African swine fever virus (ASFV) causes highly contagious swine disease, African swine fever (ASF), thereby posing a severe socioeconomic threat to the global pig industry and underscoring that effective antiviral therapies are urgently required. To identify safe and efficient anti-ASFV compounds, a natural compound library was screened by performing an established cell-based ELISA in an ASFV-infected porcine alveolar macrophage (PAM) model. In total, 6 effective anti-ASFV compounds with low cytotoxicity were identified. Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid, was the most potent inhibitor effect with an IC50 of 0.3223 μM. To further investigate the mechanism through which CEP inhibits ASFV replication, transcriptome profiles were generated in PAMs treated with CEP and/or infected with ASFV. ASFV infection dramatically altered immune response-associated gene expression. CEP treatment upregulated the expression of cholesterol biosynthesis-related genes, regardless of infection status. According to time-of-addition experiments, CEP primarily exerts its antiviral effect during the early stages of ASFV infection, specifically by inhibiting viral entry. Transcriptomic analysis suggested that CEP blocks ASFV entry through the clathrin-mediated endocytosis pathway by increasing EHD2 gene expression in macrophages. Disrupting EHD2 with small interfering RNA promoted ASFV entry into clathrin-positive vesicles. Finally, the protective effect of CEP in vivo was evaluated using ASFV-infected pigs. CEP could provide partial protection against ASFV infection, as indicated by an increase in survival time from 9.67 days to 16.67 days. Our findings imply that CEP exhibits potential antiviral activity against ASFV infection in PAMs, positioning it as a promising therapeutic strategy for ASF.

中文翻译：

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确定头孢嘌呤是非洲猪瘟病毒复制的有效抑制剂。


非洲猪瘟病毒 （ASFV） 会导致高度传染性的猪病，即非洲猪瘟 （ASF），从而对全球养猪业构成严重的社会经济威胁，并强调迫切需要有效的抗病毒疗法。为了鉴定安全有效的抗 ASFV 化合物，通过在 ASFV 感染的猪肺泡巨噬细胞 （PAM） 模型中进行已建立的基于细胞的 ELISA 来筛选天然化合物库。总共鉴定出 6 种具有低细胞毒性的有效抗 ASFV 化合物。头孢喹嘌 （CEP） 是一种双苄基异喹啉生物碱，是最有效的抑制作用，IC50 为 0.3223 μM。为了进一步研究 CEP 抑制 ASFV 复制的机制，在用 CEP 处理和/或感染 ASFV 的 PAM 中生成转录组谱。ASFV 感染显著改变了免疫反应相关基因的表达。CEP 治疗上调胆固醇生物合成相关基因的表达，无论感染状态如何。根据添加时间实验，CEP 主要在 ASFV 感染的早期阶段发挥其抗病毒作用，特别是通过抑制病毒进入。转录组学分析表明，CEP 通过增加巨噬细胞中 EHD2 基因的表达来阻断 ASFV 通过网格蛋白介导的内吞途径进入。用小干扰 RNA 破坏 EHD2 促进 ASFV 进入网格蛋白阳性囊泡。最后，使用 ASFV 感染的猪评价 CEP 在体内的保护作用。CEP 可以提供对 ASFV 感染的部分保护，存活时间从 9.67 天增加到 16.67 天。 我们的研究结果表明，CEP 在 PAM 中表现出针对 ASFV 感染的潜在抗病毒活性，使其成为一种有前途的 ASF 治疗策略。