马传染性贫血病病毒弱毒疫苗株感染性分子克隆的构建

采用ＰＣＲ方法分三段扩增出马传染性贫血病毒驴白细胞弱毒疫苗株（ＥＬＡＶ　 ＤＬＡ）的前病毒ＤＮＡ,这三个片段覆盖马传染性贫血病毒的全部基因组,ＰＣＲ产物经克隆后顺次连接,获得一个含有ＥＬＡＶ全基因（８．０Ｋｂ）的重组质粒,将其命名为ｐ８．０。将此８．０Ｋｂ ＥＩＡＶ全基因再亚克隆到含有一完整 ＥＩＡＶ ＤＬＡ株长末端重复序列的质粒中,获得一含有 ＥＩＡＶ驴白细胞弱毒前病毒全基因的重组质粒,将其命名为ｐ８．２,经核苷酸序列分析,证明ｐ８．２含有ＥＩＡＶ前病毒的全基因。用ｐ８．２转染驴白细胞,将其作为种毒进行传代,于感染该克隆毒的细胞培养上清中检测出了反转录酶,说明在驴白细胞中由ｐ８．２衍生出了ＥＩＡ病毒。驴白细胞经该克隆毒感染后,第４天出现病变,经透射电镜可观察到典型的马传染性贫血病毒粒子,进一步证明ｐ８．２具有感染性,我们获得了马传染性贫血病毒驴白细胞弱毒疫苗株的感染性分子克隆,为进一步在分子水平上阐明我国ＥＩＡＶ疫苗株的减毒机理和免疫保护机制奠定了基础。     

绵羊进行性肺炎病毒与山羊关节炎一脑炎病毒的血清学交叉反应的研究

本实验用琼脂凝胶免疫扩散试验（ＡＧＩＤＴ）和免疫印迹试验（ＩＢＡ）对实验感染绵羊进行性肺炎病毒（ＯＰＰＶ）的山羊血清与山羊关节炎—脑炎病毒（ＣＡＥＶ）抗原以及实验感染ＣＡＥＶ的绵羊血清与ＯＰＰＶ抗原的交叉反应进行了研究。４只接种ＯＰＰＶ的山羊中有一只山羊的血清可与ＣＡＥＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＣＡＥＶ的ｇｐ４４、ｐ３５和ｐ２８。２只接种ＣＡＥＶ的绵羊中有一只绵羊的血清可与ＯＰＰＶ琼扩抗原发生交叉反应,并在免疫印迹试验中可识别ＯＰＰＶ的ｇｐ４４和ｐ２８。以上的交叉反应结果表明ＯＰＰＶ与ＣＡＥＶ的抗原之间具有密切的相关性,这对于ＯＰＰＶ通过山羊和ＣＡＥＶ通过绵羊的传代研究是非常重要的,并对将来的免疫预防策略具有重要的指导意义。     

马传染性贫血野毒辽宁株前病毒全基因组核苷酸序列测定

采取马传染性贫血病毒（ＥＩＡＶ）辽宁野毒株（Ｌ株）感染马的血液,分离白细胞。提取白细胞中的ＤＮＡ,并以此为模板,利用ＰＣＲ技术分四个片段扩增 ＥＩＡＶ前病毒 ＤＮＡ。将其分别克隆到载体质粒 ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ中,经酶切鉴定后测序。对测序结果分析、拼接,得到 ＥＩＡＶ　 Ｌ株前病毒基因组全序列。     

不同启动子对重组痘苗病毒表达狂犬病病毒糖蛋白基因的影响

以血凝素（ＨＡ）基因为侧翼，将狂犬病病毒糖蛋白基因ｃＤＮＡ置于不同类型的痘苗病毒复合启动子下游，构建了４种表达质粒。重组表达质粒转染已感染ＷＲ株痘苗病毒的ＢＨＫ２１细胞，并通过鸡红细胞吸附试验，筛选、纯化出与表达质粒对应的４组ＨＡ－重组痘苗病毒：ｖＳＦＪ１－１０ＲＶｇｐ，ｖＳＦＪ２－１６ＲＶｇｐ，ｖＲＪ１－１０ＲＶｇｐ，ｖＲＪ２－１０ＲＶｇｐ。经间接免疫荧光试验（ＩＦＡ）鉴定，从４组重组病毒中每组各鉴定出１株阳性重组病毒。Ｗｅｓｔｅｒｎｂｌｏｔ检测显示，重组病毒感染的ＢＨＫ２１细胞裂解物上清中有１种蛋白与抗ＲＶＧＰ的ＭｃＡｂ发生特异反应，分子量为６９０００。在重组病毒感染早期，ＲＶＧＰ的表达量以ｖＳＦＪ１－１０ＲＶｇｐ最高；而在感染晚期，则以ｖＳＦＪ２－１６ＲＶｇｐ最高。４株重组病毒免疫小鼠，均能够不同程度地诱导小鼠产生抗ＲＶＧＰ特异性抗体。     

