Pyrimidine metabolism is crucial for the replication of viruses and the functionality of host cells. However, the cell-type-specific organization of this metabolism in the central nervous system and its dysregulation during Japanese encephalitis virus (JEV) infection remain poorly understood. Here, we aimed to characterize the cell-type-specific landscape of pyrimidine metabolism in the CNS and uncover pyrimidine metabolic reprogramming during JEV infection.

Single-cell RNA sequencing data from normal and JEV-infected mouse brains were analyzed to profile the expression of pyrimidine metabolic genes across neuronal, glial, and vascular cell types. Functional validation of these metabolic pathways on JEV replication was performed in Neuro-2a cells and primary mouse brain-derived mixed neuron/glia cultures using pharmacological inhibitors, metabolite supplementation, and virological assays, including quantitative reverse transcription polymerase chain reaction, immunoblotting, and plaque assay.

We defined a universal core program for pyrimidine homeostasis alongside specialized, function-oriented metabolic programs across neurons, glia, and vascular cells in the normal mouse brain. JEV infection triggered cell-intrinsic reprogramming of this metabolic network, with viral replication critically depending on the de novo pyrimidine biosynthesis pathway in neurons. Pharmacological inhibition of the key enzyme dihydroorotate dehydrogenase significantly suppressed JEV replication. Furthermore, JEV replication created a metabolic dependency on exogenous glutamine and enhanced glutamate oxaloacetate transaminase-mediated aspartate synthesis for precursor acquisition.

JEV rewires host brain pyrimidine metabolism in a cell-type-specific manner, revealing distinct metabolic dependencies on de novo pyrimidine biosynthesis, exogenous glutamine uptake, and glutamate oxaloacetate transaminase-mediated aspartate synthesis in neurons and highlighting potential targets for future antiviral strategies. By resolving these alterations at single-cell resolution, this study also provides a cell-type-resolved pyrimidine metabolic landscape of both the normal and JEV-infected brain.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide. JEV exhibits significant neuroinvasiveness and neurotoxicity, resulting in considerable damage to the nervous system. Japanese encephalitis is associated with high morbidity and mortality rate, seriously harming both human health and livestock production. The current lack of specific antiviral drugs means that the development of new therapeutic agents for JEV has become urgent.

Anti-JEV drugs were screened from 111 inhibitors of neurotransmitter receptor-related molecules by high content technology. The antiviral effects of clomipramine HCl were evaluated through plaque assay, realtime quantitative PCR, immunofluorescence assay and western blotting assay. Bioinformatic tools were utilized to cluster the altered signaling pathway members after clomipramine HCl treatment. Finally, the anti-JEV mechanism was deeply resolved in vivo via such molecular biology and virological detection techniques.

In this study, we screened nine compounds with significant anti-JEV activity, of which clomipramine HCl demonstrated the most potent antiviral effect and exhibited dose-dependent activity. Mechanistically, clomipramine HCl may activate endoplasmic reticulum stress and modulate the unfolded protein response, thus inhibiting the assembly stage of JEV infection.

This study highlights the importance of clomipramine HCl as a promising approach for JEV infection protection, which may lead to new host-directed antiviral approaches to such mosquito-borne viruses.