非结构蛋白在非洲猪瘟病毒感染中作用

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)感染引起的一种急性、出血性猪传染病,给疫情发生国家(地区)的养猪业造成重大经济损失.ASFV为双股DNA病毒,基因组含有150～167个开放阅读框(ORFs),可编码150～200种蛋白质,其中非结构蛋白有100余种.ASFV编码的酶、转录因子、调节宿主细胞功能蛋白和病毒免疫逃逸相关蛋白等作为重要的非结构蛋白,在病毒核苷酸代谢、DNA复制、修复、转录、蛋白修饰以及病毒与宿主细胞相互作用等过程中发挥重要作用,但仍有许多非结构蛋白的功能尚不明晰.因此,本文综述了 ASFV非结构蛋白在病毒感染中的作用,以期为ASFV非结构蛋白的进一步研究提供参考.     

非洲猪瘟病毒的免疫逃逸策略

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病。目前无商品化的ASF疫苗,一旦发病,仅能依靠快速扑杀进行防控,严重威胁我国养猪及相关行业的健康发展。ASF疫苗研发面临的主要困难是对ASFV的毒力相关基因、致病及其免疫逃逸机制知之甚少。本文对ASFV的免疫逃逸研究进行了总结,探讨了ASFV免疫逃逸基因及其编码蛋白的功能,以便加深对ASFV及其免疫逃逸策略的认知,为致病机制研究和疫苗研发提供借鉴。     

非洲猪瘟病毒的非必需基因：真的可有可无吗？

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种出血性、致死性的猪烈性传染病。ASF在全球广泛传播,给养猪业造成重大的经济损失。ASFV基因组庞大,可编码150多种蛋白,一些非必需基因编码的蛋白与调控病毒毒力、复制和免疫逃逸等相关。通过删除ASFV毒力相关的非必需基因所构建的减毒株是当前比较有前景的疫苗,然而其安全性有待提高。系统地鉴定ASFV非必需基因及其功能,不仅有助于ASF基因缺失疫苗的研发,也有益于ASFV致病机制研究。本文对目前已鉴定的ASFV非必需基因及其功能研究进行了总结分析,着重讨论了影响ASFV毒力、调控病毒复制、参与免疫逃逸的非必需基因及其编码蛋白的功能,旨在加深对ASFV病原学的认识,为新的ASFV非必需基因的鉴定和功能研究提供参考。     

非洲猪瘟病毒拮抗宿主cGAS-STING信号通路的研究进展

非洲猪瘟病毒（African swine fever virus,ASFV）拥有多种逃逸宿主免疫应答的策略,造成病毒难以被宿主清除。cGAS-STING信号通路介导的天然免疫在抗ASFV感染中发挥了重要作用,然而病毒编码的多个蛋白靶向该通路中的不同分子以拮抗宿主的I型干扰素应答。利用基因编辑技术敲除这些病毒基因后,ASFV对宿主的致病性降低,成为基因缺失疫苗的研制潜在靶点。本文对目前已知参与调控宿主cGAS-STING信号通路的病毒蛋白进行总结,旨在阐明这些蛋白免疫逃逸cGAS-STING信号通路的分子机制,加深对ASFV免疫逃逸策略的理解,以期为ASFV致病机制研究与疫苗创制提供参考。     

非洲猪瘟病毒及其衣壳蛋白CD2v研究进展

非洲猪瘟(African swine fever,ASF)是一种由非洲猪瘟病毒(African swine fever virus,ASFV)感染家猪和各种野猪(如非洲野猪、欧洲野猪等)引起的严重急性、出血性、烈性传染病,病死率可达100％.2018年我国发生首例非洲猪瘟疫情,并迅速蔓延,给我国生猪产业造成数百亿元的经...     

非洲猪瘟研究进展

非洲猪瘟是由非洲猪瘟病毒感染家猪或野猪后引发的一种急性、烈性传染病,主要通过病猪及其周围环境传播,蜱是中间宿主。1921年该病首次暴发于非洲肯尼亚,2018年8月传入我国,目前已有24个省级行政区发生疫情。非洲猪瘟病毒主要经呼吸道和消化道进入猪体内,感染靶细胞主要是单核-巨噬细胞,目前受体还不明确。非洲猪瘟病毒是单分子双链DNA病毒,长度为170~190kb,编码150~200种蛋白,包括多种免疫调控蛋白,可以抵抗机体免疫。非洲猪瘟病毒疫苗研究较多,包括灭活疫苗、减毒疫苗、亚单位疫苗和基因疫苗等,但迄今这些疫苗都不能保护家猪免受非洲猪瘟病毒感染。今后需要对非洲猪瘟病毒及其发病机制做详细系统的研究,为开发有效防治方案提供资料。     