实验感染山羊关节炎—脑炎病毒的绵羊抗体应答反应的研究

本实验用琼脂凝胶免疫扩散试验和免疫印迹试验对实验感染山羊关节炎—脑炎病毒（ＣＡＥＶ）的绵羊抗体应答反应进行了研究，用两种方法都可在接毒绵羊的血清中检测到ＣＡＥＶ的抗体。琼脂凝胶免疫扩散试验最早可于接毒后的第７周时检测到抗体，免疫印迹试验最早可于接毒后的第６周时检测到抗ＣＡＥＶ的ｇｐ１２５、ｇｐ４４、ｐ３５、ｐ２８和ｐ１４的抗体，这说明免疫印迹试验更为敏感一些。本实验的结果表明ＣＡＥＶ可在绵羊体内诱生明显的体液免疫应答反应，因此用ＣＡＥＶ通过绵羊体传代的方法可能会得到具有良好的抗原性的ＣＡＥＶ毒株，这对于人工培养ＣＡＥＶ强毒是非常重要的。此外，本实验还为ＣＡＥＶ通过绵羊体传代的研究提供了非常实用的检测手段     

伪狂犬病病毒糖蛋白gp50基因的克隆和表达

从包含伪狂犬病病毒（ＰＲＶ）闽Ａ株ＢａｍＨＩ－７片段的重组质粒ｐＰＲ１２８中分离出含有完整糖蛋白ｇｐ５０基因的２．１ｋｂＤＮＡ片段，用ＫｐｎＩ和ＳｔｕＩ酶切后，将其酶切片段分别克隆到ｐＵＣ１９载体中，构建了２．１ｋｂ片段完整测序用质粒。对其序列进行分析，发现与文献报道结果一致，证明分离的ｇｐ５０基因是正确的。将包含ｇｐ５０基因的２．１ｋｂ和１．６ｋｂＤＮＡ片段分别插入带有痘苗病毒天坛株ＴＫ基因区段的ｐＧＪＰ－５质粒Ｐ７．５启动子的下游，构建了ｐＧＢＴ５０－３６和ｐＧＢＴ５０－Ｓ２２个嵌合载体。将嵌合载体通过磷酸钙共沉淀法转染预先感染ＴＫ＋痘苗病毒天坛株的人ＴＫ－１４３细胞或ＣＶ－１细胞，进行体内同源重组。经蚀斑纯化，在ＢｄＵＲ选择压力下，通过光敏生物素标记的探针杂交，获得带有ＰＲＶｇｐ５０基因的重组痘苗病毒。用ＥＬＩＳＡ检测，重组痘苗病毒有特异性ＰＲＶｇｐ５０抗原存在。     

狂犬病病毒糖蛋白基因在原核细胞中的表达

将狂犬病病毒糖蛋白（ＲＶｇｐ）基因ＢｇｌⅡ片段（１６７５ｂｐ）分别正向插入到原核高效表达载体ｐＥＴ－１７ｂ和ｐＥＴ－１７ｂ２（用ＳａｃⅠ－ＮｄｅⅠ缺失掉ｐＥＴ－１７ｂ６０ｂｐ含起始密码子ＡＴＧ小片段）的ＢａｍＨⅠ切点，构建重组质粒ｐＥＴ－１７ｂＲＶｇｐ和ｐＥＴ－１７ｂ２ＲＶｇｐ。将其分别转化表达受体菌Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）和Ｅ，ｃｏｌｉＢＬ２１（ＤＥ３）ｐｌｙｓＳ．ＩＰＴＧ诱导表达，菌体经超声波裂解处理后ＳＤＳ－ｐＡＧＥ，染色，在分子量约６００００处可见重组质粒表达的较宽的蛋白带，以抗ＲＶｇｐＭｃＡｂ进行Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测，表明该表达蛋白为ＲＶｇｐ。通过扫描显示，表达的ＲＶｇｐ占菌体总蛋白的１０％～１４％，其中ｐＥＴ－１７ｂ２ＲＶｇｐ在Ｅ。ｃｏｌｉＢＬ２１（ＤＥ３）中的表达量最高。     

