NS4B and NS5 mutations expand the temperature range permissive for the replication of dual-host affiliated insect-specific flaviviruses in mosquito cells

The genus Orthoflavivirus within the family Flaviviridae includes mosquito-borne flaviviruses (MBFs) and dual-host affiliated insect-specific flaviviruses (dISFs). MBFs infect both vertebrates and mosquitoes, while dISFs are thought to infect only mosquitoes, despite their phylogenetic proximity to MBFs. Some dISFs have been shown to be capable of infecting mammalian cells in the setting of a suppressed immune response and subphysiological temperatures. Therefore, temperature sensitivity is considered one of the key factors restricting viral host tropism. To investigate the effect of temperature on dISFs propagation, we evaluated the growth of two dISFs, Psorophora flavivirus (PSFV) and Barkedji virus (BJV), in mosquito-derived C6/36 cells at 28 and 37 °C. While MBFs, including dengue virus (DENV) and Japanese encephalitis virus (JEV) could propagate efficiently at both temperatures, both PSFV and BJV failed to propagate at 37 °C. Serial passaging of PSFV with a gradual increase in temperature resulted in PSFV adaptation to 37 °C, and whole-genome sequencing revealed that the 37 °C-adapted PSFV acquired non-synonymous amino acid substitutions in the non-structural proteins, NS4B and NS5. We demonstrated that both NS4B and NS5 amino acid mutations in PFSV confer the ability to propagate at 37 °C, suggesting that the replication machinery contributes to the thermal restriction on dISFs. This study provides new insights into the temperature sensitivity of dISFs and their relationship to host tropism.