Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus of clinical importance, with symptoms in humans ranging from febrile illness to severe neurological manifestations. The recent 2022 Australian JEV outbreak has prompted renewed concerns regarding the potential for the virus to spread and cause disease. Although JEV vaccines are commercially available, there are no available therapeutics for symptomatic patients. This is, in part, due to the lack of understanding of the virus-host interactions of JEV.
The mechanisms by which key proteins and host factors that JEV relies on to replicate and infect have not been fully characterised. A high-throughput tool to identify these potentially essential proteins is whole genome CRISPR-Cas9 knockout screening. Herein, we perform a knockout screen in human HEK293T cells using a human CRISPR-Cas9 library. HEK293T cells were transduced with the CRISPR-Cas9 library and infected with JEV. Surviving cells were then sequenced to identify crucial genes required for JEV replication.
Bioinformatic analyses of the sgRNAs highlighted an overlap of 25 positively enriched genes between two JEV genotypes, JEVNakayama (GIII) and the 2022 Australian outbreak isolate, JEVNSW/22 (GIV). The 25 identified genes are involved in cellular pathways and components essential for flavivirus replication cycle, thus validating our experimental design. This proof-of-principle experiment will guide a CRISPR-Cas9 knockout screen in mature RENCell VM neural progenitor cells and identify for the first-time key host genes implicated in the neurocytotropism of encephalitic flaviviruses such as JEV. This will provide mechanistic insights and aid in the development of targeted therapeutics for JEV.