1株非洲猪瘟病毒自然变异毒株的鉴定

非洲猪瘟(ASF)于2018年传入我国后已流行了2年有余,我国非洲猪瘟病毒(ASFV)毒株未曾出现较大变异.最近,针对我国ASF疫情放缓以及感染猪出现的低死亡率现象,我们在ASFV生态学研究中,从主动监测的样品中分离到1株源自湖北某地的ASFV自然变异株.经基因组测序发现,其EP402R基因(CD2v)和上游相邻的EP...     

非洲猪瘟病毒传播方式及控制措施

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种急性、热性、高度传染性疫病,感染猪以发热和全身性出血为主要特征,病程短、病死率高。非洲猪瘟主要流行于非洲国家,随后相继传入西欧、南美洲、东欧,以及亚洲国家,对全球养猪业、食品安全和猪及其产品国际贸易产生了严重危害和深远影响。现结合参考文献和我国非洲猪瘟发生、控制情况,分析了非洲猪瘟病毒传播动力学、传播方式,提出防控非洲猪瘟的主要措施。     

规模化猪场非洲猪瘟采样检测关键点和常见误区

近年来，非洲猪瘟疫情对国内养猪业的冲击很大，严重影响了大中小型规模化猪场的发展和利润，也给养猪业造成了巨大的损失。然而，随着非洲猪瘟病毒自然和非自然变异株的大量出现，对ASFV传统采样和检测须得到一些新技术的支持，如“唾液/深咽拭子+血液”抗原抗体同步采样检测、多重PCR以及微滴数字PCR等。基于此，文章归纳总结了规模化猪场采样和检测的关键点和常见误区，以期为国内规模化猪场现场采样和检测方面提供理论借鉴。     

非洲猪瘟防控策略

我国对非洲猪瘟（ASF）的防控工作取得了阶段性成果,但疫情依然严峻,生猪稳产保供仍面临较大压力。文章通过了解非洲猪瘟防控现状和兽医队伍体系现状以及机构改革后非洲猪瘟防控工作存在的不足,分析了非洲猪瘟防控工作存在的问题,提出如下建议：1）重点加强中小规模养殖场（户）疫情防控工作;2）加强生猪调运监管;3）加强生猪屠宰监管。     

非洲猪瘟常态下猪日粮设计的几点思考

自2019年以来,我国生猪养殖已进入非洲猪瘟常态化时代,除生物安全升级、养殖模式改变等外,日粮设计也至关重要。文章从减法原则、猪舍内环境、提高猪机体免疫力等方面讨论了非洲猪瘟常态化下日粮设计的基本原则与关键点,以期为非洲猪瘟常态下猪的日粮生产提供参考与依据。     

三类常用消毒剂对非洲猪瘟病毒灭活效果的评价

为评价戊二醛类、酚类、含氯类常用消毒剂对非洲猪瘟病毒(ASFV)的灭活效果,参考OIE参考实验室相关操作流程,基于畜禽栏舍、运载工具、器具消毒目的,根据说明书标明的浓度范围选择低、中、高3个工作浓度,与ASFV分别在4℃和20℃条件下作用30 min,10倍连续稀释后接种猪肺泡巨噬细胞,同时加入猪红细胞,培养观察红细胞...     

非洲猪瘟病毒核酸检测技术与应用进展

自2018年8月以来,非洲猪瘟已成为我国养猪业的头号疫病,对养猪业造成了巨大损失。基于聚合酶链式反应(PCR)的核酸检测方法具有高灵敏性和高特异性,在对非洲猪瘟的诊断、感染监控和养殖复产过程中生物安全评价等方面发挥了重要的作用。文章对常用的核酸检测技术与特点、非洲猪瘟病毒核酸检测方法的应用、核酸检测新技术展望3个方面进行了总结,以期为养猪企业在非洲猪瘟核酸检测和监测方面提供帮助。     

非洲猪瘟病毒VP73结构蛋白表达及鉴定

根据GenBank登录的非洲猪瘟病毒(ASFV) VP73基因序列,人工合成VP73主要抗原表位区基因片段,克隆至pUC57载体中.设计1对引物,PCR扩增获得429 bp的VP73基因片段;将VP73基因片段使用EcoRI和SalI酶切后亚克隆至表达载体pGEX-KG,经PCR、酶切、测序鉴定挑选阳性克隆,转化至表达菌株BL21 (DM3)中,进行体外诱导表达,SDS-PAGE和Western blot分析蛋白表达及其活性.结果显示,成功构建表达重组载体pGEX-KG-VP73,对该重组载体进行诱导,有效表达了41 ku重组蛋白,表达量约占菌体总蛋白的17％,Western blot分析证实该蛋白具有良好反应原性.     

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD₅₀)/ml, 10¹ HAD₅₀/ml and 10⁶ HAD₅₀/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD₅₀/ml and 10¹ HAD₅₀/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD₅₀/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 10³ HAD₅₀/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.     

