Japanese encephalitis virus envelope protein activates the TLR4/NF‑κB pathway to induce testicular inflammation

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis in Asia and poses serious threats to reproductive health. However, the molecular mechanisms underlying JEV-induced testicular inflammation remain incompletely understood. In this study, an integrated approach involving proteomic, cellular, and animal experiments was used to investigate the role of Toll-like receptor 4 (TLR4) in JEV-triggered inflammatory responses. Proteomic analysis and in vitro assays revealed that JEV infection upregulated TLR4 expression and activated the NF‑κB signaling cascade, resulting in enhanced secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Among the viral proteins examined, only the envelope (E) protein significantly increased endogenous TLR4 levels in a dose-dependent manner. Direct interaction between the E protein and TLR4 was confirmed by co-immunoprecipitation and immunofluorescence. In a mouse model of JEV infection, the virus induced severe testicular inflammation, characterized by tissue disruption, TLR4 upregulation, and activation of the TLR4/NF-κB pathway, consistent with the in vitro observations. Collectively, these findings identify the JEV E protein as a key inducer of TLR4-mediated inflammation and provide mechanistic insights into JEV-associated testicular pathology.