口蹄疫病毒3D蛋白对表位多肽免疫效果调节作用的研究

为了研究口蹄疫病毒(FMDV)重组蛋白3D对病毒表位抗原的免疫调节功能,笔者克隆了Asia1型FMDV的3D蛋白基因,并实现了重组蛋白在E.coli的表达及纯化。将纯化的重组蛋白3D与重组抗原按     

抗Asia1型口蹄疫病毒单克隆抗体的制备及抗原捕获ELISA检测口蹄疫病毒的研究

本研究用纯化的Asial型口蹄疫病毒(Foot and mouth disease virus,FMDV)免疫BALB/c小鼠,按常规单克隆抗体技术方法,经筛选获得6株能稳定分泌抗Asial型FMDV单抗的杂交瘤细胞株。以牛抗口蹄疫病毒IgG为捕获抗体,选择一株单抗(184)用辣根过氧化物酶标记(HRP-184)作为检测抗体,建立了检测Asial型FMDV的抗原捕获ELISA方法。该方法可检出0.5859μg纯化Asial型FMDV抗原和2.5×10~3TCID_(50)病毒,对O型FMDV、牛结核病、牛肺疫、牛流热、赤羽病、牛传染性鼻炎等病毒进行检测,均为阴性,无交叉反应发生。本研究建立的FMDV抗原捕获ELISA方法,具有敏感性高、特异性强和重复性好的特点,可用于Asial型FMDV的特异性检测。     

PAGE对口蹄疫病毒结构多肽的分离及其抗原变异的研究进展

多年来,补体结合试验和病毒中和试验是鉴定口蹄疫病毒(FMDV)血清型和亚型以及抗原变异研究的传统血清学方法。近代,生物化学方法发展很快,许多研究者用聚丙烯酰胺凝胶电泳(PAGE)、电聚焦和T_1—寡核苷酸指纹图技术鉴定和区分病毒株,研究了FMDV的抗原变异,并且探索了某些地区发生口蹄疫的病毒来源。PAGE     

口蹄疫病毒亚型鉴定的研究进展

关于口蹄疫病毒(FMDV)的分类以及型和亚型的鉴定标准,Brooksby和Fontainc等(1968)曾在第19次口蹄疫国际讨论会上作了详细的论述。口蹄疫病毒在毒力和抗原性上特别容易发生变异,经过逐渐的抗原漂移过程,常常导致产生一系列无明显界限的亚型。因此,EMDV的型和亚型的鉴定以及抗原成分及其变异的研究,对流行病学调查和免疫接种是极其重要的。FMDV亚型的鉴定,既可提供侦察流行动态和发现特定病毒     

金丝桃素对口蹄疫病毒的体外抗病毒实验

本文报道了金丝桃素体外抗口蹄疫病毒（FMDV）的研究结果。采用双抗体捕获抗原法、病毒滴度测定、MTT法、组织细胞培养等方法均检测到抗病毒作用。结果显示：金丝桃素在BHK-21细胞感染FMDV前、后及同时加入，都有明显抑制细胞病变的作用。金丝桃素使FMDV的TCID50从8．5下降到6．25。双抗体捕获抗原法检测到金丝桃素有效抑制FMDVBHK-21细胞的吸附、融合，抑制率可达59．72％。研究结果提示：金丝桃素在体外有抗口蹄疫病毒作用，抑制口蹄疫病毒与宿主细胞的吸附、融合是抗病毒的重要途径之一。     

口蹄疫病毒P1结构蛋白和3A非结构蛋白研究进展

口蹄疫病毒(FMDV)P1基因所编码的结构蛋白是FMDV衣壳的主要蛋白,是其主要抗原位点所在的区域,决定着FMDV的抗原性,3A非结构蛋白能与宿主细胞结合,其特征性变化与病毒的表型变化密切相关,影响着宿主嗜性和致病力,P1和3A基因都是FMDV研究的热点。论文综述了FMDV P1和3A基因及其所编码蛋白的结构、抗原特性及其生物学功能的研究现状,为后续FMDV研究提供参考。     

金丝桃素体外抗口蹄疫病毒与宿主细胞吸附作用研究

对光活化金丝桃素体外抑制口蹄疫病毒与宿主细胞吸附作用进行实验。体外培养的BHK-21细胞加入金丝桃素,日光灯下光活化1h后感染FMDV。通过细胞病变观察、MTT法、双抗体捕获抗原法、扫描电子显微镜进行抑制FMDV与宿主细胞吸附、融合研究。结果表明金丝桃素有明显抑制细胞病变的作用。双抗体捕获抗原法检测金丝桃素有效抑制FMDV和BHK-21细胞的吸附、融合,抑制率可达59．72％。扫描电镜观察,金丝桃素组与病毒对照组相比,BHK-21细胞FMDV颗粒明显减少。提示金丝桃素在体外有显著抑制口蹄疫病毒与宿主细胞的吸附、融合作用。     

口蹄疫病毒对树突状细胞感染能力的研究

树突状细胞（dendritic cell,DC）是重要的抗原递呈细胞,本研究发现通过口蹄疫病毒（foot and mouth diseasevir US,FMDV）感染DC细胞后可以有效感染小鼠骨髓来源DC,而且在感染后48h,FMDV对未成熟DC（imDC）的感染能力稍高于成熟DC（mDC）.     

口蹄疫诊断技术的研究进展

口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引起的偶蹄动物的一种烈性传染病。FMDV是小     

口蹄疫病毒整合素受体研究进展

口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)感染宿主细胞首先是病毒与被感染细胞表面的病毒受体结合,通过网格蛋白介导的内吞途径,酸化的内吞泡运输进入细胞内,然后衣壳迅速解体,释放出基因组RNA,细胞受体决定口蹄疫病毒宿主特异性和组织特异性.对口蹄疫病毒细胞受体的研究将有助于揭示口蹄疫病毒的感染机制、复制过程、致病机理和疫病预防及治疗等,细胞受体的研究已经成为目前口蹄疫病毒研究中的重点领域之一,论文就近年来FMDV整合素受体研究现状进行了综述,并对其发展进行了展望.     

The relationship between cellular immune response to foot-and-mouth disease virus Asia 1 and viral persistence in Indian cattle (Bosindicus)

Foot-and-mouth disease (FMD), the most contagious animal disease, is associated with persistent viral infection in ruminants, despite the induction of systemic immune response. The present study was performed to decipher the relation between the persistent FMD virus (FMDV) infection and cellular immune response in Indian cattle (Bosindicus) following experimental inoculation of FMDV Asia 1. Persistent viral infection (carriers) was detected by antigen capture RT-PCR on the oesophageal-pharyngeal fluid. Viral excretion was found to be intermittent and strongly variable among the persistently infected Indian cattle. Lymphocyte proliferative (LP) response, assessed as reactivity of peripheral blood mononuclear cells to FMDV Asia 1 antigen (Ag) was of low magnitude indicating a weak primary cellular immune response following infection. LP response to FMDV Ag was higher among the non-carriers than carriers of FMDV Asia 1. An enhanced LP response was associated with the lack of virus shedding in the OPF. The findings of this study are suggestive of relationship between cellular immune response and virus excretion during persistence of FMDV Asia 1 in infected cattle.     

Foot-and-mouth disease virus in the llama (Lama glama): diagnosis, transmission, and susceptibility

Foot-and-mouth disease virus (FMDV) was shown to be transmitted from either cattle to llamas, llamas to swine (interspecies), or llamas to llamas (intraspecies). Response to FMDV varied greatly in the 6 llamas studied; 3 llamas developed generalized clinical disease with mild pyrexia, 2 after intradermolingual inoculation, and 1 after exposure to a calf infected with FMDV serotype A24. Another contact llama developed vesicular lesions on all 4 extremities but no oral lesions. Two contact llamas, in separate study groups, did not seroconvert or develop clinical signs of FMDV infection. All 4 llamas showing clinical disease developed virus-neutralizing antibodies against FMDV A24 and antibodies against the virus-infection-associated antigen. Virus-neutralizing antibody titers remained elevated for over 200 days postinoculation or exposure. Antibodies to virus-infection-associated antigen were detected several days after virus-neutralizing antibody appeared and became weaker 100-125 days post-FMDV exposure in 3 of the 4 clinically affected llamas. One inoculated llama was still positive for virus-infection-associated antigen at 360 days after inoculation. Foot-and-mouth disease virus A24 was not detected from esophageal-pharyngeal fluid specimens beyond 8 days postexposure using in vitro techniques.     