规模猪场防控非洲猪瘟的生物安全措施

猪场的生物安全措施对该场的疫病预防和控制起到了非常重要的作用。文章介绍了在非洲猪瘟背景下,猪场需要在人员生物安全意识、猪群健康、猪场入口处、猪场出口处、猪场车辆管理等方面需要采取的一系列生物安全措施。供大家参考,以期提升猪场的生物安全管理水平,提高猪场疫病预防控制能力。     

非洲猪瘟五级生物安全防控法的实践

2018年8月,辽宁暴发国内首例非洲猪瘟(ASF)疫情,短短数月,疫情席卷全国,养猪业一片哀鸿。这注定了整个养猪业的未来是艰辛且坎坷的。笔者作为一名集团公司区域兽医负责人,负责40 000头自繁自养母猪群的健康管理,通过我们的不懈努力,2019年全年,4万头自繁自养母猪群、13个养殖场未发生一例疫情。在一年多的防控非洲猪瘟斗争中,笔者也总结了一套特色的非洲猪瘟防控的体系方案。     

Entry of African swine fever virus into Vero cells and uncoating

African swine fever virus (ASFV) enters Vero cells by adsorptive endocytosis [Valdeira, M.L., Geraldes, A., 1985. Morphological study on the entry of African swine fever virus into cells, Biol. Cell. 55, 35–40]. Electron microscopy of a lysosomotropic drug-controlled penetration indicated that this step takes place in the endosomes, after fusion between the viral envelope and the limiting membrane of the endosome. Inhibition studies with colcemid, cytochalasin B, sodium azide, dinitrophenol, lysosomotropic weak bases, and the ionophore monensin, showed that the virus uptake is largely independent of cytoskeletal and lysosomal function, but dependent on oxidative phosphorylation. Some protease inhibitors inhibited viral replication at an early step, indicating that the initiation of infection depends on a viral proteolytic cleavage.     

非洲猪瘟检测中的常见问题与分析

实验室检测是非洲猪瘟防控中的重要一环，但实验室检测不是仪器与试剂盒的简单叠加，是“人、机、料、法、环”多方面结合后的有效输出。在实验室广泛普及的同时，实验室的管理、质控、结果分析、报告解读及临床指导是重中之重。结果的正确解读及与临床信息的对接是其价值所在，临床及实验室的有效结合是综合诊断的基础。     

非洲猪瘟的防控关键技术

非洲猪瘟传入我国以来,给我国养猪业造成了巨大的损失。非洲猪瘟可防可控,文章从消灭传染源、切断传播途径、保护易感猪群方面介绍了构建猪场生物安全体系的防控技术。     

Patterns of alveolar macrophage activation upon attenuated and virulent African swine fever viruses in vitro

The pattern of porcine alveolar macrophage (AM) activation upon classical stimuli of two strains of African swine fever (ASF) viruses, an attenuated ASFV-BA71V and virulent ASFV-Georgia2007 were investigated. In an in vitro experiment ASFV-Georgia2007-infected AM showed M1 polarization pattern different from the one induced by classical stimuli. Altered morphology, appearance of binuclear cells, decreased synthesis of IFN-alpha as well as IFN-epsilon was observed compared with attenuated ASFV-BA71V, and decreased synthesis of IFN-omega compared with intact cells. However, CD68 level did not significantly differ between alveolar macrophage populations infected by ASFV-Georgia2007 and control group, while both LPS/IFN-gamma stimulation and non-pathogenic ASFV-BA71V virus increased the level of CD68 soluble receptor.AM infection with ASFV-Georgia2007 resulted in remarkable DNA proliferation whereas LPS/IFN-gamma and ASFV-BA71V induced less expressed DNA proliferation in activated cells. The higher value of nitric oxide was obvious in the cells infected with ASFV-BA71V, compared to ASFV-Georgia2007 and LPS/IFN-gamma activated cells.In conclusion, pattern of activation of alveolar macrophages induced by ASFV-Georgia2007 virus differs from the one expressed in LPS/IFN-gamma- and ASFV-BA71V-activated cells. ASFV-BA71V and LPS/IFN-gamma share similar antiviral response of porcine AM. Therefore we assume that wild type virulent ASFV can partially down regulate antiviral response of AM and conclude that evolutionary decrease of virulence in ASFV is related to alterations of control of the host cell antiviral response.     

臭氧在非洲猪瘟防控上的合理利用

非洲猪瘟病毒抵抗力强、危害严重,臭氧须合理利用,才能发挥其在猪场非洲猪瘟防控消毒中的相应效果。     

养殖场非洲猪瘟防控措施及实践

2018年8月3日,农业农村部发布辽宁沈阳发生全国首例非洲猪瘟。至今,全国32个省、自治区、直辖市、特别行政区公布共发生149起非洲猪瘟疫情,超过116万头猪只被扑杀。面对前所未有的非洲猪瘟,目前尚无有效的药物和疫苗预防,养猪行业面临种种危机。文章对非洲猪瘟及其危害做了简要介绍,并提出了防控非洲猪瘟的措施和建议。     

非洲猪瘟病毒p72蛋白杆状病毒表达系统的构建及其抗原性分析

为研究真核表达非洲猪瘟病毒(ASFV)主要结构蛋白p72,本研究从含ASFV p72基因全序列的重组质粒pGEX-6p-p72中扩增出1 941 bp的p72全长基因,将其插入于pFastBac HTa杆状病毒载体中,构建了重组质粒pFastBac HTa-p72,转化至感受态细胞DH10Bac中,获得重组杆粒rBacmid-p72。再将其转染至sf9昆虫细胞,获得重组杆状病毒。Western blot和夹心法ELISA分析表明ASFV p72基因在昆虫细胞sf9中获得了正确表达,重组p72蛋白可以被特异性抗ASFV血清、p72单克隆抗体识别,表明该蛋白特异性强、活性稳定,具有良好的抗原性。为进一步研究p72蛋白的结构、功能和免疫学特性奠定基础。     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。