Development of Differential Detection Chikungunya Virus in East/Central/South Africa Lineage

Abstract

Chikungunya fever (CHIKF) is a mosquito-borne disease endemic to tropical regions. It is caused by the Chikungunya virus (CHIKV) and infects over one million people per year. CHIKV was first reported in Thailand in 1960 and a huge outbreak occurred in 2008-2009, which belonged to the East/Central/South Africa (ECSA) lineage (E1: A226V mutation). In this study, we analyzed the CHIKV E1 and E2 protein sequences in Thailand isolates and developed a method for detecting wild-type and mutant forms of the highly invasive ECSA lineage using one step multiplex PCR assay. A phylogenetic tree revealed that CHIKV found in Thailand during 2018–2019 represented the ECSA lineage and contained a double mutation (A226V and K211E) of the E1 protein. An additional E1 residue change, V264A, in the E2 protein of the ECSA lineage was also observed. Therefore, we designed specific primers and optimized multiplex PCR conditions to detect E1:K211E, E1:A266, and E2:V264A. The multiplex PCR results indicated that a PCR mixture containing 50, 100, or 200 ng/µl of DNA template, 1.5 mM MgCl2, 10 mM Tris-HCL (pH 9.1), 0.2 mM dNTP, 0.25 µM of primer mix, and 2.5 units of DNA polymerase amplified A226, K211E, and V264A in one reaction. The results indicate that this assay can readily detect the ECSA strain in one reaction, which will facilitate the routine, high-throughput diagnosis of CHIKV.