Broad reactivity of anti-E1 protein monoclonal antibodies (mAbs) towards three genotypes of CHIKV.

Plasmids were constructed to encode the full-length CHIKV envelope protein (E3-E2-6K-E1) from each of the three viral genotypes: ECSA (CP10 strain), WA (37997 strain), or Asian (CK12-686 strain; Wild type). Site-directed mutagenesis was used to introduce mutations at position 350 of the 6K-E1 proteins in all three genotypes, resulting in ECSA and WA bearing E350D (6K-E1 E350D) and Asian bearing D350E (6K-E1 D350E). Indirect immunofluorescence test was performed by using the indicated anti-E1 mAbs and Alexa Fluor 488-conjugated secondary antibody towards HEK293T cells transfected with each of these six versions of plasmids encoding CHIKV envelope proteins. CK47 was used as a reference for 350E-restricted reactivity, while CK119 was used as the transfection control. Alexa Fluor 488 signals in each photo were quantified by NIS-Elements software (Nikon Corporation, Tokyo, Japan). ++++ (very strong; mean fluorescence intensity/pixel ≥ 75), +++ (strong; ≥ 35), ++ (medium; ≥ 26), + (weak; ≥ 15). Images shown are representative of results obtained from two independent experiments and were taken under 40x objective magnification using an IX71 microscope (Olympus, Tokyo, Japan).