RNASeq analysis of Aedes albopictus mosquitoes during chikungunya virus infection

Abstract

Chikungunya virus (CHIKV), preferentially transmitted by Aedes mosquitoes, is an emerging pathogen around the world and causes significant morbidity in patients. A single amino acid mutation in the envelope protein of CHIKV has led to shift in vector preference towards Aedes albopictus, an invasive mosquito. Previous studies have shown that after infection, mosquitoes mount an antiviral immune response. However, molecular interactions during the course of infection at different tissues and time-points remain largely uncharacterised. Here we performed whole transcriptome analysis on dissected midguts and head/thorax of CHIKV (Indian Ocean strain) infected Aedes albopictus to identify differentially expressed genes compared with uninfected controls. For this, RNA was extracted at two days post-infection (D2) from pooled midguts and eight days post-infection (D8) from heads and the anterior 1/3rd of the thorax. We identified 25 and 96 differentially expressed genes from the D2 and D8 samples respectively (p-value <0.05). Custom de novo transcriptomes were assembled for the reads that did not align with the reference genome and an additional 225 and 4771 differentially expressed genes from D2 and D8, respectively, were identified. Twenty-two of the identified transcripts, possibly involved in immunity, were validated by qRT-PCR. Interestingly, we also detected changes in viral diversity, as shown by number of mutations in the viral genome, with increase in number of mutations in the midgut compared with mammalian host (Vero cell culture), followed by reduction in the number of mutations in head and thorax at D8, indicating a possible genomic bottleneck. Taken together, these results will help in understanding Aedes Albopictus interactions with CHIKV and can be utilised to reduce the impact of this viral infection.\n\nAuthor SummaryChikungunya virus has caused several outbreaks around the world in the last decade. Once a relatively unknown virus, it now causes seasonal infections in tropical and some temperate regions. This change in epidemiology is attributed to vector switch from Aedes aegypti to Aedes albopictus, an invasive pest leading to spread and causing infections in temperate regions. Although recent research has identified mosquito factors influencing infections, our understanding of interaction between chikungunya virus and its vector is limited. Using whole transcriptome sequencing of chikungunya infected mosquitoes, we identified differentially expressed genes in the midgut and head and thorax, over the course of mosquito infection. We also detected changes in the viral genome during mosquito infection and a possible genetic bottleneck event with reduction in viral variants at the head and thorax region of mosquito in the later stages of infection. These results will lead to improving our understanding of mosquito-virus interactions with Aedes albopictus as a vector and in turn lead to development of novel disease control strategies.