Application of real-time RT-PCR in vector surveillance and assessment of replication kinetics of an emerging novel ECSA genotype of Chikungunya virus in Aedes aegypti

Abstract

Chikungunya has emerged as one of the most important arboviral infection of global significance. Expansion of Chikungunya virus endemic areas can be ascribed to naive population, increasing vector population and adaptability of virus to new vector. In this study, a SYBR Green I based quantitative RT-PCR assay was developed. The assay was found to be 10-fold more sensitive than conventional RT-PCR and no cross reactivity was observed with related alphaviruses and flaviviruses. The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Aedes aegypti, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Ae. aegypti for CHIKV, which revealed 100% infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Ae. aegypti revealed highest titre at day 6 post infection in midgut and at day 10 post infection in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes.