Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes

Abstract

Mosquito-borne viruses cause significant levels of morbidity and mortality in humans and domesticated animals. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although the vertebrate infection is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. An antiviral RNAi response has been shown to limit the replication of RNA viruses in flies. However, the importance of the RNAi pathway as an antiviral defense in mammals is unclear. Differences in the immune responses of mammals and mosquitoes may explain why these viruses are not generally associated with pathology in the invertebrate host. We identified virus-derived small interfering RNAs (viRNAs), 21 nt in length, in Aedes aegypti infected with the mosquito-borne virus, Sindbis (SINV). viRNAs had an asymmetric distribution that spanned the length of the SINV genome. To determine the role of viRNAs in controlling pathogenic potential, mosquitoes were infected with recombinant alphaviruses expressing suppressors of RNA silencing. Decreased survival was observed in mosquitoes in which the accumulation of viRNAs was suppressed. These results suggest that an exogenous siRNA pathway is essential to the survival of mosquitoes infected with alphaviruses and, thus, the maintenance of these viruses in nature.

Fig. 1.

Small RNAs in Ae. aegypti infected with SIN viruses (96 h). (A) Distribution of small RNA sequences (18–24 nt) in libraries prepared from mosquitoes infected with dsSINV or dsSINV-B2 (FHV), or from uninfected mosquitoes. (B) Length of small RNA sequences (18–24 nt) matching the dsSINV or dsSINV-B2 (FHV) genomes (100% match). (C) Distribution of 21-nt viRNA-generating loci in dsSINV and dsSINV-B2 (FHV) genomes. Vertical axis indicates the number of times a nucleotide position in the viral genome was represented in a unique viRNA sequence (normalized by smaller sequence set). Blue peaks indicate viRNAs derived from virus (+) strands (s viRNAs). Red peaks indicate viRNAs derived from virus (−) strands (as viRNAs).

Fig. 2.

viRNA-mediated restriction of SINV infection in Ae. aegypti. (A) Detection of viRNAs in mosquitoes after infection with SIN viruses (96 h). The 2S ribosomal RNA (rRNA) indicates equivalent loading of total RNA in each lane. (B) Accumulation of SINV genomic (49S), subgenomic (26S), and 2nd subgenomic (S2) RNAs in mosquitoes intrathoracically injected with a recombinant SINV virus. (C) Survival curves for mosquitoes intrathoracically injected with recombinant SIN virus (1 day old). Survival curves for uninjected, uninfected mosquitoes (1 day old) are shown for comparison. Error bars indicate the standard deviation among 3 replicate cohorts (50 mosquitoes per cohort).

Fig. 3.

Decreased survival of An. gambiae after infection with recombinant ONN virus expressing a suppressor of RNA silencing. An. gambiae were intrathoracically injected with virus (1 day old), and mortality was monitored daily. Survival curves for uninjected, uninfected mosquitoes are shown for comparison. Error bars indicate the standard deviation among 3 replicate cohorts (50 mosquitoes per cohort).