非洲猪瘟病毒的分子生物学研究进展

非洲猪瘟是一类动物传染病,致死率高达100%,在我国虽未发现该病,但一旦发生会给畜牧养殖业带来巨大经济损失。文中概述了非洲猪瘟病毒的分类、形态、基因组特征、主要结构蛋白,以及分子生物学诊断技术的研究进展。为进一步研究该病毒的复制机理、毒力、致病机理及疫苗的开发提供参考依据。     

非洲猪瘟病毒p54蛋白单克隆抗体的制备与鉴定

2018年8月我国发现非洲猪瘟(African swine fever,ASF),并在多地蔓延,由于尚无疫苗和有效的治疗方法,病死率高达100%,给养猪业带来巨大的损失。目前控制该病,主要依靠快速、准确的诊断方法,尽早发现、处理,防止扩散。为制备特异性的非洲猪瘟病毒(African swine fever virus,ASFV)p54蛋白单克隆抗体,本研究利用大肠杆菌表达ASFV p54蛋白C端胞内区片段,构建能表达ASFV p54蛋白的工程菌E.coli BL21/pET-p54,经IPTG诱导表达可溶性的p54蛋白。用纯化后的可溶性蛋白免疫BALB/c小鼠,将免疫小鼠的脾细胞与骨髓瘤细胞融合,经4次筛选和3次亚克隆获得了21株分泌ASFV p54蛋白单克隆抗体的细胞株。通过ELISA测定腹水效价,结果均在1︰40 000～1︰5 120 000范围之内。以免疫组织化学染色法鉴定单克隆抗体,结果有9株可以特异性检出ASFV抗原。对免疫组化阳性的单克隆抗体进行亚类鉴定,结果 5株为IgG1,4株为IgG2a。以免疫印迹试验(Western Blot)和间接免疫荧光法(Indirect ...     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

Regulation of antiviral immune response by African swine fever virus (ASFV)

African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality approaching 100% in domestic pigs. ASF is an endemic in countries in sub-Saharan Africa. Now, it has been spreading to many countries, especially in Asia and Europe. Due to the fact that there is no commercial vaccine available for ASF to provide sustainable prevention, the disease has spread rapidly worldwide and caused great economic losses in swine industry. The knowledge gap of ASF virus (ASFV) pathogenesis and immune evasion is the main factor to limit the development of safe and effective ASF vaccines. Here, we will summarize the molecular mechanisms of how ASFV interferes with the host innate and adaptive immune responses. An in-depth understanding of ASFV immune evasion strategies will provide us with rational design of ASF vaccines.     

非洲猪瘟病毒的生物学特性与疫苗研制的难点

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种猪烈性传染病,是全球养猪业的"头号杀手",强毒株引发的超急性和急性感染死率高达100%。2018年8月ASF首次传入我国,截止2019年6月6日,已有32个省份累计暴发137起疫情,给我国社会、经济构成巨大威胁。ASF疫苗的研制始于20世纪60年代,但均以失败而告终,其主要原因是对ASFV生物学特性缺乏深入的研究。有效控制当前ASF疫情扩散、研制安全有效的疫苗将是我国面临的巨大挑战。本文对ASFV形态与基本结构、传播途径、致病机制、基因组及编码蛋白、入侵机制、免疫逃逸等生物学特性进行了概述,并分析了当前疫苗研制面临的难点,以期为我国有效控制ASF疫情及病原研究提供参考。     

非洲猪瘟防控及疫苗研发：挑战与对策

非洲猪瘟是由非洲猪瘟病毒引起的一种接触传染性、广泛出血性猪烈性传染病,最急性和急性感染死亡率高达100%。自2018年8月我国发生首起非洲猪瘟疫情后,3个多月内,已有18个省份累计暴发69起,给我国养猪业造成了沉重打击。从目前非洲猪瘟全球流行态势及世界各国防控经验来看,我国非洲猪瘟防控和根除面临的形势不容乐观,亟需安全有效的疫苗用于该病的防控。文中结合当前非洲猪瘟病原学最新研究成果,系统总结了非洲猪瘟防控策略、疫苗研究进展及其面临的挑战,重点分析了疫苗研发历程、存在的问题、未来发展方向以及商业化应用所面临的关键科学问题,以期为我国非洲猪瘟防控及病原和疫苗研究提供借鉴。     

