**Background:** Zika virus (ZIKV) infection during the first trimester of pregnancy causes severe fetal brain damage, including microcephaly, while infection in later trimesters results in milder neurodevelopmental abnormalities. The mechanisms underlying this trimester-specific vulnerability remain unclear. Neuronal progenitor cells undergo metabolic shifts during differentiation—from glycolysis-dominant metabolism in quiescent progenitors to mitochondrial glucose and fatty acid oxidation in differentiating cells. This study hypothesized that ZIKV differentially hijacks the metabolism of cortical progenitors at different maturation stages, contributing to the distinct brain damage patterns observed across trimesters.
**Methods:** Cortical neuronal progenitors (hi-NPCs) were differentiated from three independent hiPSC lines using a modified 2D protocol. Cells were harvested at two time points—early (less differentiated, quiescent-like) and late (more differentiated, immature neurons). Metabolic characterization was performed using radioactive tracing of glycolytic flux (5-³H-glucose), glucose oxidation (¹⁴CO₂ capture), and oleic acid oxidation (³H₂O release). Long-term glucose consumption and lactate release were measured over 72 hours. Cells were infected with ZIKV (African strain MP1751, MOI 1) for 2 hours. Viral replication was assessed by qPCR (intracellular ZIKV RNA), flow cytometry (NS1 and Envelope proteins), and plaque assay (extracellular virions). Cell viability was measured by LDH assay. Glycolytic gene expression (HK-1, GLUT-1, GAPDH) was quantified by qPCR. Mitochondrial parameters (membrane potential, size, abundance, distribution) were analyzed by imaging flow cytometry (Amnis ImageStream) in Env+ve, Env-ve, and non-infected cells. Lipid droplet homeostasis (size, abundance, distribution) was assessed by BODIPY staining and imaging flow cytometry. Fatty acid oxidation (PDK2, ACADM, HADHA) and biosynthesis (ACACA, FASN) gene expression were quantified by qPCR.
**Key Results:** Early hi-NPCs exhibited significantly higher glycolytic flux (P=0.0286) and glucose consumption (P<0.0001) but lower fatty acid oxidation (P=0.0286) compared to late hi-NPCs, confirming distinct metabolic maturation stages. Late hi-NPCs accumulated significantly more ZIKV RNA than early hi-NPCs (late:early ratio 1.77 at 48 h.p.i., 1.98 at 72 h.p.i.) and released more infectious virions (late:early ratio 2.38 at 48 h.p.i., 2.52 at 56 h.p.i.). Despite lower viral replication, early hi-NPCs showed significant cell death at 72 h.p.i. (P≤0.0031), while late hi-NPCs showed no significant viability changes. ZIKV infection significantly increased glycolytic capacity exclusively in early hi-NPCs at 24 h.p.i. (P=0.0167), 48 h.p.i. (P=0.0022), and 56 h.p.i. (P=0.0004), with significantly increased lactate release at 48 h.p.i. (P=0.045) and 56 h.p.i. (P=0.006). Mitochondrial analysis revealed that Env+ve early hi-NPCs had significantly reduced mitochondrial size (P=0.0328) and abundance (P=0.0091) at 24 h.p.i., while Env+ve late hi-NPCs showed reduced mitochondrial size (P=0.0047) and abundance (P=0.002) only at 56 h.p.i. Mitochondrial distribution was reduced in both early and late Env+ve cells at 24 h.p.i. (P=0.0251 and P=0.0071, respectively). Lipid droplet analysis showed that Env+ve early hi-NPCs had significantly increased lipid droplet area (P≤0.0242) and abundance (P≤0.0189) at 24 h.p.i., while late hi-NPCs showed no lipid droplet alterations. Gene expression analysis revealed that PDK2 was significantly increased in early vs. late hi-NPCs at 72 h.p.i. (P=0.0153). ACADM showed greater expression in late hi-NPCs at 24 h.p.i. (late:early ratio 1.27) but greater in early hi-NPCs at 72 h.p.i. (early:late ratio 1.62). HADHA was significantly higher in late hi-NPCs at 24 h.p.i. (late:early ratio 1.26). For lipid biosynthesis, ACACA and FASN showed greater expression in late hi-NPCs at 24 h.p.i. (ratios 1.64 and 1.43, respectively) but greater in early hi-NPCs at 48 h.p.i. (ratios 1.33 and 1.12, respectively).
**Clinical Implications:** This study provides mechanistic insight into why ZIKV infection causes more severe fetal brain damage during the first trimester. Early-stage (quiescent-like) cortical progenitors, which predominate in the first-trimester fetal brain, are metabolically distinct—they rely heavily on glycolysis and are vulnerable to ZIKV-induced metabolic hijacking, including increased glycolytic capacity, mitochondrial fragmentation, and lipid droplet accumulation. These disruptions may lead to the significant cell death observed in early hi-NPCs, potentially explaining the microcephaly and severe neurodevelopmental defects seen in congenital Zika syndrome. In contrast, late-stage progenitors (resembling second/third-trimester cells) rely more on mitochondrial oxidation and show different metabolic vulnerabilities—including delayed mitochondrial stress without significant cell death—which may correlate with the milder neurodevelopmental abnormalities (e.g., epilepsy, autism-like features) observed when infection occurs later in pregnancy. The findings highlight that therapeutic strategies targeting metabolism may need to be tailored to the developmental stage of infection. The study also reconciles conflicting literature on ZIKV-induced metabolic changes by demonstrating that effects on fatty acid oxidation and biosynthesis are time-dependent and maturation-stage-specific.