Development of pseudotyped virus assays for the serological study of Japanese encephalitis flavivirus

• Main Author: Mather, Stuart Thomas
• Published: University of Kent 2017
• Subjects: 616.9
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.721658

Japanese encephalitis virus (JEV) is one of the primary global causes of viral encephalitis, with approximately 68,000 clinical cases and 20,000 deaths attributed to the virus annually. Between 30% and 50% of survivors suffer from debilitating neurological sequelae. Despite being a vaccine-preventable disease, no antiviral treatments are licensed and commercially available to counteract JEV infection. In order to quantify the neutralising antibody response raised against antigenic epitopes on flavivirus prME glycoproteins, conventional serological assays such as the plaque reduction neutralisation test (PRNT) can be employed. However, these assays often necessitate the handling of pathogenic wild-type virus in expensive high-biosafety laboratories, restricting the scope of their application, particularly in resource-deprived areas. Chimeric, replication-deficient pseudotype viruses can offer a solution to this problem, as they mimic wild-type virus entry mechanisms, enabling their use in pseudotype virus neutralisation assays (PVNAs). PVNAs bypass high biosafety requirements and permit vaccine evaluation and serosurveillance studies with no risk of inadvertent infection. This project focuses on the production of functional pseudotype viruses displaying the prM and E surface glycoproteins of the JEV flavivirus, for utilisation in serological neutralisation assays. Subcloning of the prME gene into an appropriate eukaryotic expression vector and insertion mutagenesis to produce prME with 15- and 24-residue upstream signal peptides are shown, before production of JEVpp with either HIV or MLV cores is attempted, via the conventional multi-plasmid co-transfection approach or the utilisation of constitutive gag-pol expressing cell lines. The impact of additional plasmid-derived furin protease expression and low glucose culture medium, as well as the construction of JEV/VSV chimeric prME glycoproteins and the introduction of Kozak consensus sequences upstream of the prME gene, to enhance the efficiency of JEVpp generation is also explored. Finally, the infectivity of lentiviral pseudotype viruses following lyophilisation, storage and reconstitution is confirmed, thus enabling their affordable global distribution.