马立克氏病病毒囊膜糖蛋白B抗原I型特异性和群共同性决定簇的…

用能表达马立克氏病病毒（ＭＤＶ）糖蛋白Ｂ（ｇＢ）的重组杆状病毒感染的Ｓｆ９细胞免疫小鼠，制备针对ＭＤＶｇＢ的单克隆抗体，以Ｉ型马立克氏病病毒ＧＡ株感染的鸡胚成纤维细胞作为检测抗原，同时以免疫荧光试验（ＩＦＡ）和酶联免疫吸附试验（ＥＬＩＳＡ）来筛选杂交瘤细胞，结果获得了ＩＦＡ和ＥＬＩＳＡ均为阳性的１株单克隆抗体细胞株，定义名ＢＡ４和ＢＤ８。在ＩＦＡ和免疫沉淀试验中，单抗ＢＤ８与Ｉ，ⅡⅢ型ＭＤＶ均呈阳     

马立克氏病病毒囊膜糖蛋白B抗原Ⅰ型特异性和群共同性决定簇的单克隆抗体

用能表达马立克氏病病毒（ＭＤＶ）糖蛋白Ｂ（ｇＢ）的重组杆状病毒感染的Ｓｆ９细胞免疫小鼠，制备针对ＭＤＶｇＢ的单克隆抗体。以Ⅰ型马立克氏病病毒ＧＡ株感染的鸡胚成纤维细胞作为检测抗原，同时以免疫荧光试验（ＩＦＡ）和酶联免疫吸附试验（ＥＬＩＳＡ）来筛选杂交瘤细胞，结果获得了ＩＦＡ和ＥＬＩＳＡ均为阳性的２株单克隆抗体细胞株，定名为ＢＡ４和ＢＤ８。在ＩＦＡ和免疫沉淀试验中，单抗ＢＤ８与Ⅰ、Ⅱ、Ⅲ型ＭＤＶ均呈阳性反应；单抗ＢＡ４只对Ⅰ型ＭＤＶ（包括ＣＶＩ９８８疫苗株）呈阳性反应。免疫沉淀反应进一步确证２株单抗识别的是ＭＤＶ糖蛋白Ｂ抗原。     

马传染性贫血病毒驴白细胞弱毒株LTR启动子功能的研究

为了证明我国马传染性贫血病毒（ＥＩＡＶ）驴白细胞弱毒株的长末端重复序列（ＬＴＲ）是否能够启动基因的表达,我们将该ＬＴＲ克隆于含ＣＡＴ基因的载体中,获得重组质粒ｐＣＡＴ－ＬＴＲ,用ｐＣＡＴ－ＬＴＲ分别转染驴胎皮肤细胞（ＦＤＤ）、胶质母细胞瘤（ＴＪ－９０５）细胞、驴白细胞及接种ＥＩＡＶ弱毒的ＦＤＤ及驴白细胞,结果以ＥＩＡＶ弱毒的ＬＴＲ为启动子,ＣＡＴ在这几种细胞中均获得了不同程度的表达,证实了我国ＥＩＡＶ弱毒株的ＬＴＲ在ＦＤＤ、ＴＪ－９０５及驴白细胞中确实具有一定的启动子功能,并能被反式激活表达,为进一步研究ＥＩＡＶ弱毒复制及表达调控奠定了基础。     

Proviral Genomic Variation of EIAV Envelop gp 90 Gene Observed in Experimentally Infected Horse

The present experiment was designed to observe the genetic variation of equine infectious anemia virus (EIAV) envelop gp 90 gene in infected horse. One horse was infected experimentally with P337-V70 strain and showed no clinical signs after being infected at twice with the same virus strain. Seventeen proviral sequences covering principal neutralizing domain (PND) of EIAV gp 90 gene were obtained from the buffy coat and liver of the horse through PCR amplification and cloning. Comparative analysis of the sequences revealed that some sequences contained the nucleotide insertions in the PND region. The insertions might be generated by direct repeat and strand displacement of sequence segment in their PND gene, showing different lenghts.     

