Dengue virus replicates and accumulates in Aedes aegypti salivary glands

Dengue virus (DENY) is an RNA virus transmitted among humans by mosquito vectors, mainly Aedes aegypti. DENY transmission requires viral dissemination from the mosquito midgut to the salivary glands. During this process the virus undergoes several population bottlenecks, which are stochastic reductions in population size that restrict intra-host viral genetic diversity and limit the efficiency of natural selection. Despite the implications for virus transmission and evolution, DENY replication in salivary glands has not been directly demonstrated. Here, we used a strand-specific quantitative RT-PCR assay to demonstrate that negative-strand DENY RNA is produced in Ae. aegypti salivary glands, providing conclusive evidence that viral replication occurs in this tissue. Furthermore, we showed that the concentration of DENY genomic RNA in salivary glands increases significantly over time, indicating that active replication likely replenishes DENY genetic diversity prior to transmission. These findings improve our understanding of the biological determinants of DENY fitness and evolution.