Enhanced Molecular Surveillance of Chikungunya Virus

Outbreaks of the emerging arbovirus chikungunya virus (CHIKV) affect millions of individuals in Asia, Africa, and Latin America. Vector competence can be changed dramatically by single amino acid exchanges located predominantly within the CHIKV E1 and E2 envelope proteins, which are associated with enhanced transmissibility by anthropophilic Aedes mosquitoes. Commonly used reference assays for molecular surveillance cover only a few adaptive mutations within the envelope domains and have not been validated for all CHIKV genotypes. The recognized landscape of CHIKV adaptive mutations is thus likely incomplete. We designed two nested reverse transcription-PCR (RT-PCR) assays that cover hot spots of viral adaptation to vectors within the E1 and E2 genomic domains. Primers were designed in conserved genomic regions to allow broad usability across CHIKV genotypes. The sensitivity of both assays was at least equivalent to E1- and E2-based reference assays and robust among CHIKV genotypes at 51.4 IU/reaction (E1, 95% confidence interval [CI], 39.8 to 78.9) and 4.0 IU/reaction (E2, 95% CI, 2.0 to 7.4). Upon analysis of the complete known CHIKV genomic diversity, up to 11 nucleotide mismatches with CHIKV variants occurred under oligonucleotide binding sites of reference assays, potentially limiting assay sensitivity, whereas no critical mismatches occurred in the new assays. Specificity testing with nine alphaviruses representing all serocomplexes showed amplification of Mayaro virus and O’nyong-nyong virus by the E1-based assay, but not by the E2-based assay. The high sensitivity and specificity of the new E2-based assay may allow its diagnostic usage in resource-limited settings. The combined new assays allow improved molecular epidemiological surveillance of CHIKV globally.