Chikungunya virus (CHIKV) is a positive sense RNA virus of Alphavirus genus, transmitted by Aedes mosquitoes. Traditionally, it has been hypothesized that arboviruses are constrained genetically due to the fitness trade-off, when adapting simultaneously for infection and replication in mosquitoes as well as mammals. During mosquito infection, virus crosses midgut barrier before its replication in the body. Previous studies have identified genomic bottleneck events during mosquito infection. Here, using next-generation sequencing, we determined CHIKV follows similar fate during Aedes albopictus infection.
Initially, Vero cell-derived (mammalian) CHIKV (Isolate 06113879) was sequenced using Ilumina MiniSeq and virus genome was assembled and annotated (GeneBank MH229986). Adult female Aedes albopictus mosquitoes were infected with this CHIKV isolate by blood-feeding. Two days post-infection (D2), RNA was extracted from pooled midguts (MG) of 6 mosquitoes. Eight days post-infection (D8), RNA was extracted from the pooled heads and the top 1/3rd of the thorax (HT) of 6 mosquitoes. RNAseq libraries were prepared from three D2 infected pools and two D8 infected pools using the Nugen Ovation Universal kit v2 and sequenced on a single lane on an Illumina Hiseq 2500. Viral reads were extracted using SAMtools v1.3.1 and aligned to the previously generated consensus sequence and variant calling was performed using Varscan2 v2.3.9.
Our results show that compared to Vero-derived CHIKV, there was a significant increase in the number of mutations in the viral genome in the mosquito midgut 2 days post-infection. This was followed by a significant reduction in the number of mutations in the viral genome from the head and thorax 8 days post-infection. Interestingly, region in non-structural protein (NS)-3 encoding nucleotides was found to be ‘hot-spot’ for mutations. These results indicate possible genomic bottleneck events responsible for viral adaptation to the mosquito host system as it travels from a mammalian host system.