口蹄疫146S病毒粒子抗原对微血管内皮细胞分泌IL-1的影响

白介素1（IL-1）是免疫与炎症反应启动的重要调节因子。为了研究微血管内皮细胞（microvascular endothelial cells,MVECs）在口蹄疫病毒（foot and mouth disease virus,FMDV）免疫反应中的作用,本试验采用ELISA方法检测了FMDV 146S抗原对体外培养大鼠心肌膜MVECs分泌IL-1α和IL-1β的影响。研究结果发现,正常情况下大鼠心肌膜MVECs能够低水平的分泌IL-1α和IL-1β,FMDV 146S刺激后其分泌量显著增加。结果表明,MVECs通过上调IL-1分泌,在FMDV抗原的免疫反应中发挥重要的调控作用。     

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.     

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.     

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection

ABSTRACT: Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in na?ve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.     

Ultrastructural changes and antigen localization in tissues from foot-and-mouth disease virus-infected guinea-pigs

Foot-and-mouth disease virus (FMDV)-induced ultrastructural changes in guinea-pig tongue, heelpad, mammary and liver tissues were examined using scanning and transmission electron microscopy. FMDV infection caused cell rounding and the release of virus in membrane limited vesicles in the animal tissues similar to that seen in other work in cell cultures. Microfilaments were present which may be responsible for cell rounding. Immunoperoxidase labeling revealed the attachment of the virus-infection associated (VIA) antigen to the smooth vacuoles of mammary and liver tissues, and to milk fat globules. The electron microscope immunoperoxidase procedure increased the sensitivity of detection sufficiently to allow the visualization of VIA antigen in tissues not previously shown to have the antigen. It is postulated that the release of the smooth vacuoles from the liver cells stimulates the animal's immune response to the VIA antigen.     

嵌合口蹄疫病毒抗原表位的重组塞内卡病毒A的构建及其鉴定

塞内卡病毒A（SVA）与口蹄疫病毒（FMDV）引起猪的临床症状难以区分,并且以SVA作为载体插入FMDV抗原表位外源基因的研究,目前还没有报道。为了构建一种嵌合FMDV抗原表位的重组SVA毒株并进行鉴定,本研究利用基因克隆技术将O型FMDV的B细胞表位与白蛋白信号肽（human albumin signal peptide,HAS）基因串联插入到SVA基因组中,成功构建了重组质粒,转染细胞后拯救出重组病毒rSVA-HAS-OB。结果显示：RT-PCR扩增与基因测序结果表明目的基因正确插入;细胞间接免疫荧光和Western blot鉴定了嵌合抗原蛋白的有效表达;遗传稳定性分析表明重组病毒在25代次的传代过程中仍稳定存在B细胞表位;病毒蚀斑表型和一步生长曲线结果表明,重组病毒与亲本毒株具有相似的增殖特性。本研究成功构建并拯救出能够表达FMDV抗原表位的重组SVA毒株,为以SVA为基础的嵌合病毒及疫苗的研究提供了理论和实验基础。     

猪A型口蹄疫病毒特异性IgA抗体间接ELISA检测方法的建立与初步应用

为建立一种检测口蹄疫病毒（foot-and-mouth disease virus,FMDV）特异性IgA抗体的检测方法,本研究以原核表达系统表达纯化的FMDV结构蛋白VP1作为包被抗原,以鼠抗猪IgA单克隆抗体为二抗,辣根过氧化酶标记的羊抗鼠IgG抗体为三抗,建立猪A型FMDV特异性IgA抗体间接ELISA检测方法。确定抗原包被浓度为3.50 μg/mL,二抗与三抗的最佳稀释度为1∶10 000,二抗和三抗作用时间均为30 min。所建立的方法与抗猪瘟病毒、猪繁殖与呼吸道综合征病毒等病原的特异性IgA抗体间无交叉反应,A型口蹄疫感染样品的阳性检出率在90%以上,批内和批间重复性试验的变异系数介于3.16%~9.76%。该方法为监测FMDV特异性IgA抗体水平变化规律及猪的黏膜免疫效果评价及口蹄疫的早期诊断提供了一种新方法。