Classical swine fever virus in plasma and peripheral blood mononuclear cells of acutely infected swine

The distribution of classical swine fever virus (CSFV) in plasma, monocytes, T and B lymphocytes in peripheral blood was monitored during experimentally induced acute classical swine fever infection in piglets. Six piglets were infected with 10(3.8) TCID50 of virus and blood samples taken up to 18 days post-inoculation (p.i.). Infectious virus was detected in monocytes, T and B lymphocytes to similar titres in five of the six infected piglets. Infectious virus was detected earlier in plasma than in any of the mononuclear cell subpopulations. No significant difference was observed in the period of time in which virus could be isolated from the three cell subpopulations. While a progressive lymphopenia developed, a marked B cell depletion was observed. However, B cells were apparently replaced by non-IgM-bearing mononuclear cells, as the proportion 'total lymphocyte/total leucocytes' remained unaltered throughout the experiment. Virus titres in plasma and peripheral blood mononuclear cells showed a tendency to increase as the disease progressed to its outcome.     

9株猪瘟分离毒株的致病特性

采用9株临床表现强、中、低致病特点的猪瘟野毒分离株第3代细胞毒作为种毒,按3mL/头剂量分别注射猪瘟抗原及抗体阴性猪,再用上一代传代猪发病后的血毒作为下一代接毒用种毒接种试验猪1头或2头,或上一代传代猪发病后,同圈放入1头或2头试验猪进行同居感染.如此进行,GDGZ1/95、BJCY1/96和JL1/94传至8代;FJFQ1/99传至7代;HeBHH1/95、HeNBY1/96、BJTX3/96、GXBH1/98和HeNXH2/98传至3代.结果显示:上述分离株传至3～5代过程中均表现出毒力增强的趋势,从3～5代传代到8(7)代的过程中毒力进一步增强并保持稳定,且均超过了标准石门强毒株的发病特点.所有试验猪均出现较典型的猪瘟临床症状,解剖后均表现出不同程度的病理变化,死后或解剖后的各种脏器经HCFA检查均为强阳性,这种现象进一步证实了猪是猪瘟病毒的敏感动物,各毒株之间毒力没有明显差异.对其中7株分离株传代血毒部分代次E2基因主要区域进行序列分析,结果仅GDGZ1/95株从F1～F8代中的F6代有2个核苷酸的差异,引起1个相应氨基酸的变异,其余毒株的不同代次没有碱基发生变异,初步说明猪瘟病毒基因型表现相对的稳定性.     

CSFV的RT-PCR检测在控制与净化猪瘟中的应用

应用反转录-聚合酶链反应(RT-PCR)检测健康猪扁桃体猪瘟病毒以监控与净化猪瘟.2006年用RT-PCR对广西某存栏250头种猪场的母猪扁桃体连续进行3次猪瘟病毒检测,检出并清除带毒猪,猪群中猪瘟病毒的带毒猪明显下降.结果表明,RT-PCR检测猪扁桃体可应用于猪场猪瘟的控制与净化.     

猪瘟病毒单克隆抗体及其应用

自1985年wenvoort G C等首次应用细胞培养猪瘟病毒接种小鼠的方法建立并制备13株单克隆抗体以来,猪瘟单克隆抗体不仅越来越多的应用于猪瘟的鉴别诊断,而且还用于猪瘟病毒的分子生物学研究.论文对猪瘟病毒的单克隆抗体在猪瘟的鉴别诊断和猪瘟病毒抗原结构蛋白、保护性抗原蛋白、抗原变异以及Ez囊膜糖蛋白抗原表位分析等方面进行综述.     

Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs

Classical swine fever virus (CSFV) compromises the host immune system, causing indirect leucopoenia and disruption of in vitro T cell stimulation capacity. In order to explore the potential role of dendritic cells (DC) in such phenomena, the activation of conventional DC (cDC) and plasmacytoid DC (pDC) in blood and secondary lymphoid organs of infected pigs was investigated in the early time course post-inoculation (pi), together with viral components dissemination and cytokine production in serum. Whereas CD11R1+CD172a+ cDC frequencies were markedly reduced in blood and spleen, analysis of CD4+CD172a+ pDC numbers revealed a rapid turn-over of this DC subset in tissues pi. Both subsets matured and were activated after infection, as demonstrated by down-regulation of CD1a, up-regulation of the co-stimulation molecule CD80/86 and expression of cytokines. cDC essentially expressed tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10, whereas pDC produced alpha interferon (IFN-alpha) and IL-12. IFN-alpha and TNF-alpha productions revealed an enhancement of innate anti-viral immune responses. Detection of antigen activated B lymphocytes in tonsil T-cell areas at 72 h pi, subsequently to the transient translocation of the viral E2 protein within germinal centres at 48 h pi, indicates the initiation of humoral response. This response was also evidenced by an important IL-10 production in serum one week pi. IL-12 expression in organs, as well as transient detection of IL-18 and IFN-gamma in serum, reflected the initiation of cellular immune responses. However, the uncommonly high levels of TNF-alpha and IFN-alpha produced by DC and measured in serum early post-infection, together with IL-10 expression in spleen, could play a role in the disruption of immune system cells, either inducing apoptosis or impairing DC functionalities themselves.     

猪瘟病毒及其致病机制研究进展

猪瘟(CSF)是猪的一种高度接触性传染病,该传染病可分为急性、亚急性、慢性、非典型性和不明显型.急性CSF由强毒株引发,一般导致高发病率和死亡率,而弱毒病毒感染则表现不明显.由于疫苗的广泛应用,有效地控制了猪瘟的大流行,减少了急性死亡.但从20世纪80年代以后,临床症状不典型且病程变长的非典型性猪瘟(或慢性猪瘟)成为该病的主要发生形式,持续感染普遍存在,疫苗的预防效果明显下降,使猪瘟防制遇到了新的困难.以目前人类对猪瘟的认识水平,尚难以从分子水平解释这一新变化的成因,这是因为对猪瘟病毒致病机理及其分子基础的认识深度不够.就此,文章综述了猪瘟及猪瘟病毒研究进展,主要涉及CSFV生物学特性、致病机制及其防控,希望能为猪瘟防控提供新的思路和对策.     

猪瘟病毒持续性感染的分子机制

猪瘟病毒的持续性感染是典型急性猪瘟感染以外的一种感染形式,在这类感染中,感染猪并不表现明显的临床症状,但是却长期携带病毒成为重要的传染源。研究表明,猪瘟病毒是通过对细胞溶解性感染的调节,引起树突状细胞无应答,诱导淋巴细胞凋亡,抑制I型干扰素产生和囊膜糖蛋白E2变异等多个方面来逃脱机体的免疫应答,从而建立持续性感染。     

我国猪瘟病毒分子流行病学研究进展

猪瘟是严重危害我国养猪业的烈性传染病.近年来,我国在猪瘟病毒的分子流行病学研究方面取得了较大进展.基于E2、E0基因对我国猪瘟病毒流行毒株分群显示,基因Ⅱ群占主导地位,基因Ⅰ群次之,没有发现基因Ⅲ群.我国多数猪瘟病毒流行毒株E2基因越来越向远离HCLV株的方向变异,这一趋势可能会对我国以疫苗免疫为主的防控策略构成威胁.流行毒株E0基因与疫苗株相比发生了一定程度的变异,但在RNase活性有关的关键位点上高度保守,没有发现变异位点.     

