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Disruption of cellular calcium homeostasis by duck Tembusu virus facilitates viral replication via AMPK pathway activation.

Ziding Yu,Xiaoyong Chen,Wen-Jian Zhuo,Zhenxing Ma,Zejun Xu,Kuanhui Liu,Julong Wang,Ting Li,Guangshuang Zhu,Ben Wang,Ran Xiong,Chao Li,Haiyan Zhang,Jingliang Su

Published 2026 in Frontiers in Cellular and Infection Microbiology

ABSTRACT

Background The flavivirus Duck Tembusu virus (DTMUV) exhibits high pathogenicity and transmissibility, posing a severe threat to the poultry industry in China and Southeast Asia. Although the molecular mechanisms of DTMUV pathogenesis remain unclear, its potential to disrupt intracellular calcium ion (Ca²+) homeostasis, a known driver of viral replication, has not been investigated. Here, we investigated the role and underlying mechanism of Ca²+ homeostasis in DTMUV infection. Methods Fluo-4AM staining and flow cytometry of duck embryo fibroblasts (DEFs) were used to detect cytoplasmic Ca²+. To modulate Ca²+ levels and AMP-activated protein kinase (AMPK) activity, we used voltage-gated calcium channel (VGCC) blockers (verapamil, diltiazem hydrochloride), a Ca²+ chelator (BAPTA-AM), and an AMPK inhibitor (Compound C). Viral entry, genomic RNA replication (targeting the DTMUV NS5 gene), viral yield, and release were evaluated via qRT-PCR and plaque assays. AMPK activation was detected using western blotting with anti-phospho-AMPKα (Thr172) and anti-AMPKα antibodies. Statistical analyses were performed using Student's t-test or two-way analysis of variance. Results DTMUV infection significantly increased cytoplasmic Ca²+ in DEFs at 6, 8, 10, and 12 hours post-infection. This elevation was suppressed by treatment with verapamil or diltiazem hydrochloride, indicating that DTMUV induces extracellular Ca²+ influx via VGCCs. Functional assays showed that reducing cytoplasmic Ca²+ via treatment with VGCC blockers or BAPTA-AM specifically inhibited DTMUV RNA replication, but not viral entry or release, decreasing progeny virus production. Further mechanistic analysis revealed that DTMUV infection activates the AMPK pathway in a Ca²+-dependent manner, with Compound C-mediated AMPK inhibition dose-dependently suppressing viral RNA replication and progeny yield. Conclusions DTMUV disrupts host Ca²+ homeostasis to activate AMPK, which promotes viral RNA replication. This study provides novel insights into DTMUV pathogenesis and identifies Ca²+-AMPK signaling as a potential anti-DTMUV target.

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