Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often phylogenetically related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called “superinfection exclusion”, whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISFs) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimise vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.

Up to 40% of the world population now live in areas where dengue mosquito vectors coexist with humans. Aedes aegypti are vectors for zoonotic diseases that affect hundreds of millions of individuals per year globally. We recently identified the eggshell organizing factor 1 (EOF1) protein using systematic RNA interference (RNAi) screening of mosquito lineage-specific genes. It was shown that eggs deposited by RNAi-EOF1 A. aegypti and A. albopictus mosquitoes were non-melanized, fragile, and contained nonviable embryos. Motivated by this discovery, we performed RNAi screening of eggshell proteins to determine putative downstream target proteins of intracellular EOF1. We identified several eggshell proteins as essential for eggshell formation in A. aegypti and characterized their phenotypes in detail by molecular and biochemical approaches. We found that Nasrat, Closca, and Polehole structural proteins, together with the Nudel serine protease, are indispensable for eggshell melanization and egg viability. While all four proteins are predominantly expressed in ovaries of adult females, Nudel mRNA expression is highly upregulated in response to blood feeding. Furthermore, we identified four secreted eggshell enzymes as important factors for controlling the processes of mosquito eggshell formation and melanization. These enzymes included three dopachrome converting enzymes and one cysteine protease. All eight characterized eggshell proteins were required for intact eggshell formation. However, their surface topologies in response to RNAi did not phenocopy the effect of RNAi-EOF1. Still, it remains unclear how EOF1 influences eggshell formation and melanization. The use of proteomic analysis of eggshell proteins from RNAi-EOF1 assisted in the identification of additional proteins that could be regulated in EOF1 deficient eggshells.

Insect-specific viruses (ISVs) include viruses that are restricted to the infection of mosquitoes and are spread mostly through transovarial transmission. Despite using a distinct mode of transmission, ISVs are often closely related to arthropod-borne viruses (arboviruses) that are responsible for human diseases and able to infect both mosquitoes and vertebrates. ISVs can also induce a phenomenon called “superinfection exclusion”, whereby a primary ISV infection in an insect inhibits subsequent viral infections of the insect. This has sparked interest in the use of ISVs for the control of pathogenic arboviruses transmitted by mosquitoes. In particular, insect-specific flaviviruses (ISF) have been shown to inhibit infection of vertebrate-infecting flaviviruses (VIFs) both in vitro and in vivo. This has shown potential as a new and ecologically friendly biological approach to the control of arboviral disease. For this intervention to have lasting impacts for biological control, it is imperative that ISFs are maintained in mosquito populations with high rates of vertical transmission. Therefore, these strategies will need to optimize vertical transmission of ISFs in order to establish persistently infected mosquito lines for sustainable arbovirus control. This review compares recent observations of vertical transmission of arboviral and insect-specific flaviviruses and potential determinants of transovarial transmission rates to understand how the vertical transmission of ISFs may be optimised for effective arboviral control.