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Palm Creek virus (PCV) is an insect-specific flavivirus that can interfere with the replication of mosquito-borne flaviviruses in Culex mosquitoes, thereby potentially reducing disease transmission. We examined whether PCV could interfere with arbovirus replication in Aedes (Ae.) aegypti and Ae. albopictus mosquitoes, major vectors for many prominent mosquito-borne viral diseases. We infected laboratory colonies of Ae. aegypti and Ae. albopictus with PCV to evaluate infection dynamics. PCV infection was found to persist to at least 21 days post-infection and could be detected in the midguts and ovaries. We then assayed for PCV–arbovirus interference by orally challenging PCV-infected mosquitoes with Zika and chikungunya viruses. For both arboviruses, PCV infection had no effect on infection and transmission rates, indicating limited potential as a method of intervention for Aedes-transmitted arboviruses. We also explored the hypothesis that PCV–arbovirus interference is mediated by the small interfering RNA pathway in silico. Our findings indicate that RNA interference is unlikely to underlie the mechanism of arbovirus inhibition and emphasise the need for empirical examination of individual pairs of insect-specific viruses and arboviruses to fully understand their impact on arbovirus transmission.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo., Author summary Defective viral genomes (DVGs) are produced during virus replication. On their own they cannot replicate, but some of them can compete with wild-type virus for viral and/or cellular resources. For chikungunya virus, interference by DVGs has not been described. Here, we use a new approach based on experimental evolution and computational analysis to characterize all DVGs generated in a virus population and identify those with the highest antiviral potential. We confirm their antiviral activity in both mammalian and mosquito host environments and show that some can broadly interfere with other strains or related alphaviruses. Finally, we show that DVGs can inhibit virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo. Author summary Defective viral genomes (DVGs) are produced during virus replication. On their own they cannot replicate, but some of them can compete with wild-type virus for viral and/or cellular resources. For chikungunya virus, interference by DVGs has not been described. Here, we use a new approach based on experimental evolution and computational analysis to characterize all DVGs generated in a virus population and identify those with the highest antiviral potential. We confirm their antiviral activity in both mammalian and mosquito host environments and show that some can broadly interfere with other strains or related alphaviruses. Finally, we show that DVGs can inhibit virus dissemination in mosquitoes. DARPA INTERCEPT program [HR0011-17-2-0023]; Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" [ANR-10LABX-62-IBEID]; Equipe FRM grant from the French Foundation for Medical Research [EQU201903007777]; France's defence procurement agency (DGA) This work was funded by the DARPA INTERCEPT program managed by Dr. Jim Gimlett, Dr. Brad Ringeisen and Dr. Seth Cohen, and administered though DARPA Cooperative Agreement #HR0011-17-2-0023 to M.V. and M-C. S. (the content of the information does not necessarily reflect the position or the policy of the U.S. government, and no official endorsement should be inferred). This work was also funded by the Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant ANR-10LABX-62-IBEID) to M.V. and M-C.S., and the Equipe FRM grant #EQU201903007777 from the French Foundation for Medical Research to M.V. L. I.L. was funded by a doctoral fellowship from France's defence procurement agency (DGA). None of the funders indicated above had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a continent-wide survey of DENV susceptibility using a panel of field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti . DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain., Competing Interests: Competing interests Authors declare no competing interests.