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Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway

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Abstract

Japanese encephalitis virus (JEV) infection can cause brain tissue lesions characterized by neuronal death, and apoptosis is involved in JEV-induced neuronopathy. In the present study, mouse microglia were infected with JEV, and pyknosis with dark-staining nuclei of infected cells was detected using Hoechst 33342 staining. TUNEL staining showed that JEV infection promoted the apoptosis of BV2 cells, and the apoptosis rate was significantly increased at 24-60 hours postinfection (hpi) (P < 0.01) and was the highest at 36 h (P < 0.0001). Western blot results showed that the expression of the Bcl-2 protein in JEV-infected cells was downregulated significantly at 60 hpi (P < 0.001), whereas that of the Bax protein was observably upregulated at 60 hpi (P < 0.001). At the same time, the level of cytochrome c (Cyt c) was significantly increased (P < 0.001), and the expression levels of two apoptosis-related proteins, namely, cleaved caspase-3 (P < 0.01) and caspase-9 (P < 0.001), were elevated significantly. Immunofluorescence staining showed that the amount of Cyt c increased with time after infection. After BV2 cells were infected with JEV, the expression of RIG-1 increased significantly from 24 hpi to 60 h (P < 0.001). The expression of MAVS increased significantly at 24 h (P < 0.001) and decreased gradually from 24 h to 60 hpi. The expression of TBK1 and NF-κB (p65) was not significantly changed. The expression of p-TBK1 and p-NF-κB (p-p65) increased significantly within 24 h (P < 0.001) and decreased from 24 to 60 hpi. The expression levels of IRF3 and p-IRF3 peaked at 24 hpi (P < 0.001) and decreased gradually from 24 to 60 hpi. However, the expression levels of JEV proteins showed no significant change at 24 and 36 hpi but were markedly elevated at 48 and 60 hpi. Interference with the expression of the RIG-1 protein in BV2 cells resulted in a dramatic increase in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05), whereas the pro-apoptotic protein Bax, cleaved caspase-9, and especially cleaved caspase-3 were downregulated (P < 0.05), and viral protein expression was notably reduced (P < 0.05). These results indicate that JEV induces apoptosis through mitochondrial-dependent apoptosis pathways, interfering with the expression of RIG-1 in BV2 cells can inhibit viral replication and inhibit apoptosis.

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Funding

This work was supported by grants from the National Key Research and Development Program of China (SQ2022YFD1800105) and the National Natural Sciences Foundation of China (32030107) to CG and SC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. Mingxing Gao and Zelin Liu contributed equally to this study.

Authors and Affiliations

  1. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, People’s Republic of China

    Mingxing Gao, Zelin Liu, Xiaoyan Guo, Jinhua Zhang, Guofu Cheng, Xueying Hu, Wanpo Zhang & Changqin Gu

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Correspondence to Changqin Gu.

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Gao, M., Liu, Z., Guo, X. et al. Japanese encephalitis virus induces apoptosis by activating the RIG-1 signaling pathway. Arch Virol 168, 169 (2023). https://doi.org/10.1007/s00705-023-05780-z

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