Duck Tembusu virus (DTMUV) is an enveloped, single-stranded positive-sense RNA virus belonging to the genus Flavivirus within the family Flaviviridae. The genome of DTMUV is approximately 11,000 nucleotides (nt) in length, containing one open reading frame (ORF) that encodes three structural proteins (C, PrM, E) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Tang et al., 2012). The Tembusu virus (TMUV) prototype strain MM1775 was first isolated from mosquitoes in 1955 (Platt et al., 2016). In 2010, a DTMUV outbreak occurred in southeastern China, characterized by encephalitis and a sharp decline in egg production in duck and goose flocks (Qiu et al., 2011). Subsequently, diseases associated with this virus were also reported in Southeast Asian countries (Homonnay et al., 2014; Ninvilai et al., 2018). In recent years, accumulating studies have confirmed that several DTMUV strains, characterized by substantial antigenic variation and belonging to a unique genetic cluster, have begun circulating in chicken flocks (Yu et al., 2021; Yan et al., 2022). DTMUV exhibits high pathogenicity and transmissibility, with infection in avian hosts leading to a marked decline in egg production and triggering neurological symptoms, typically encephalitis. Currently, this virus is widely distributed across China and Southeast Asia, causing enormous economic losses to the regional poultry industry. Although vaccination has historically been a key strategy for DTMUV control, effective prevention requires elucidating its pathogenic mechanisms to identify potential antiviral targets.

The calcium ion (Ca²⁺) is one of the most ubiquitous and versatile signaling molecules in eukaryotic cells. As an intracellular second messenger, Ca²⁺ is involved in regulating almost all cellular physiological processes, including gene transcription, differentiation, proliferation, and kinase activation (Berridge et al., 2000, 2003). Under normal cellular physiology, intracellular Ca²⁺ homeostasis—maintaining stable cytoplasmic Ca²⁺ concentrations—is tightly regulated by the coordinated interplay of channels, transporters, and pumps (Berridge et al., 2003). As a stimulant, viruses often disrupt intracellular Ca²⁺ homeostasis by hijacking Ca²⁺-mediated signaling transduction pathways to facilitate the infection cycle. For instance, upon infecting host cells, duck enteritis virus (DEV) and porcine reproductive and respiratory syndrome virus (PRRSV) increase cytoplasmic Ca²⁺ concentrations, inducing autophagy mediated by the calmodulin-dependent protein kinase II (CaMKK-II) and adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway to promote their own replication (Yin et al., 2018; Manicassamy et al., 2023). Within the genus Flavivirus, host cell infection by West Nile virus (WNV) and dengue virus (DENV) also induces an increase in cytoplasmic Ca²⁺ concentration. Importantly, pharmacological inhibition of this Ca²⁺ elevation significantly reduces the production of viral particles, suggesting that the infection-induced elevation in cytoplasmic Ca²⁺ is essential for the replication of these flaviviruses (Scherbik and Brinton, 2010; Dionicio et al., 2018).

Despite the well-documented association between Ca²⁺ homeostasis and flavivirus infection, its role in DTMUV infection remains uncharacterized. Here, we report the first investigation of this association, demonstrating that DTMUV infection significantly increases cytoplasmic Ca²⁺ concentrations in host cells. Furthermore, this elevation in Ca²⁺ activates the AMPK signaling pathway, a critical mediator of DTMUV RNA replication. These findings lay a solid foundation for elucidating the molecular mechanisms of DTMUV pathogenesis and offer new insights into potential targets for anti-DTMUV strategies.

As a ubiquitous intracellular second messenger, Ca²⁺ plays a pivotal regulatory role in virtually all cellular processes throughout the cell cycle (Zhou et al., 2009). A growing body of evidence points to the active involvement of Ca²⁺ and Ca²⁺-related proteins in the viral life cycle, playing a critical role in viral pathogenesis (Casciano and Bouchard, 2018; Fujioka et al., 2018; Gu et al., 2016; Lee et al., 2024). In this study, we found that DTMUV infection disrupts Ca²⁺ homeostasis in host cells by increasing cytoplasmic Ca²⁺ levels. This elevation in cytoplasmic Ca²⁺ activates the AMPK pathway, promoting viral RNA replication and proliferation. To our knowledge, this is the first report linking Ca²⁺, the AMPK pathway, and DTMUV replication.

