Small plaque size variant of chikungunya primary isolate showed reduced virulence in mice.

BACKGROUND: Plaque size is a common feature of viral characterization. Small plaque size is used as a marker of attenuation for live-attenuated vaccine development. OBJECTIVE: To investigate whether the naturally occurring plaque size variation reflects virulence of the variants of chikungunya virus (CHIKV). METHODS: We selected and purified a variant with small plaque size from the primary isolate. The viral variant was tested for the plaque morphology, in vitro growth kinetics and mouse neurovirulence in comparison with the parental wild type. RESULTS: The small plaque size variant showed stable homogenous small plaques after 4 plaque purifications. The small plaque virus grew slower and to the lower titer when compared with wild type virus. After 21 days of infection, mice that received small plaque virus showed 98% survival rate while 74% of mice survived after infected with wild type virus. CONCLUSION: The small plaque size variant of CHIKV can be obtained by plaque purification and the virus displays decreased virulence.

The synergistic effect of nsP2-L618, nsP3-R117, and E2-K187 on the large plaque phenotype of chikungunya virus.

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.