Recent findings indicate that additional rodent species also carry LASV and infection and transmission experiments involving different LASV strains in M. natalensis revealed previously that strains originating from M. natalensis (homologous strains) establish persistent infections, while those from other sources (heterologous strains) are cleared rapidly. The objective of this project was to pinpoint viral factors that limit the host range of LASV. To achieve this, an in vitro system with M. natalensis cells was established in the beginning of this project and characterized regarding the expression of receptors required for LASV cell entry and the release of infectious progeny viruses. This system allowed to study host barriers in vitro and thus reduced the number of animals used during these studies significantly. The growth kinetics of LASV strains originating from different rodents were assessed on two cell lines derived from M. natalensis, namely Mastomys kidney epithelial cells (MasKEC) and Mastomys embryonal fibroblasts (MasEF). The growth of the heterologous Kak428 strain was attenuated compared to the Mastomys-derived Ba366 strain. As these growth kinetics resembled the growth of previous in vivo experiments, the next step was to assess the role of each viral protein on the virus-host barrier. Therefore, chimeric viruses were generated and used both for in vitro and in vivo experiments. When the glycoprotein (GP), L protein (L) or nucleoprotein (NP) of a heterologous strain was inserted into the genetic background of a homologous strain, the growth was strongly impaired, and the virus was cleared rapidly from the animals. The strains exhibiting attenuated growth on MasKEC cells were further characterized. Serial in vitro infection experiments on MasKEC were employed to adapt the heterologous LASV strain to Mastomys natalensis, and potential host-adaptive mutations were identified by Next Generation Sequencing. Following serial passaging on MasKEC, rapid adaptation occurred, resulting in accelerated replication and elevated peak titers for the heterologous strain. The mutations occurring during this process were accumulating in the GP, L and NP genes and mapped to their respective 3D protein structures. Additionally, some virus variants after ten passages of adaptation in cell culture, were used for in vivo experiments and revealed varying levels of adaptation. By using virus-life cycle modelling systems, the different life cycle steps were elucidated further. During entry, an impairment of the heterologous strain was noted and in replicon assays to assess the replication and transcription, the NP of this strain seemed to be more restricting than the L protein. The latter assay was also used to characterize the NP mutations identified during the adaptation experiments further. In summary, an in vitro model for LASV infection of natural host cells was established, and it closely mirrors the in vivo observations. Moreover, LASV demonstrated the ability to adapt to new host cells quickly, and potential host-adaptive mutations within the virus genome were characterized in replicon assays, entry assays and in in vivo experiments.