Differential effect of acute versus persistent insect-specific flavivirus infection on superinfection exclusion of West Nile, Zika and chikungunya viruses in RNAi-competent and -deficient mosquito cells

Millions of people are annually infected by mosquito-transmitted arboviruses including dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV) and chikungunya virus (CHIKV). Insect-specific flaviviruses (ISFs), which only infect mosquitoes and cannot replicate in vertebrates, can offers a potential one health strategy to block the transmission of arboviruses by reducing the mosquito's susceptibility for subsequent arbovirus infections through superinfection exclusion (SIE),. Most SIE studies focus on acute ISF infections in RNAi-deficient <i>Aedes albopictus</i> C6/36 cells. Because ISFs are known to persistently infect mosquitoes, acute infections in C6/36 cells may not accurately reflect natural interactions between ISFs and arboviruses. To study the underlying mechanisms for SIE, we persistently infected C6/36 and RNAi-competent <i>Aedes aegypti</i> Aag2 cells with the ISF Binjari virus (BinJV) and a BinJ-ZIKV chimera that contains the ZIKV prME structural genes. SIE of WNV, ZIKV and CHIKV by BinJV was more pronounced in acute than in persistently infected cells and much stronger in acutely infected C6/36 cells compared to Aag2 cells. The viability of RNAi-deficient mosquito cells was severely reduced upon acute ISF infection, which correlated to the observed SIE. However, persistently infected mosquito cells still inhibited subsequent arbovirus replication. Moreover, RNAi-competent Aag2 cells were better protected against ZIKV superinfection when they were pre-infected with BinJ-ZIKV as compared to BinJV. Therefore, acute ISF infections and strong cytopathic effects in RNAi-deficient cells augment SIE, while in persistently infected cells SIE is established through RNAi-dependent and independent mechanisms. This highlight the importance of using more representative <i>in vitro</i> models.