Dual-fluorescence reporter-based assay for rapid quantitative evaluation of antiviral activity against Venezuelan equine encephalitis virus

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus primarily infecting equines, causing severe illness or death. The virus can also infect humans causing severe neurological complications. There are no antivirals or human vaccines available. Although major outbreaks have been relatively rare in recent years, there is a need for antiviral drugs to combat this virus, both as a precaution against epidemics and for biological defense. In this study, we developed a rapid, fluorescence-based antiviral assay using recombinant VEEV TC-83 expressing the reporter Azurite. Two different strategies were compared (i) the nsP3–Azurite fusion construct (VEEV-nsP3-Azurite), in which the reporter was inserted in-frame within the nsP3 gene, and (ii) the extra subgenomic promoter construct (VEEV-SINVpr-Azurite), in which the Azurite reporter was expressed under the control of an additional Sindbis virus (SINV) subgenomic promoter as an independent open reading frame. While VEEV-nsP3-Azurite rapidly lost fluorescence in cell culture, the clone selection allowed the VEEV/SINVpr-Azurite maintained reporter expression for at least two passages, enabling enrichment to ~ 50% Azurite-positive plaques. Viral replication kinetics of VEEV/SINVpr-Azurite closely matched parental TC-83 in BHK-21 J. For quantitative readout, a BHK-21 J cell line expressing nuclear-localized miRFP670 was used to enable automated nuclear segmentation and cell counting during high-content image analysis. Optimized assay conditions resulted in robust viral reporter signal (Z′ >0.5) within 18 h post-infection. Validation with reference antivirals demonstrated dose-dependent inhibition comparable to the activity against the WT virus, providing a reliable assay for antiviral evaluation.