Single-cell and bulk RNA sequencing reveal lncRNAs driving neuroimmune responses in Japanese encephalitis

Japanese encephalitis virus (JEV), a neurotropic flavivirus, is a leading cause of viral encephalitis in Asia and remains a major public health concern. Long non-coding RNAs (lncRNAs) have emerged as important regulators of immune and neurological diseases; however, their roles in viral encephalitis remain largely unknown. Here, single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq), in vivo validation, and functional analyses were integrated to comprehensively characterize lncRNA dynamics during Japanese encephalitis (JE). ScRNA-seq analysis revealed widespread cell type–specific lncRNA expression patterns during JEV infection. By integrating differential expression analysis with disease-stage information, 108 concordantly differentially expressed lncRNAs (CoDElncRNAs) were identified, showing monotonic changes from healthy to mild to severe disease states across multiple brain cell populations. Among these, nine pan-cellular CoDElncRNAs—including AW112010, AW011738, Gm12216 (CARINH), Gm20559, 4930599N23Rik, G530011O06Rikx, 4933412E12Rik, Mir142hg, and Zfas1—were consistently upregulated across multiple CNS-resident cell types. Co-expression network inference revealed immune-linked lncRNA modules enriched in cytokine signaling, interferon responses, antigen presentation, and cell development pathways, underscoring the potential contribution of lncRNAs to neuroinflammatory regulation. Bulk RNA-seq and RT–qPCR recapitulated cell-level findings at the tissue scale, providing a multidimensional understanding of lncRNA-mediated mechanisms underlying viral encephalitis. In addition, several circulating lncRNAs were detectable in serum samples from infected mice, highlighting their potential as minimally invasive biomarkers. Functional analyses further identified Zfas1 as a regulator of microglia-associated inflammatory responses, where siRNA-mediated knockdown significantly attenuated JEV-induced cytokine and chemokine expression. This study provides a cell-resolved lncRNA landscape in viral encephalitis, highlighting their role in neuroinflammation and offering new avenues for biomarker and therapeutic development for neurotropic viral infections.