Under normal cellular physiological conditions, intracellular Ca²⁺ homeostasis—defined as the maintenance of a stable concentration of cytoplasmic free Ca²⁺—is tightly regulated by the coordinated interplay of multiple membrane transporters and regulatory proteins. These key mediators include plasma membrane-localized Ca²⁺ channels, Na⁺-Ca²⁺ exchangers, plasma membrane Ca²⁺-ATPases; mitochondrial Ca²⁺-H⁺ exchangers, and a repertoire of cytoplasmic Ca²⁺-binding proteins that act as Ca²⁺ buffers, fine-tuning cytoplasmic Ca²⁺ dynamics (Berridge et al., 2003). Viral infection, as an external stimulus, frequently disrupts intracellular Ca²⁺ homeostasis. RNA viruses such as HIV type 1, human T-lymphotropic virus type 1 (Ding et al., 2002), and hepatitis C virus (HCV) (Griffin et al., 2002), as well as DNA viruses including hepatitis B virus (Casciano and Bouchard, 2018) and herpes simplex virus types 1 and 2 (Cheshenko et al., 2003), have been shown to elevate cytosolic Ca²⁺ levels and perturb cellular Ca²⁺ homeostasis. This disruption is typically mediated by specific viral proteins that either facilitate the influx of extracellular Ca²⁺ into cells or induce the release of Ca²⁺ from intracellular stores. In this study, we found that VDCC blockers could significantly inhibit the increase in cytoplasmic Ca²⁺ concentration induced by DTMUV infection, but failed to completely abolish this effect. These results indicate that VDCCs are not the sole pathway mediating the elevation of cytoplasmic Ca²⁺ levels in DEFs upon DTMUV infection. Recent studies have reported distinct mechanisms of Ca²⁺ homeostasis disruption by closely related flaviviruses. For instance, WNV induces an influx of extracellular Ca²⁺ into infected cells by activating plasma membrane calcium channels (Scherbik and Brinton, 2010). In contrast, DENV disrupts intracellular Ca²⁺ homeostasis through two mechanistically distinct pathways: enhancement of plasma membrane permeability to Ca²⁺ and activation of the store-operated calcium entry (SOCE) pathway, which is governed by intracellular calcium stores (Dionicio et al., 2018). The aforementioned studies have demonstrated that in flavivirus-infected cellular models, both extracellular calcium influx and the release of calcium from intracellular stores disrupt cytoplasmic calcium homeostasis, resulting in elevated cytoplasmic Ca²⁺ concentrations. Therefore, it is reasonable to hypothesize that, beyond the VDCCs-mediated Ca²⁺ influx demonstrated in the present study, DTMUV infection may also trigger the release of Ca²⁺ from intracellular calcium stores via activating channels such as inositol 1,4,5-triphosphate receptors (IP₃R) and ryanodine receptors (RyR) on the mitochondrial membrane, thereby elevating the cytoplasmic Ca²⁺ level. Further investigation is warranted to clarify these mechanisms in greater detail.

Additionally, it remains unclear how DTMUV infection activates L-type calcium channels on the plasma membrane. One possibility involves lipid rafts, which are cholesterol- and sphingolipid-rich microdomains that serve as unique signal transduction platforms. Multiple studies have demonstrated that lipid rafts and caveolar microdomains of the plasma membrane are enriched in proteins that regulate calcium signaling, including those that facilitate L-type calcium channel activation and calcium influx (Pani and Singh, 2009; Balijepalli et al., 2006). Furthermore, these lipid rafts are critically involved in the cellular entry of certain flaviviruses, including DENV and WNV (Osuna-Ramos et al., 2018). As a member of the Flavivirus genus, DTMUV is hypothesized to utilize lipid raft microdomains of the plasma membrane as a functional platform for aggregating key signaling molecules, thereby activating L-type calcium channels and mediating Ca²⁺ influx. Based on this proposed regulatory mechanism, subsequent studies should prioritize systematic, in-depth investigations to elucidate its molecular basis.

