Characterization of clinical respiratory syncytial virus isolates and the effect of protease inhibitors on early viral entry

Respiratory syncytial virus (RSV) is the principal viral cause of serious lower respiratory tract infections in infants and is the primary cause of hospitalization during the first year of life. RSV disease severity in young children displays great variety, ranging from mild cases with common cold-like symptoms to severe cases in need of hospitalization, respiratory support, additional hydration and tube-feeding. The factors attributing to the observed differences in disease severity can be divided into host factors, such as genetic and environmental background, and virus-intrinsic factors. Multiple studies have indicated that genetic variations in the host inflammatory immune response to RSV are responsible for the wide range of disease severity. In contrast, the role of factors intrinsic to the virus itself in contributing to disease has only partially been researched. No vaccine or therapeutic has been market approved to prevent or treat RSV infections and associated disease, therefore, treatment is currently limited to supportive care. Research on vaccines has been severely hampered by a legacy of vaccine enhanced respiratory disease. Blocking RSV entry by small molecules is an attractive therapeutic pathway, but fully understanding the entry mechanisms of RSV into the host cell is important for their development. Two theories currently describe the RSV fusion event, fusion at the cell surface and fusion within endosomes. Questions remain about the event that triggers the fusion of the viral envelope with the cell membrane and two main propositions have been made: spontaneous conversion of the metastable prefusion F protein conformation to the stable postfusion F protein conformation, or post-translational modifications by furin-like proteases. Firstly, this thesis focuses on the isolation and phenotypical and genetic characterization of recent clinical isolates. RSV was isolated from nasal samples derived from sick children and characterized in vitro. These experiments showed that the clinical isolates differ from laboratory strains isolated in 1961, and from each other on several characteristics. Additionally, these clinical isolates were then continuously cultured to assess their adaptation, which was found limited after 30 passages in most cases. Secondly, this thesis aimed to gain more insight in the role of host proteases during the RSV entry process. RSV infected cells were first treated with several protease inhibitors to examine their effects on viral entry and the replication cycle of RSV. Serine protease AEBSF was found to inhibit the RSV infection during early entry, and the process was further elucidated using electron microscopy showing that fusion was inhibited.

Jaar van publicatie:2019

Trefwoorden:Doctoral thesis

Toegankelijkheid:Open