非洲猪瘟在俄罗斯的流行与研究现状

非洲猪瘟(African swine fever,ASF)由非洲猪瘟病毒(ASFV)引起,是家猪和野猪的一种高度接触性、致死性传染病,可表现为最急性、急性、亚急性和慢性四种形式。猪感染后以发热、高病毒血症和出血性病变为特征。有的毒株可引起高发病率和高死亡率。自2007年ASF传入格鲁吉亚以来,该病在高加索地区(包括俄罗斯)逐步蔓延,造成多地大量家猪和野猪病死,经济损失惨重。2017年3月,ASF突然在远东地区伊尔库茨克州出现,疫点距中国北方最大陆路口岸满洲里仅约1 000 km,使得传入中国的风险空前提高。为此,本文对该病10年间在俄罗斯的流行状况和研究情况进行总结,以期为我国对该病的防控提供参考。     

非洲猪瘟病毒C717R蛋白诱导小鼠炎症应答

【目的】通过炎症应答系统筛选,发现非洲猪瘟病毒(African swine fever virus, ASFV) C717R蛋白可诱导炎症反应,本研究旨在首次鉴定C717R蛋白功能,通过构建C717R重组慢病毒并感染BALB/c小鼠,探究其对炎症应答产生的影响。【方法】通过炎症小体表达系统筛选出诱导炎症应答的C717R蛋白,并构建C717R重组慢病毒。利用C717R重组慢病毒感染小鼠,使C717R在小鼠组织中表达。经实时荧光定量、蛋白质免疫印迹等方法检测C717R慢病毒包装、蛋白表达以及促炎细胞因子TNF-α、IL-1β、IL-6、IFN-β的变化。【结果】C717R蛋白在小鼠组织中正常表达。酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)检测发现,表达C717R蛋白的小鼠,血清中促炎细胞因子TNF-α、IL-1β、IL-6、IFN-β及IFN-γ分泌水平显著升高。实时荧光定量检测表达C717R蛋白的小鼠组织证实,C717R、TNF-α、IL-1β、IL-6的mRNA转录水平显著上调;蛋白质免疫印迹证实,C717R可诱导小鼠不同组织caspase-1和IL-1β的成熟。组织病理切片结果显示,表达C717R蛋白的BALB/c小鼠和对照组和相比,肝脏、心脏、肺脏等器官炎性细胞浸润程度较对照组更严重。【结论】ASFV的C717R蛋白表达诱导BALB/c小鼠产生炎症应答,为鉴定和阐明C717R蛋白介导的促炎新机制提供了重要依据。     

Embryo transfer as a means of controlling the transmission of viral infections: V. the in vitro exposure of zona pellucida-intact porcine embryos to African swine fever virus

African swine fever virus (ASFV) was detected on or in zona pellucida-intact porcine embryos that had been exposed to 10^6.6 hemadsorption dose 50%/ml (HAdD₅₀/ml) of ASFV for 18 hours, washed and then cultured. Ninety-five percent of the embryos retained infectious virus after washing. Treating the embryos with papain, EDTA or ficin had no effect on the retained virus, whereas treating them with trypsin or pronase reduced the number of embryos carrying detectable virus (30% instead of 95%) and lowered the amount of virus on the embryos. It has not yet been determined whether ASFV enters the embryonic cells but the evidence suggests that most of the virus, and possibly all of it, is bound to the zona pellucida.     

African swine fever virus cysteine protease pS273R inhibits pyroptosis by noncanonically cleaving gasdermin D

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N_1–107). Interestingly, unlike the effect of GSDMD-N_1–279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N_1–107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.     

非洲猪瘟病毒内囊膜蛋白p17与宿主互作蛋白的初步鉴定

非洲猪瘟病毒(African swine fever virus,ASFV)的感染导致猪的死亡率高达100%,给养猪业造成毁灭性灾难。因此,开展针对ASFV感染复制的研究有着重大的意义。目前发现ASFV有超过150个开放阅读框,其中D117L基因编码的内囊膜蛋白p17参与病毒二十面体结构的形成,但是对p17调控宿主细胞功能的机制知之甚少。研究通过免疫沉淀技术联合蛋白质谱分析,初步筛选出与ASFV p17潜在的宿主互作蛋白。通过进一步免疫共沉淀技术和激光共聚焦实验确认了p17与线粒体外膜蛋白TOMM70(translocase of outer mitochondrial membrane 70)、热休克蛋白HSPA8(heat shock 70 kDa protein 8)的互作。该研究为进一步探索p17在ASFV感染过程中的功能提供了重要信息。