Current evidence suggests that Ca²⁺ plays a critical role in nearly every stage of the viral life cycle. Here, we demonstrated that the L-type calcium channel blocker diltiazem hydrochloride and the cytoplasmic calcium chelator BAPTA-AM reduce DTMUV yield by inhibiting the viral RNA replication step, without affecting viral entry or release. In previous studies on the interaction between cellular Ca²⁺ and viruses, pharmacologically reducing intracellular Ca²⁺ concentrations using calcium channel blockers or calcium chelators effectively inhibited viral RNA replication in Zika virus (ZIKV) (Bezemer et al., 2022), PRRSV (Manicassamy et al., 2023), porcine deltacoronavirus (PDCoV) (Bai et al., 2020), and respiratory syncytial virus (RSV) (Chen et al., 2024). Such findings are highly consistent with those of the present study. Within the Ca²⁺-mediated intracellular signal transduction network, AMPK is activated by CaMKK-II and functions as a key intracellular energy sensor, regulating critical physiological processes including cellular metabolism and autophagy (Salt et al., 2024; Herzig and Shaw, 2017). Therefore, from a mechanistic perspective, this study focused on AMPK as a central target for investigation. Our results revealed a coupling between the DTMUV infection-induced elevation of cytoplasmic Ca²⁺ and AMPK activation. Furthermore, treatment with an AMPK inhibitor significantly reduced DTMUV production by suppressing viral RNA replication—effects consistent with those observed upon treatment with VSCCs or a cytoplasmic Ca²⁺ chelator. These data indicate that cellular Ca²⁺ primarily regulates DTMUV replication via activation of the AMPK signaling pathway. This mechanism aligns with findings from studies of other viruses. For example, DEV infection increases cytoplasmic Ca²⁺ levels to activate the CaMKK-II–AMPK pathway and induce autophagy of DEFs; blocking this pathway with specific inhibitors significantly suppresses DEV replication (Yin et al., 2018). Similarly, PRRSV promotes its own replication via the same Ca²⁺–CaMKK-II–AMPK–autophagy axis (Manicassamy et al., 2023). Drawing on the rationale of the aforementioned studies, we investigated whether AMPK activation mediated by elevated cytoplasmic Ca²⁺ could induce autophagy in DEFs. We found that the expression level of microtubule-associated protein 1 light chain 3-II (LC3-II) in DEFs was significantly upregulated at 12 h post-infection with DTMUV. Interstingly, treatment of DEFs with BAPTA-AM and Compound C did not significantly alter the LC3-II expression level induced by DTMUV infection (Supplementary Material). Previously, Hu et al. confirmed that DTMUV infection can induce a complete autophagic response in DEFs by demonstrating the degradation dynamics of p62, the turnover of LC3-II throughout the infection cycle, and the formation of autolysosome-like vesicles observed via transmission electron microscopy (TEM) (Hu et al., 2020). In the present study, LC3-II was employed as the sole marker for autophagy detection, and experimental observations were restricted to a single time point at 12 hours post DTMUV infection. As a result, dynamic changes in autophagy throughout the entire course of DTMUV infection could not be fully captured. Thus, the available data are insufficient to elucidate whether autophagy modulates DTMUV replication via the calcium-AMPK signaling pathway, and the underlying molecular mechanisms merit further investigation. In addition, another potential mechanism is associated with oxidative stress, a biological state characterized by the disruption of the balance between the accumulation and clearance of intracellular reactive oxygen species (ROS). Accumulating evidence has confirmed that oxidative stress, Ca²⁺ signaling, AMPK activity, and autophagy form a highly interconnected regulatory network (Michalak et al., 2025). Among the Flavivirus genus, such as HCV, DENV, and Japanese encephalitis virus (JEV) have all been shown to induce ROS production, thereby triggering oxidative stress in infected cells (Korenaga et al., 2005; Guzman et al., 2014; Ghoshal et al., 2007). A close bidirectional interaction exists between ROS and Ca²⁺ signaling: on the one hand, ROS can modulate intracellular calcium signaling; on the other hand, Ca²⁺ signaling is a prerequisite for ROS generation (Görlach et al., 2015). For instance, studies have demonstrated that ROS facilitate calcium ion uptake in vascular smooth muscle cells via L-type and T-type voltage-gated calcium channels (Tabet et al., 2004); conversely, Ca²⁺ influx in neutrophils promotes ROS production by activating the recruitment of Ca²⁺-dependent S100A8/A9 proteins (Bréchard et al., 2013). Furthermore, in viral infection models, ROS can promote AMPK activation, thereby regulating autophagy and metabolism-related processes (Zhao et al., 2025). At the level of viral life cycle regulation, oxidative stress has been verified to modulate the capping of flaviviral RNA, which in turn enhances viral proliferation (Gullberg et al., 2015). Taken together, based on existing research findings and the data from the present study, we hypothesize that cellular oxidative stress may be involved in the regulation of viral replication via the Ca²⁺-AMPK axis induced by DTMUV infection, and this hypothesis warrants systematic verification in subsequent studies. Besides, it is worth noting that the Ca²⁺-CAMKK-II axis is not the sole pathway mediating the activation of AMPK during viral infection. As another upstream kinase of AMPK, liver kinase B1 (LKB1) also has the capacity to activate AMPK. For instance, the NS1 protein of dengue virus type 2 (DENV-2) could act as an assembly scaffold to facilitate the AMPK-LKB1 interaction, thereby inducing AMPK activation (Wu et al., 2023). Therefore, subsequent studies should investigate the role of LKB1 in AMPK activation induced by duck Tembusu virus (DTMUV) infection.

