Preparation and application of chikungunya pseudovirus containing double reporter genes

Chikungunya virus (CHIKV), a highly infectious and rapidly spread viral pathogen, is classified as a pathogenic agent at the biosafety level 3. Operation of live authentic CHIKV needs a specific laboratory with the P3 or above containment, which greatly confines the CHIKV-associated studies. To establish an evaluation system of CHIKV that can be utilized in a BSL2 laboratory, we constructed a pseudovirus (PsV) system of CHIKV containing double reporter genes (ZsGreen1 and luciferase). The fluorescent ZsGreen1 is a convenient and cheap reporter for monitoring the efficiency of transfection and titration of PsV. The enzyme luciferase is a sensitive reporter for the application of PsV to neutralization assay or drug screening. The CHIKV PsV produced in this study, with a titer of up to 3.16 × 10⁶ TU/ml, was confirmed by Western blotting and transmission electronic microscopy (TEM). Finally, we developed a microneutralization assay with the CHIKV PsV produced in this study, which was successfully applied to evaluate neutralizing activities of convalescent sera from CHIKV-infected patients. In summary, we have established a convenient and sensitive double-reporter CHIKV pseudovirus system, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.