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。     

非洲猪瘟病毒跨膜蛋白CD2v的结构与功能研究进展

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASF V)感染家猪和野猪,引起的高度传染性和致死性动物传染病,不同品种和年龄阶段的猪均易感,发病率和死亡率可高达100％.ASFV是全球生猪产业的重点疫病之一,目前无商品化疫苗.ASFV是一种大而复杂的双链DNA双层囊膜病毒.ASFV主要跨膜蛋白CD2v是介导血细胞吸附(Hemadsorption,HAD)和鉴定病毒血清型的关键蛋白,它参与ASFV宿主免疫应答、免疫逃逸和病毒复制等生理生化过程,且是ASFV疫苗和抗病毒分子研发的潜在重要靶点.探究CD2v在ASFV感染中的作用及机制渐成热点.因此,本文对ASFV CD2v的结构及其生物学功能研究报道进行分析综述,将为阐明ASFV致病机理和疫苗研发等提供重要参考.     

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...     

非洲猪瘟病毒多样性

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起猪的一种急性、高度致死性传染病.该病在全世界多个地方流行,导致该病流行的原因之一是缺乏有效的预防及治疗药物、疫苗等.尽管ASFV基因总的突变率相对其基因组来说较低,但是,与其它病毒相比,其基因突变总数则相当巨大.研究发现ASFV的多个基因具有高突变率的特性,表现为基因多样性,此外,由于该病毒为核质大DNA病毒,编码大量蛋白,其抗原也表现为多样性.本文总结了 ASFV基因多样性和抗原多样性,分析其发生原理并综述了最新研究成果,以期为研究ASFV病毒遗传演化、开发疫苗及指导疫情防控提供思路.     

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。     

猪瘟病毒及其疫苗研究进展

猪瘟病毒及其疫苗研究进展陆宇,陈建国,丁明孝（北京大学生命科学学院,北京１００８７１）ＲｅｓｅａｒｃｈＰｒｏｇｒｅｓｓｏｎＨｏｇＣｈｏｌｅｒａＶｉｒｕｓａｎｄｉｔｓＶａｃｃｉｎｅＬｕＹｕ;ＣｈｅｎＪｉａｎｇｕｏ;ＤｉｎｇＭｉｎｇｘｉａｏ（Ｃｏｌｌｅｇ...     

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。     

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.     

Recombinant Newcastle disease virus expressing African swine fever virus protein 72 is safe and immunogenic in mice

African swine fever(ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus(ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus(r NDV) expressing ASFV protein 72(p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, r NDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in r NDV/p72 was stably maintained through ten passages. Mice immunized with r NDV/p72 developed high titers of ASFV p72 specific Ig G antibody, and had higher levels of Ig G1 than IgG2 a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that r NDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.     

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。     

非洲猪瘟病毒VP73基因克隆及在大肠杆菌中的高效表达

参照Genebank中非洲猪瘟VP73基因序列,人工合成的VP73全基因克隆至pMD 18-T克隆载体质粒中,采用PCR扩增得到1188 bp的VP73基因;将VP73基因片段亚克隆插入pBAD/Thio表达载体,经测序鉴定,筛选获得VP73基因正向插入、有正确读码框的阳性克隆,成功构建了非洲猪瘟病毒VP73基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP73蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002 %的L-Arabinose进行诱导,4 h后表达量最高,表达蛋白为融合蛋白,分子量约60 kDa,表达产量约占菌体总蛋白的30％。Western blotting和ELISA检测表明,表达的融合蛋白能与非洲猪瘟阳性血清发生特异性反应,说明表达获得的产物为非洲猪瘟病毒VP73融合蛋白,且具有良好的反应原性,这为应用该表达蛋白抗原制备ASFV免疫血清学诊断试剂和疫苗研究奠定了基础。     

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.     

Transovarial transmission of African swine fever virus in the argasid tick Ornithodoros moubata

The aim of this study was to determine filial infection prevalence of experimentally infected colony Ornithodoros moubata Walton (Ixodoidea: Argasidae) ticks for African swine fever virus (ASFV). Three groups of ticks were used: an uninfected control group, one group orally infected with the VIC T90/1 isolate and another group orally infected with the LIV 13/33 isolate of ASFV. The results show that filial infection prevalences were not constant but were highly variable between egg batches from different ticks and between successive egg batches from the same tick. Filial infection prevalences ranged from 1.8% to 31.8% for ticks infected with the VICT90/1 isolate and from 1.2% to 35.5% for ticks infected with the LIV 13/33 isolate. A similar pattern was noted after the third feed. Immunohistochemisty showed that virus replicates in the developing larval cells and not in the yolk sac cells or within the outer layers of the eggs. The results show that ASFV can replicate to a high titre (10(5.1)log10HAD50) within the larval cells of the developing egg.     

Mechanism of collapse of endoplasmic reticulum cisternae during African swine fever virus infection

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.     

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。