Generation of flaviviral replicon expressing stable cell lines

Abstract

Flaviviruses are a group of arboviruses that are considered re-emerging pathogens which can cause severe illnesses in vulnerable populations. Although a significant issue for the healthcare system, there are no effective treatments available, and flavivirus countermeasures rely on preventative actions like the control of vector spread and vaccination. The majority of flaviviruses are biosafety level 3 (BSL3) agents, which necessitate special precautions when working with infectious viruses. This complicates the development of antivirals and vaccines. To address this issue, when investigating the antiviral mechanism of inhibitors at lower biosafety levels, subgenomic replicons can be used to study viral replication events without virus entry or assembly. In this work, we generated stable cell lines based on flavivirus replicon RNAs, including reporter marker genes and the puromycin N-acetyltransferase (PAC) gene. The resulting puromycin-resistant stable cell lines persistently expressed non-structural proteins and fluorescent or luminescent reporter proteins. These stable cell lines can be used for high-throughput screening of potential replication inhibitors for the development of novel antiviral therapies against flaviviruses.