Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.

SUMMARY: Chikungunya (CHIK) is a mosquito-borne viral disease. In the 2004 CHIK outbreak in Kenya, diagnosis was delayed because of the lack of accurate diagnostics. Therefore, this study aimed to develop and evaluate an in-house IgM-capture enzyme linked immunosorbent assay (ELISA) (in-house ELISA) for the detection of chikungunya virus (CHIKV) infections. Anti-CHIKV antibodies were raised in rabbits, purified and conjugated to horseradish peroxidase. These anti-CHIKV antibodies and cell-culture derived antigen were used to develop the ELISA. To validate the in-house ELISA, 148 patient sera from the 2005 Comoros CHIK outbreak were tested with centers for disease control and prevention (CDC) IgM-capture ELISA (CDC ELISA) and focus reduction neutralization test (FRNT) as reference assays. The in-house ELISA had a sensitivity of 97.6z and specificity of 81.3z compared to the CDC ELISA and a sensitivity of 91.1z and specificity of 96.7z compared to FRNT. Furthermore, 254 clinically suspected dengue patient samples from Eastern Kenya, collected in 2013, were tested for CHIKV IgM using the in-house ELISA. Out of the 254 samples, 26 (10.2z) were IgM positive, and of these 26 samples, 17 were further analyzed by FRNT and 14 (82.4z) were positive. The in-house ELISA was able to diagnose CHIKV infection among suspected dengue cases in the 2013 outbreak.

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