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Diseases caused by the genus  Flavivirus , including dengue virus (DENV) and Zika virus (ZIKV), have a serious impact on public health worldwide. Due to serological cross-reactivity among flaviviruses, current enzyme-linked immunosorbent assay (ELISA) for IgM/G cannot reliably distinguish between infection by different flaviviruses. In this study, we developed a reporter-based neutralization assay using single-round infectious particles (SRIPs) derived from representative flaviviruses. SRIPs were generated by transfection of human embryonic kidney 293 T cells with a plasmid encoding premembrane and envelope (prME) proteins from DENV1–4, ZIKV, Japanese encephalitis virus, West Nile virus, yellow fever virus, Usutu virus, and tick-borne encephalitis virus, along with a plasmid carrying DENV1 replicon containing the luciferase gene and plasmid for expression of DENV1 capsid. Luciferase activity of SRIPs-infected cells was well correlated with number of infected cells, and each reporter SRIP was specifically neutralized by sera from mice immunized with each flavivirus antigen. Our high-throughput reporter SRIP-based neutralization assay for multiple flaviviruses is a faster, safer, and less laborious diagnostic method than the conventional plaque reduction neutralization test to screen the cause of primary flavivirus infection. The assay may also contribute to the evaluation of vaccine efficacy and assist in routine surveillance and outbreak response to flaviviruses.

High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon