Identifying optimal capsid duplication length for the stability of reporter flaviviruses

Mosquito-transmitted flaviviruses cause widespread disease across the world. To provide better molecular tools for drug screens and pathogenesis studies, we report a new approach to produce stable NanoLuc-tagged flaviviruses, including dengue virus serotypes 1-4, Japanese encephalitis virus, yellow fever virus, West Nile virus, and Zika virus. Since the reporter gene is often engineered at the capsid gene region, the capsid sequence must be duplicated to flank the reporter gene; such capsid duplication is essential for viral replication. The conventional approach for stabilizing reporter flaviviruses has been to shorten or modify the duplicated capsid sequence to minimize homologous recombination. No study has examined the effects of capsid duplication length on reporter virus stability. Here we report an optimal length to stabilize reporter flaviviruses. These viruses were stable after ten rounds of cell culture passaging, and in the case of stable NanoLuc-tagged Zika virus (ZIKV C38), the virus replicated to 10⁷ FFU/ml in cell culture and produced robust luciferase signal after inoculation in mosquitoes. Mechanistically, the optimal length of capsid duplication may contain all the <i>cis</i>-acting RNA elements required for viral RNA replication, thus reducing the selection pressure for recombination. Together, these data describe an improved method of constructing optimal reporter flaviviruses.