Identification and functional characterisation of Aedes albopictus genes against Chikungunya virus

Chikungunya virus (CHIKV) of Alphavirus genus, 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 infections in temperate regions. Although recent research has identified mosquito factors influencing infections, our understanding of interaction between CHIKV and its new vector is limited.

Using whole transcriptome sequencing of CHIKV infected mosquitoes, we studied differential expression of genes in the midgut and head and thorax, the two critical barrier sites of the mosquito at two time points. We identified several up and down regulated transcripts in the mosquito host genome in response to the viral infection. Two days post-infection, in the midgut tissue of the mosquitoes, 250 differentially expressed transcripts (25 when the next-generation sequencing (NGS) reads were aligned to the published reference genome and 225 when the reads were aligned to a de novo custom transcriptome we generated) were identified. From the head and thorax tissue of the mosquitoes, 8 days post-infection, 159 differentially expressed transcripts (96 when the NGS reads were aligned to the published reference genome and 63 when the reads were aligned to the de novo custom transcriptome) were identified. Twenty-seven of the targets (13 from 2dpi/midgut and 14 from 8dpi/head & thorax) identified to be differentially expressed were validated separately via qRT-PCR. Seven transcripts found to be differentially expressed in midguts of Ae. albopictus two days post-infection were also assessed for changes in expression in midguts of Ae. aegypti two days post-infection. Apart from differential expression in genes, we also identified down regulation of long non-coding RNAs that may also have functional relevance. The comparison between Ae. albopictus and Ae. aegypti also showed that the expression patterns of the same targets are different between the two species of mosquitoes after CHIKV infection.

From the targets we validated, two were selected for further functional studies. Niemann-Pick 2 (NPC2) gene homologue was found to be significantly upregulated in the midguts of both Ae. albopictus and Ae. aegypti mosquitoes, two days post-infection with CHIKV. Known cytoplasmic lipid transporters, NPC family proteins had previously been implicated in pathogenesis of several viruses including dengue, Ebola and HIV. In fact, NPC2 protein was found to be essential for successful replication of CHIKV in human fibroblasts. To characterise the role of NPC2 during CHIKV in Ae. albopictus mosquito, the gene was over expressed in C6/36 mosquito cells 24 hours prior infection with the virus. The infectivity titres of extracellular mature virus and intracellular viral RNA levels were compared between wildtype cells and cells over expressing the protein. The expressed NPC2 protein and the virus were also labelled using antibodies and studied under confocal microscopy. While significant differences were not observed in the viral RNA levels or infectivity titres, confocal microscopy showed partial co-localisation of NPC2 protein and the virus.

Inhibitor of Bruton¿s tyrosine kinase (BTKi) was identified to be significantly upregulated 8 days post-infection in the head and thorax of Ae. albopictus mosquitoes. To assess its functional significance, BTKi was knocked-down using double-stranded RNA in RML12, a mosquito cell line. While no significant difference in viral RNA levels or infectivity titers was detected, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. BTK is a pro-inflammatory cytoplasmic Tec kinase and is known to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis. The upregulation of BTKi a known anti-inflammatory protein post viral infection and increase in cellular apoptosis when the gene is knocked down may suggest a possible conserved mechanism at play between mosquitoes and mammals.

We also studied changes in the viral genome during mosquito infection. We 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 8 dpi, indicating a possible genomic bottleneck.

Taken together, these results will help in understanding Ae. albopictus interactions with CHIKV and can lead to development of novel disease control strategies.