The Chikungunya virus (CHIKV) is a positive-sense RNA alphavirus that circulates in nature by alternating replication between mammalian hosts and mosquito vectors, primarily Aedes aegypti and Aedes albopictus. Remarkably, while CHIKV infection causes significant disease in mammals, mosquitoes remain asymptomatic, reflecting the presence of powerful disease tolerance mechanisms within the vector. Previous studies have revealed that a key mechanism underlying this tolerance is the synthesis of viral DNA (vDNA) using the CHIKV RNA genome as a template. This conversion is mediated by endogenous retrotransposon-encoded reverse transcriptases (RTs) within the mosquito. Pharmacological inhibition of this process using classical reverse transcriptase inhibitors such as zidovudine (AZT) abrogates vDNA synthesis and impairs disease tolerance, leading to increased viral susceptibility. However, the molecular pathways governing vDNA biogenesis, as well as the distinct impacts of different classes of reverse transcriptase inhibitors in mosquitoes, remain poorly characterized.

To address these gaps, we investigated the mechanisms of vDNA synthesis both in vivo and in vitro. We mapped the reverse-transcribed regions of the CHIKV genome and characterized the kinetics of vDNA synthesis following infection. In parallel, we evaluated the comparative efficacy of five RT inhibitors—AZT, d4T, 3TC, nevirapine, and tenofovir—representing nucleoside analogs, nucleotide analogs, and allosteric inhibitors, in blocking vDNA synthesis in both mosquitoes and mosquito-derived cells. For each compound, we assessed cellular toxicity, viral loads, cell viability, and cytopathic effects, alongside quantification of vDNA inhibition. Finally, we performed transcriptomic analysis to dissect the broader impact of RT inhibition on mosquito antiviral immune mechanisms, cellular metabolism, and stress responses during CHIKV infection.

Our findings elucidate core molecular features of vDNA synthesis and demonstrate distinct inhibitor effects, advancing understanding of mosquito tolerance to viral disease and offering potential avenues for vector-targeted control strategies.