养殖与饲料

[目的]评价黄酮醇对非洲猪瘟病毒(ASFV)的体外抑制活性，并初步探讨其潜在作用靶点，为抗ASFV候选药物的筛选与开发提供参考依据。[方法]以猪肺泡巨噬细胞(PAMs)为细胞模型，采用细胞活力检测评价黄酮醇的细胞毒性；以Ⅱ型ASFV SY-1和Ⅰ/Ⅱ型重组ASFV 819为研究对象，检测不同浓度黄酮醇处理后病毒的复制水平，计算半数细胞毒性浓度(CC₅₀)、半数抑制浓度(IC₅₀)及选择指数(SI)；通过时间阶段试验分析黄酮醇的主要作用阶段，并采用AutoDock Vina进行黄酮醇与ASFV pS273R蛋白的分子对接，分析预测结合能、结合构象及其与结合位点氨基酸残基之间的相互作用。[结果]黄酮醇处理PAMs 48 h后，10μmol/L及以下浓度组细胞活率均高于90%。10μmol/L黄酮醇对ASFV SY-1和819在PAMs上的复制均具有显著抑制作用。在PAMs中，黄酮醇的CC₅₀为16.71μmol/L,IC₅₀为0.669μmol/L,SI为24.98。时间阶段试验结果显示，黄酮醇主要在病毒与细胞共孵育阶段及感染后处理阶段发挥抑制作用。分子对接结果显示，黄酮醇与pS273R蛋白的结合能为-25.9 kJ/mol，可与Asn187形成氢键，并与Pro194和His168形成π相互作用。[结论]黄酮醇在不明显影响PAMs细胞活力的浓度范围内能够抑制ASFV SY-1和819的复制，具有较好的体外抗ASFV活性。分子对接结果提示黄酮醇可能与pS273R蛋白发生相互作用，pS273R可能是其潜在作用靶点之一，但该推测仍需通过酶活实验、结合实验及关键位点突变实验进一步验证。

[Objectives]The in vitro antiviral activity of flavonols against African swine fever virus(ASFV) was studied and the potential molecular targets of flavonols were identified to provide reference for the screening and development of candidate anti ASFV drugs. [Methods]Porcine alveolar macrophages(PAMs) were used as a cell model to evaluate the cytotoxicity of flavonols via cell viability assay. The replication level of the virus in ASFV genotype II strain SY-1 and recombinant genotype I/II strain 819 after treatment with different concentrations of flavonols was detected. The 50% cytotoxic concentration(CC₅₀), 50% inhibitory concentration(IC₅₀), and selectivity index(SI) were calculated. The main stage of flavonols action was analyzed through a time-of-addition assay. AutoDock Vina was used for molecular docking of flavonols with ASFV pS273R protein to analyze and predict the binding energy, binding conformation, and interactions with binding site amino acid residues.[Results] The cell viability of PAMs after 48 hours of treatment with flavonols in the 10 μmol/L and below concentration groups was higher than 90%. 10 μmol/L flavonols significantly suppressed the replication of ASFV SY-1 and 819 in PAMs. The CC₅₀, IC₅₀, and SI of flavonols in PAMs was 16.71 μmol/L, 0.669 μmol/L, and 24.98, respectively. The results of a time-of-addition assay showed that flavonols acted mainly during the stage of virus-cell coincubation and post-infection. The results of molecular docking showed that the binding energy between flavonols and pS273R protein was-25.9 kJ/mol, which can form hydrogen bonds with Asn187 and π-interactions with Pro194 and His168.[Conclusions]Flavonols can inhibit the replication of ASFV SY-1 and 819 within a concentration range that does not significantly affect the cell viability of PAMs, with good in vitro antiviral activity against ASFV. The results of molecular docking indicated that flavonols may interact with pS273R protein, which may be one of its potential targets. However, this speculation still needs to be further validated through enzyme activity assays, binding assays, and site-directed mutagenesis.