逆向遗传学技术在猪瘟病毒研究中的应用

猪瘟是猪的最严重疾病之一,其病原 是猪瘟病毒,基因组为单股正链RNA。猪瘟 弱毒疫苗在猪瘟的免疫防治中发挥了巨大作 用,但由于弱毒疫苗免疫的动物不能从血清 学上与自然感染动物区分,使其应用受到很 大限制,研制标记疫苗是解决这个问题的重 要途径。由于RAN的结构不稳定成为阻碍 RNA病毒研究的主要障碍,所以病毒分子生 物学是新型猪瘟疫苗研究的必要手段。近年 来兴起的逆向遗传研究将RNA病毒的基因 组转化为cDNA,文章就猪瘟病毒逆向遗传 研究的意义、方法、概况及其在猪瘟新型疫苗 研究中的应用进行了全面综述。     

猪瘟病毒与猪圆环病毒2型混合感染的检测

对广西南宁市、贵港市、崇左市共5个规模化猪场送检病猪的组织器官样品(脾脏和淋巴结),应用已建立的检测猪瘟病毒(CSFV)的RT-PCR技术和检测圆环病毒2型(PCV-2)的PCR技术,快速准确地扩增出了CSFV和PCV-2特异的目的基因片段,从而证实为猪瘟病毒和猪圆环病毒2型混合感染。     

猪瘟病毒低毒力毒株FJFQ株的分离鉴定

从福建某猪场分离到 1 株病毒,其在PK 15细胞上的毒价为 106.5 TCID50/mL,该病毒能被猪瘟病毒高免血清所中和(效价为1∶8)。通过 RT -PCR 扩增出猪瘟病毒约250 bp的E2蛋白主要抗原编码区序列,其与几株已发表毒株序列的核苷酸及氨基酸同源性分别为79.9%~87.9%,77.7%~86.6%,与Alfort 株同属于基因二群。经本动物传3代均不表现明显的临床症状。用猪瘟兔化弱毒疫苗免疫后以此分离毒作强攻进行免疫保护相关实验,结果免疫组猪在攻毒前及攻毒后扁桃体 HCFA检测均为阴性,对照组猪扁桃体HCFA于攻毒后1周开始出现阳性结果,且一直持续到试验结束。用分离株免疫本动物后再攻石门毒, 2 头试验猪中 1 头死亡,1头出现临床症状。初步说明,所分离的病毒为猪瘟病毒(命名为CSFV- FJFQ株),可能是一株低毒力毒株,且其免疫原性不好。     

猪瘟病毒E2蛋白抗原表位的表达与免疫活性研究

以猪瘟病毒全长基因组质粒为模板,PCR扩增出E2囊膜糖蛋白上A/D抗原区B细胞表位878aa～938aa;PCR扩增片段连接到pET-32a( )载体构建pET-AD原核表达质粒,将阳性质粒转化到BL21大肠埃希菌中诱导表达,以Ni-NTA亲和柱纯化,纯化后的蛋白进行Western blot鉴定。结果表明,重组质粒pET-AD在BL21大肠埃希菌中可以高效表达,表达的蛋白质经纯化后仍然有反应原性。     

猪瘟病毒Erns基因的克隆及原核表达

从猪瘟病毒石门株血液样品中提取总RNA,通过R-PCR对Erns基因进行cDNA扩增,获得了696 bp的片段.将该片段克隆于T-easy载体后进行序列分析,确认PCR产物为猪瘟病毒Erns基因,从阳性克隆中提取质粒,经Bam H Ⅰ和Hin d Ⅲ双酶切,回收产物亚克隆到pET-32a表达载体中,提取质粒后转化BL21(DE3)感受态细胞,并筛选出阳性克隆,经IPTG诱导后通过SDS-PAGE检测出Erns基因的表达.     

