一种非洲猪瘟病毒假病毒细胞感染模型的建立及应用

目前国内外大多数针对非洲猪瘟病毒(African swine fever virus, ASFV)的研究须在生物安全三级实验室(biosafety level 3 laboratories, BSL-3 labs)中进行，因此针对该病毒的感染过程、中和抗体逃逸机制、药物研发等研究受到了一定限制。鉴于此，本研究选择ASFV包膜蛋白中与其进入细胞紧密相关的蛋白p12、CD2v、p30、p54和pE248R，构建表达这5种包膜蛋白的真核表达质粒，利用水疱性口炎病毒(vesicular stomatitis virus, VSV)假病毒包装体系，制备多种ASFV假病毒。以荧光素酶报告基因实验(luciferase assay)检测假病毒感染水平；选择1个包膜蛋白为代表，使用蛋白质印迹法(Western blot，WB)检测其在假病毒中的表达情况；采用芫花素检测其对所建立的ASFV假病毒(p30-pE248R-ASFV-PsV)的抑制活性。结果显示，VSV包装体系以及p30、pE248R包膜蛋白质粒的组合制备方法所包装出的假病毒具有较优的感染活性，适合用于建立细胞感染模型。ASFV的包膜蛋白pE248R被有效整合到VSV-ΔG rLuc颗粒中，并包装出ASFV假病毒。芫花素可浓度依赖性地抑制ASFV假病毒感染Vero细胞，其半数抑制浓度(half maximal inhibitory concentration, IC50)为4.05±0.88 μmol/L。本研究通过建立基于ASFV假病毒的细胞感染模型，筛选获得了1种可感染已报道的一些ASFV敏感细胞的假病毒。该假病毒无复制性，可在生物安全级别较低的实验室中进行操作，并且带有海肾荧光素酶报告基因，有望用于ASFV入侵抑制剂的高通量筛选及中和活性的初步评价，为研发抗ASFV药物提供了一个安全、方便的研究模型。

The establishment and application of an ASFV pseudovi rus infection cell model

African swine fever virus (ASFV) poses a serious threat to animal health, food safety, and even the global economy. To date, there is still no ASFV vaccine or therapeutic available. For biosafety reasons, most researches on ASFV must be conducted in biosafety level 3 (BSL-3) laboratories. This is the main obstacle for the study on ASFV, including its pathogenesis and escape mechanism, and the development of ASFV vaccines and therapeutics. In this study, we selected some ASFV envelope proteins that are reported to participate in the process of viral entry into the host cell and used the vesicular stomatitis virus (VSV) pseudovirus packaging system to produce several ASFV pseudoviruses. We found that one of these ASFV pseudoviruses could effectively infect some ASFV-susceptible cells, and thus established an ASFV pseudovirus infection cell model, which could be used in laboratories with a lower biosecurity level. As the pseudovirus contains a Renilla luciferase reporter gene, this ASFV pseudovirus could be used for screening of ASFV entry inhibitors and neutralizing antibodies, thus providing a safe and convenient model for research and development of antiviral drugs against ASFV infection.