# CD4 Molecule Plays an Important Role in the Inflammatory Response Induced by Japanese Encephalitis Virus Infection

## TL;DR

This study shows that the CD4 molecule helps Japanese encephalitis virus spread and causes inflammation in cells, offering new insights into how the virus causes disease.

## Contribution

The study reveals that CD4 regulates JEV proliferation and STAT1 signaling independently of viral replication.

## Key findings

- CD4 knockdown inhibits JEV replication, adsorption, and internalization in TM3 cells.
- CD4 modulates STAT1 phosphorylation and inflammatory cytokine production independently of viral replication.
- Restoring CD4 expression reverses the inhibitory effects of CD4 knockdown on JEV and STAT1 signaling.

## Abstract

TM3 cells were utilized as a model to investigate the regulatory effects of CD4 on Japanese encephalitis virus (JEV) infection, the STAT1 signaling pathway, and inflammatory factor expression. Our results demonstrate that CD4 knockdown inhibits the early adsorption, internalization, and proliferation of JEV, while concurrently downregulates phosphorylated STAT1 (p-STAT1) and inflammatory cytokines. Although treatment with the STAT agonist RO8191 failed to fully restore these signaling indicators in CD4-knockdown cells, rescuing CD4 expression significantly reversed the inhibitory effects. Studies have confirmed that, on one hand, CD4 molecules positively regulate JEV proliferation, while on the other hand, CD4 modulates STAT1 activation, and this regulation extends beyond the impact of viral replication, providing a theoretical basis for understanding the pathogenic mechanisms of JEV in the reproductive system and its prevention and control.

Japanese encephalitis virus (JEV) is an important flavivirus that causes zoonotic and arboviral diseases. Infection with JEV not only induces acute central nervous system (CNS) infectious diseases but also leads to reproductive disorders. Currently, research on the pathogenic mechanism of JEV has mainly focused on CNS inflammation caused by infection, while studies on the pathogenic mechanism of JEV targeting the reproductive system are relatively scarce. This study used TM3 cells as a model to investigate the regulatory role of the CD4 molecule in JEV infection, the STAT1 signaling pathway, and inflammatory factors. Firstly, we found that CD4 knockdown significantly inhibited JEV replication in TM3 cells. Further virus adsorption and internalization experiments confirmed that CD4 knockdown specifically impaired the early stages of JEV invasion into cells. Additionally, CD4 knockdown also drastically attenuated JEV infection-induced STAT1 phosphorylation (p-STAT1) and the production of downstream inflammatory factors. To distinguish whether CD4 affects p-STAT1 through an indirect effect of reduced viral load or its direct involvement in signal transduction, we performed experiments using RO8191, a specific agonist of the STAT1 signaling pathway. The results showed that RO8191 treatment increased the expression levels of p-STAT1 protein and inflammatory factor mRNA in both normal cells and CD4 knockdown cells, but the recovery amplitude in the CD4 knockdown group was significantly lower. In contrast, CD4 complementation significantly elevated the expression levels of p-STAT1 protein and inflammatory factor mRNA. In conclusion, this study demonstrates that the CD4 molecule positively regulates JEV proliferation in TM3 cells, while also modulating STAT1—a key factor in the STAT signaling pathway—and downstream inflammatory cytokines. Notably, this regulatory effect operates independently of viral replication. These findings provide a theoretical foundation for further elucidation of JEV pathogenic mechanisms and offer a scientific basis for the prevention and control of JEV.