马传染性贫血病毒阿根廷流行毒株前病毒gag和gp90基因在马体内的变异分析

为建立美洲马传贫毒株和我国弱毒疫苗株鉴别诊断PCR，对接种马传贫阿根廷流行毒株后马4个发热期（23、40、51和70d）外周血单核细胞前病毒DNA gag基因和gP90基因的核苷酸序列进行了监测，经过序列分析发现4个发热期中前病毒gag基因核苷酸序列变化不明显，变异率在1％之内，表明前病毒gag基因在马体内比较保守，可以作为设计鉴别诊断引物的区域；gp90区的基因变化比较大，核苷酸序列变异率在8％～10％之间，通过对几个发热期核苷酸序列推导氨基酸的分析，可以划分出A1、A2、A3和A44个可变区，但其中A1和A4变异都很稳定，在马体发病的整个过程中均只发生1次变异，而A2区第3个发热期变异后，在第4个发热期又回复了突变。对马传染性贫血病毒阿根廷代表毒株前病毒gag和gp90基因核甘酸序列的动念检测结果表明，马传贫病毒在马体整合为前病毒之后比较稳定，这就为建立PCR鉴别诊断方法提供了依据。     

马传染性贫血驴强毒gp90基因的克隆和序列分析

以EIAV驴强毒株D-AmRNA为模板，利用RT-PCR技术，扩增了约1．4kb的gp90基因。将其克隆后进行了测序。测序结果表明所扩增的1338个核苷酸片段含有完整的gp90基因全序列。核苷酸和氨基酸序列比较分析结果表明：D-A EIAV与国内分离株辽系强毒L株差异率仅有1．8％，而与国外毒株(克隆1369，WENVl7、WENVl6、PSPEIAVl9)核苷酸差异率在35．5％～37．2％之间；D-A株与国内分离株L株氨基酸水平差异率在2．9％，而与国外毒株氨基酸水平上的差异率在42．6％—46．0％；D-AEIAV有19个N-连接糖基化位点，L株、WENVl7和WENVl6是18个，克隆1369、WENVl6和WENVl7亲本毒株PSPEIAVl9是12个。     

马传染性贫血病毒囊膜基因gp90V3区糖化回复突变感染性克隆的构建

为阐明我国马传染性贫血病毒（Equine infectious anemia virus,EIAV）弱毒疫苗致弱及免疫保护的分子机制,作者分析了疫苗株及其亲本强毒株共34个囊膜基因序列。结果发现疫苗株在膜基因gp90V3区的潜在N-连接糖基化位点出现稳定的碱基替换,使该位点消失。为研究囊膜糖基化的作用,以疫苗株全长感染性克隆pLG—FD3—8为亲本,利用反向遗传技术对该突变位点进行糖基化序列回复操作,构建全基因感染性克隆pLGFDg5。将pLGFDg5转染驴胎皮肤细胞（FDD）,通过逆转录酶活性和RT-PCR方法评价其感染性。结果表明,将pLGFDg5在FDD细胞中盲传3代后,可在细胞培养上清中检测到逆转录酶活性,用RT—PCR检测到EIAV保守基因片段,在电镜下可见典型的EIAV粒子。在FDD细胞上的病毒复制动力学分析显示,与其亲本克隆pLGFD3—8的衍生病毒pLGFD3-V相比,回复突变克隆衍生的pLGFDg5-V复制速度较慢,获得较低的病毒载量。体外抗体中和试验表明,与其亲本pLGFD3-V相比,引入潜在的糖基化位点g5降低了衍生病毒对中和抗体的敏感性。这一结果为N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究提供了重要依据。     

Antibodies and PMBC from EIAV infected carrier horses recognize gp45 and p26 synthetic peptides

