Development of Universal Primer Sets for Zika Virus Envelope Gene Amplification and Sequencing.

The Zika virus (ZIKV) remains an important public health concern owing to its association with neurological disorders and congenital abnormalities. Genetically, ZIKV is classified into two major lineages-African and Asian-and continues to evolve, with evidence of genetic diversification following its introduction into the Americas. The study of the envelope (E) gene of ZIKV is a key tool for understanding viral biology, phylogenetic analysis, and developing antiviral drugs and vaccine candidates. However, few studies have focused on designing primers capable of efficiently detecting and amplifying this essential gene. In this study, we developed universal primer sets capable of amplifying and sequencing the full-length ZIKV E gene across genetically diverse lineages and strains. Primers were designed based on representative ZIKV sequences available in public databases and validated using viral isolates and in vitro-spiked plasma samples. The assay demonstrated high specificity and analytical sensitivity, with the analytical detection limit established using serially diluted DNA amplicons from the target region. Furthermore, ZIKV was successfully detected in plasma samples spiked at 1 × 103 PFU/mL, which enabled complete sequencing of the E gene. These findings highlight the utility of our primers for molecular epidemiological studies, strengthening arbovirus surveillance and improving diagnostic capacity in regions where multiple arboviruses co-circulate, using a simple and reliable assay.