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

## TL;DR

This study shows that Duck Tembusu virus disrupts calcium balance in cells, which helps the virus replicate by activating the AMPK pathway.

## Contribution

The study reveals a novel Ca²+-AMPK signaling mechanism that facilitates Duck Tembusu virus replication.

## Key findings

- DTMUV increases cytoplasmic Ca²+ in duck embryo fibroblasts via voltage-gated calcium channels.
- Reducing Ca²+ or inhibiting AMPK suppresses DTMUV RNA replication and progeny virus production.
- AMPK activation by DTMUV is Ca²+-dependent and promotes viral replication.

## Abstract

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.

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.

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.

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.