Equine infectious anemia virus (EIAV) is a lentivirus causing a persistent infection in horses characterized by recurrent febrile episodes and high levels of viremia associated with a novel antigenic strain of the virus. The virus contains two envelope glycoproteins, gp90 and gp45, and four internal proteins, p26, p15, p11 and p9. Considering that the most infected horses are able to restrict EIAV replication to very low levels and that gp45 and p26 contain highly conserved epitopes among lentiviruses, it would be necessary to identify those conserved epitopes stimulating cellular and humoral responses. The aims of this study were to determine if the synthetic peptides identified as gp45 (aa 523-547) and p26 (aa 318-346) representing two highly conserved and immunodominant regions of EIA virus are recognized by PBMC and antibodies to EIAV adult mixed-breed naturally infected carrier horses, and if these peptides are able to induce immune responses in mice. Antibodies from 100% of carrier horses, evaluated by ELISA, recognized both peptides; PBMC from 80% of carrier horses, evaluated by lymphoproliferation assay, recognized, at least, one peptide. Furthermore, immunization with 100 microg of each peptide elicited humoral and cellular responses in BALB/c mice, antibodies appeared at 48 or 63 days of immunization with gp45 or p26, respectively. Although the kinetics of gp45- and p26-specific antibody responses were similar, percentage of positivity was higher for gp45. The lymphoproliferation assay, evaluated by BrdU uptake, was higher in mice immunized with gp45 or p26 than in the control group (P<0.05). Based on our findings, we consider that both peptides could be included in an effective vaccine design to induce long-term immunological memory.     

The determination of in vivo envelope-specific cell-mediated immune responses in equine infectious anemia virus-infected ponies

Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA carrier horse with no clinical signs infected 7 years ago and 4 related experimental ponies infected 6 months previously were used in this study. Forty-four 20-mer peptides, representing the entire surface unit protein (gp90) of EIAV, were combined into 14 peptide pools and intradermally injected into the neck of EIAV-infected horses. An identical volume of saline alone was injected into a fifteenth site as a negative control. After 48h, those sites with palpable infiltrations were measured prior to the collection of 2mm and 4mm punch biopsies. Total RNA was extracted from each 2mm biopsy for determination of CD3 and interferon-γ (IFN-γ) mRNA expression by real-time PCR. The 4mm skin biopsies were formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) staining for CD3, CD20, CD25 and MAC387 (macrophage marker). Peripheral blood mononuclear cells (PBMC) were obtained prior to the injection and tested for in vitro reactivity against the same peptides. Histological examination showed that some of the envelope peptides elicited a lymphocytic cellular infiltration at the injection site, as evidenced by positive staining for CD3. Gp90 peptide-specific increases in CD3 and IFN-γ gene expression were also detected in the injection sites. Furthermore, differences were found between in vivo and in vitro responses to gp90 specific peptides. These results demonstrate a novel method for detecting in vivo cell-mediated immune responses to EIAV-specific peptides that is readily applicable to other host/pathogen systems.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

中国马传染性贫血病毒DLV株前病毒gp90基因在马体内的变异分析

分别以EIAV辽宁马强毒株（EIAVliao）前病毒DNA和马传染性贫血驴白细胞弱毒疫苗株（EIAVDLV）前病毒DNA为模板,从免疫接种后不同时期马血清中利用nested—PCR技术,扩增了约1．4kb的gp90基因。将其克隆后进行了测序,测序结果表明,免疫EIAVDLV后的第1时期至第5时期,与EIAV—liao比较核苷酸序列平均差异率分别为3．2956％、3．1456oA、3．36％、3．1856％和3．6456％。EIAVliao有20个N-连接糖基化位点,EIAVDLV平均是17．2个,其中有11个N-连接糖基化位点是高度保守的。     

血管瘤禽白血病病毒FJ0610株的分离与鉴定

为了解禽白血病病毒在商品蛋鸡中的流行情况,试验采用病理剖检、聚合酶链式反应(PCR)以及间接免疫荧光试验(IFA)等方法对送检的疑似禽白血病病毒感染的商品蛋鸡进行了病毒分离与鉴定,并对分离株的致瘤相关基因gp85基因进行测序,与国内外各亚群禽白血病病毒进行对比。结果表明:分离、鉴定到1株J亚群血管瘤禽白血病病毒,命名为FJ0610;分离株gp85基因核苷酸序列同源性在11.5%～94.5%之间,其中与血管瘤禽白血病毒株ZH-08株同源性最高,而与E亚群毒株同源性最低;基于gp85核苷酸序列的系统进化分析表明FJ0610株的gp85序列与ZH-08株的亲缘关系最近。