Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is the etiological agent of chikungunya fever. CHIKV re-emerged from 2004 onwards, and subsequently caused major outbreaks in many parts of the world including the Indian Ocean islands, Asia, and the Americas. In this study, a large plaque variant of CHIKV isolated from patient in Thailand was subjected to repeated cycles of plaque-purification in Vero cells. The resulting virus produced homogenous large plaques and showed a more pathogenic phenotype than the parental wild-type CHIKV. Whole genome analysis of the large plaque virus in comparison to parental isolate revealed a number of mutations, leading to the following amino acid changes: nsP2 (P618→L), nsP3 (G117→R), and E2 (N187→K). Eight recombinant CHIKVs were constructed to determine which amino acids mediated the large plaque phenotype. The results showed the recombinant virus which contains all three mutations, rCHK-L, produced significantly larger plaques than the other recombinant viruses (p < 0.01). Moreover, the plaque size of the other recombinant virus tended to be smaller if they contained only one or two of the large plaque associated mutations in the viral genome. In conclusion, the combination of all three residues (nsP2-L618, nsP3-R117, and E2-K187) is required to produce the large plaque phenotype of CHIKV.
Keywords: Chikungunya virus; Large plaque-associated mutations; Plaque morphology; Plaque-purification.