Supplementary Material for: Bacillus Calmette-Guérin Confers Neuroprotection in a Murine Model of Japanese Encephalitis

<b><i>Objective:</i></b> Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. <b><i>Methods:</i></b> Suckling mice were inoculated with 10⁶ CFU of BCG and at 18 days postinoculation were challenged with 100 LD₅₀ of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. <b><i>Results:</i></b> The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. <b><i>Conclusion:</i></b> These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.

**研究目的：** 日本脑炎（Japanese encephalitis, JE）是由日本脑炎病毒（JE virus, JEV；黄病毒科Flaviviridae）感染引发的致残性疾病，幸存者多遗留神经系统后遗症，多数感染者可导致死亡。JEV感染所致脑炎的核心病理机制为炎症介导的神经退行性病变。卡介苗（Bacillus Calmette-Guérin, BCG）已被应用于结核病的免疫预防以及多种恶性肿瘤的辅助治疗，且在帕金森病、阿尔茨海默病的实验模型中展现出明确的神经保护活性。本研究旨在评估卡介苗在小鼠日本脑炎模型中的神经保护作用。
**实验方法：** 将乳鼠接种10⁶ CFU的卡介苗，于接种后18天以100 LD₅₀的JEV进行攻毒。以磷酸盐缓冲液（PBS）接种的小鼠作为空白对照组。分别于攻毒后第2、4、6、8天处死小鼠，取脑组织匀浆后提取总RNA。采用一步法实时反转录聚合酶链反应（one-step real-time RT-PCR）检测肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）及诱导型一氧化氮合酶（iNOS）的相对基因表达水平。
**实验结果：** 与单纯JEV攻毒组相比，卡介苗预接种组（BCG+JEV）小鼠的神经病理症状出现时间显著延迟，生存期显著延长，且在攻毒后第2、4、6天的TNF-α、IL-6及iNOS基因表达水平均显著下调。
**研究结论：** 本研究结果表明，卡介苗接种可在小鼠日本脑炎模型中发挥神经保护作用，因此卡介苗作为日本脑炎治疗的辅助干预手段值得进一步深入研究。鉴于卡介苗在众多JEV流行国家中已作为结核病疫苗获批使用，该策略有望对这些国家的公共卫生负担产生显著积极影响。