Development of a Synthetic Genomics-Based Venezuelan Equine Encephalitis Virus Expressing Secreted Gaussia Luciferase for High-Throughput Antiviral Screening

Abstract

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that causes severe neurological disease in humans and animals. Despite its public health importance, no approved antiviral therapy is currently available, and conventional methods for quantifying infectious virus are labor-intensive and poorly suited for high-throughput antiviral screening. Furthermore, research on VEEV is complicated by the restricted availability and international transport of infectious viral materials. Here, we established a full-length infectious cDNA clone of VEEV entirely from chemically synthesized DNA fragments and rescued recombinant reporter viruses expressing mCherry2, ZsGreen, or the secreted Gaussia luciferase mutant 23 (GLucM23). The reporter viruses retained replication kinetics comparable to those of wild-type VEEV, while the fluorescent reporters enabled direct visualization of infection and virus-induced cytopathic effects. GLucM23-expressing VEEV allowed rapid, sensitive, and non-destructive quantification of viral replication directly from culture supernatants, enabling repeated longitudinal measurements without disrupting infected cells. We further demonstrated the utility of this system by screening approximately 3,000 FDA-approved compounds to identify inhibitors of VEEV replication. Together, these findings establish a versatile synthetic genomics-based reporter virus platform for quantitative studies of VEEV replication and scalable antiviral drug discovery.