Dengue virus (DENV) is a mosquito-borne RNA virus that causes serious illness in humans, ranging from mild fever to severe clinical manifestations, with dengue virus type 2 (DENV-2) being the most virulent among its four serotypes. Despite extensive research, no specific antiviral therapy is currently available, making the host-directed method an appealing therapeutic approach. Evidence shows that DENV manipulates host kinase-driven phosphorylation pathways to control viral pathogenesis. Using the kinase–substrate phosphomotif approach, we predicted phosphorylation sites across the DENV proteome and their potential human kinases. The predicted kinase–substrate interactions were systematically integrated with DENV-2-induced human phosphoproteome datasets, protein–protein interactions, and experimentally-validated viral phosphosites. The therapeutic relevance of the identified host kinases was corroborated by the impact of their inhibitors on DENV-2 infection. Among the 359 potential human kinases predicted to phosphorylate DENV-2 proteins, based on human phosphoproteome and kinase–viral protein interaction analyses, CDK9 emerged as a central hub kinase. Molecular docking analyses further revealed that the host kinases CDK9, EEF2K, HASPIN, and TNNI3K form stable interactions with the viral capsid and NS5 proteins. Additionally, a conservation analysis suggested that the predicted phosphorylation sites are evolutionarily conserved across DENV-2 strains. Computational prediction tools supported the predicted kinase–substrate interactions, underscoring the role of host kinases as key regulators of DENV infection, which may act as potential therapeutic targets. This study highlights the interplay between dengue viral and host proteins, providing insights into host-directed therapeutic strategies for DENV-2 infection and their potential to address the current lack of effective antiviral interventions.

West Nile virus (WNV) is a mosquito-borne pathogen of escalating epidemiological importance and a growing global health concern, driven by the climate-associated expansion of its Culex mosquito vectors. Although WNV is an extensively studied flavivirus, most host–pathogen interaction studies focus on static and structural aspects rather than dynamic and functional ones. Delineating phosphorylation-mediated interactions between WNV proteins and human kinases bridges a critical gap by providing important insight into the molecular mechanisms underlying infection. In this study, we investigated potential phosphorylation-mediated interactions between WNV proteins and human kinases using an integrative computational framework combining motif prediction, phosphoproteomic data analysis and structural docking. Key interactions were predicted between viral proteins and regulatory kinases within the AKT-ERK pathway and the AMPK-mediated autophagy, including major network kinases such as RAF1, IKBKB, and ULK1. In addition, experimentally validated phosphorylation sites in viral proteins were found to be associated with multiple candidate host kinases, including MAP2K7 and MAP2K9, suggesting complex regulatory networks. Integration with phosphoproteomic datasets supported the relevance of multiple predicted kinases, including those associated with antiviral responses and translational regulation. Protein–protein docking demonstrated stable, energetically favorable interactions between selected host kinases and viral proteins, particularly the viral polymerase (NS5), helicase (NS3), and NS1. The findings of this study establish a framework for future research on the development of host-directed antiviral strategies.