猪瘟病毒石门株E2基因的RT-PCR克隆及鉴定

本文采用ＲＴ－ＰＣＲ技术从感染猪血中成功扩增了我国猪瘟病毒强毒石门株Ｅ２基因,大小为１１８４ｂｐ,与预期大小一致。经巢式ＰＣＲ和酶切鉴定证实所扩增的片段为Ｅ２基因特异性片段。将扩增的Ｅ２基因克隆到Ｐ＾ＧＥＭ－Ｔ载体,采用限制性内切酶鉴定和ＰＣＲ技术了阳性重组子。     

我国近期猪瘟分子流行病学动态

利用反转录聚合酶链式反应 (RT PCR)及测序技术 ,测定 2 0 0 2年分离于我国甘肃、湖南、海南、青海等省的 9株猪瘟野毒的E2基因序列 ,并与C 株疫苗毒进行同源性比较 ,绘制CSFV系统发生树。结果表明 ,近期分离于我国的 9株野毒与C 株核苷酸序列间的同源性在80 .7%～ 99.1 %,氨基酸同源性在 89.3%～98.9%。9株野毒分属于 2个不同的组群 ,其中 5株与我国经典的石门强毒和C 株疫苗毒同属于Subgroups1 .1 ,另 4株属于与C 株有较大差异的Subgroup2 .1。     

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.     

母猪猪瘟病毒抗体与仔猪母源抗体衰减规律的研究

为了研究仔猪猪瘟病毒(CSFV)母源抗体的衰减规律,本研究随机选择36头待产母猪,产后采血,选择每头母猪对应的仔猪连续采血至8周龄,分离血清,通过ELISA方法检测CSFV抗体阻断率,所测结果利用EXCEL拟合指数曲线,计算分析母猪CSFV抗体水平与仔猪母源抗体衰减规律。结果显示,36头母猪中母猪抗体阻断率80%以上6头,母猪抗体阻断率70%~79%的11头,母猪抗体阻断率60%~69%的8头,母猪抗体阻断率50%~59%的5头,母猪抗体阻断率49%以下的6头。3周龄仔猪抗体阻断率整体与母猪抗体阻断率相近且高度相关,相关系数为99%。母猪抗体阻断率在80%以上时,母源抗体对所产仔猪8周龄时仍然具有保护力;母猪抗体阻断率在70%~79%时,母源抗体对所产仔猪7周龄时不再具有保护力;母猪抗体阻断率在60%~69%时,母源抗体对所产仔猪5周龄时不再具有保护力;母猪抗体阻断率在50%~59%时,母源抗体对所产仔猪4周龄时不再具有保护力;母猪抗体阻断率49%以下时,母源抗体对所产仔猪3周龄时不再具有保护力。以上结果表明仔猪母源CSFV抗体随着仔猪周龄的增加逐渐衰减,由试验得出仔猪猪瘟疫苗首免日龄的计算公式:y=1.19x-46.55(x为母猪CSFV抗体阻断率,y为首免日龄)。因此,母猪抗体阻断率在80%以上时,仔猪母源抗体在60日龄不再具有保护力,仔猪猪瘟疫苗首免为56日龄;母猪抗体阻断率在70%~79%时,母源抗体对仔猪的保护持续到50日龄,仔猪猪瘟疫苗首免为42日龄;母猪抗体阻断率在60%~69%时和50%~59%时,母源抗体对仔猪的保护持续至36日龄和29日龄,仔猪猪瘟疫苗首免分别为28日龄和21日龄。本研究中所得母猪CSFV抗体水平计算母源抗体衰减变化,为选择母猪首免日龄,提高仔猪猪瘟疫苗免疫效果提供了科学依据。     

猪瘟病毒E2基因主要抗原区的克隆及其原核表达

为研究猪瘟病毒E2蛋白的抗原表位在机体免疫应答中的作用和特点,试验参照猪瘟兔化弱毒疫苗株(HCLV)的基因组序列,设计、合成了1对引物,应用RT-PCR方法扩增出长为786 bp的基因片段,命名为zE2,将zE2基因克隆到原核表达质粒pET 32a中,经酶切鉴定后转化大肠杆菌Rosetta(DE3),于37℃、1.0 mmol/L IPTG条件下诱导表达,大肠杆菌菌体裂解产物再经SDS-PAGE和Western-blot分析。结果表明:该基因片段得到较高表达,融合蛋白的分子质量约为48 ku,主要以包涵体形式存在,且具有一定的免疫学活性。     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.