Metabolite profiles distinguish exposure to Dengue and Zika flaviviruses in human induced pluripotent stem cells (hiPSCs)

Flaviviruses such as Dengue virus (DENV), Zika virus (ZIKV) and West Nile Virus (WNV) pose growing public health threats, exacerbated by asymptomatic infections and non-vector transmission. Current diagnostics, including nucleic acid tests and serology, are limited by transient viremia and antibody cross-reactivity, underscoring the need for improved detection methods. We utilized untargeted metabolomics to differentiate DENV and ZIKV infections in human induced pluripotent stem cells (hiPSCs), a model relevant to study virus-host interactions, drug screening, and therapeutic safety. LC-MSbased profiling revealed virus-specific metabolic reprogramming during both acute and long-term infections. DENV3 induced early and sustained activation of metabolic features, while ZIKV-MR766 triggered transient suppression. Long term infection with DENV2, DENV3, or ZIKV-PRV resulted in distinct metabolic signatures in hiPSCs, with ZIKV-PRV showing the greatest divergence. Shared metabolite changes across conditions included amino acids (e.g., tryptophan, glutamate), lipids, and nucleosides. Functional studies demonstrated that tryptophan metabolism regulates infection dynamics: inhibiting serotonin biosynthesis reduced viral load, whereas blocking kynurenine synthesis enhanced viral replication. These findings position metabolomics as a viable approach for flavivirus detection and highlight metabolic pathways as potential therapeutic targets. ImportanceAccurate diagnosis of flavivirus infections remains challenging due to short periods of viremia, high rates of asymptomatic infection, and extensive serologic cross-reactivity among related viruses such as DENV and ZIKV. These limitations complicate clinical diagnosis, surveillance, and screening of human donor tissues. In this study, we demonstrate that untargeted metabolomics can distinguish ZIKV and DENV infections by identifying virus-specific host metabolic signatures during both acute and long-term infection. Using human induced pluripotent stem cells as a clinically relevant model, we show that metabolic reprogramming persists even in the absence of overt cytopathic effects and can reveal functional pathways that regulate viral replication. Our findings highlight host metabolite profiling as a complementary diagnostic strategy that may improve detection of flavivirus exposure, particularly in asymptomatic individuals or settings where conventional molecular and serologic tests are insufficient.