Flaviviruses typically establish an optimal replication environment by comprehensively modulating these metabolic processes of the host. On one hand, flavivirus infection can enhance the glycolytic pathway and pentose phosphate pathway of host cells, a phenomenon observed with viruses such as dengue virus DENV and JEV (Allonso et al., 2015; Li et al., 2021). The glycolysis process promoted by viral infection regulates multiple physiological processes, including increasing the reserves of adenosine triphosphate (ATP) and nucleotides to provide essential energy supply and precursor substances for material synthesis during viral replication. In addition, flavivirus replication requires the formation of invaginated structures on the endoplasmic reticulum membrane, known as replication compartments (RCs) (Gillespie et al., 2010). Flavivirus infection usually reshapes the host membrane system by promoting the synthesis of specific lipids to ensure the complete formation of RCs. For instance, WNV infection recruits 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme in cholesterol biosynthesis, to RCs, thereby enhancing cholesterol synthesis and accumulation in the RC region. Inhibition of this process blocks WNV replication (Mackenzie et al., 2007). The non-structural protein 3 (NS3) of DENV relocalizes fatty acid synthase (FASN) to viral replication sites and promotes cellular fatty acid synthesis, thus facilitating the establishment of RCs (Heaton et al., 2010). Intracellular lipid droplets also serve as important targets for host metabolic regulation following flavivirus infection. Early in DENV infection, lipid droplet reabsorption into the endoplasmic reticulum can be observed, a process that may provide lipid raw materials for endoplasmic reticulum expansion and RC formation (Peña and Harris, 2012). In the late stage of infection, DENV induces lipophagy, a type of selective autophagy, through which lipids in lipid droplets are mobilized for β-oxidation and energy production (Heaton and Randall, 2010). As a central regulator of intracellular energy homeostasis, the activated AMPK modulates various cellular metabolic pathways, including lipid homeostasis maintenance and glycolysis, via phosphorylating downstream key targets (Herzig and Shaw, 2017). Based on the aforementioned findings regarding flaviviruses, we postulate that the activation of the AMPK pathway induced by DTMUV infection is likely involved in the reprogramming of host cellular metabolic programs, thereby establishing a favorable intracellular environment for viral replication. Further studies are required to elucidate the detailed molecular mechanism underlying this process.

In summary, our data demonstrate that DTMUV infection significantly disrupts Ca²⁺ homeostasis in host cells. Dysregulation of intracellular Ca²⁺ homeostasis activates the AMPK signaling pathway, which is a critical prerequisite for DTMUV to complete viral RNA replication. These findings provide a theoretical basis for elucidating the pathogenic mechanism of DTMUV and identifying novel antiviral targets.