共查询到18条相似文献，搜索用时 343 毫秒
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非洲马瘟(African horse sickness,AHS)由非洲马瘟病毒(African horse sickness virus,AHSV)感染马科动物而引起的一种非接触性传播的虫媒病毒病,为世界动物卫生组织法定报告的动物疫病,目前主要流行于亚撒哈拉非洲等地区。库蠓(Culicoides midges)是非洲马瘟的主要媒介昆虫,其在疫区的生长繁殖直接影响着该病的流行状况。非洲马瘟弱毒苗和灭活苗已经商品化并得到广泛地应用,新型基因工程疫苗,如亚单位疫苗、活病毒载体疫苗等,正在研发当中并有望将来进入疫苗市场,作者着重对ASH疫苗的研究现状进行了评述。 相似文献
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《中国预防兽医学报》2020,(6)
为建立非洲马瘟病毒(AHSV)可视化RT-LAMP检测方法,本研究根据AHSV外衣壳蛋白(VP7)基因保守序列设计2对特异性引物,通过反应条件优化建立了AHSV可视化RT-LAMP检测方法。结果显示,本实验所建立RT-LAMP方法的最佳反应条件为62℃1 h;利用该方法检测进口灭活冻干疫苗中的9个血清型AHSV以及马流感病毒、马传贫病毒、马动脉炎病毒和蓝舌病病毒的RNA,结果显示所有血清型AHSV RNA检测均为阳性,其它病毒RNA检测为阴性,该方法特异性较强;该方法最低可检测到3.2×10~(-9)ng/μL的RNA,敏感性是普通RT-PCR方法的1 000倍。利用建立的RT-LAMP方法和普通RT-PCR方法对35份马血和5份驴血样品同时进行检测,结果显示二者阳性和阴性符合率均为100%。本研究在国内外首次建立了AHSV 9种血清型的可视化RT-LAMP检测方法,其具有特异性强、敏感性高、快速和高通量检测的优点,为非洲马瘟(AHS)快速的可视化现场诊断提供可行方法。 相似文献
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《畜牧兽医学报》2015,(4)
VP7蛋白是非洲马瘟病毒(AHSV)群特异性蛋白,其编码基因S7基因常被用作AHSV RT-PCR和实时荧光RT-PCR检测的靶标基因。本研究旨在构建耐RNase的内含AHSV部分核酸序列的病毒样颗粒。首先合成S7基因保守序列,然后克隆到含有噬菌体包膜蛋白基因的带组氨酸纯化标签的假病毒表达载体pNH-MS2his上,成功构建原核表达载体pNH-MS2his-VP7。将重组质粒pNH-MS2his-VP7转化BL21(DE3),并进行诱导表达及镍离子亲和层析纯化后,得到含AHSV部分RNA片段的病毒样颗粒。试验证实该病毒样颗粒均匀性和稳定性良好,可作为AHSV PCR检测的质控品和标准品使用。 相似文献
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为了建立非洲马瘟病毒(AHSV)环介导等温扩增(loop-mediated isothermal amplification,LAMP)快速检测方法,基于AHSV VP7基因保守序列,设计2对特异性扩增引物。通过对反应体系中各组分浓度,反应温度及时间的优化,建立了快速、灵敏的AHSV RT-LAMP检测方法,并对该方法特异性、灵敏度、重复性进行探索。结果表明,在63℃恒温下反应45min,便可进行高效率的特异性扩增。反应产物经琼脂糖凝胶电泳和染料可视化鉴定,能够快速有效检测AHSV。用建立的方法检测马属动物易感的4种疫病病原,结果均为阴性,证实具有较高的特异性。灵敏度是RT-PCR的1 000倍。建立了AHSV RT-LAMP检测方法,具有快速、特异、灵敏、操作简单、设备要求低的特点,适合用于现场AHSV快速检测。 相似文献
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Fernández-Pinero J Fernández-Pacheco P Rodríguez B Sotelo E Robles A Arias M Sánchez-Vizcaíno JM 《Research in veterinary science》2009,86(2):353-108
A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID50 per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS). 相似文献
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非洲马瘟病毒VP7基因拼接、表达及重组ELISA方法的建立与初步应用 总被引：2，自引：0，他引：2
采用人工拼接的方式拼接了非洲马瘟病毒（AHSV）含有绝大多数线性抗原表位的VP7编码基因片段,克隆于pET-30a构建重组质粒pET-30a-VP7,将pET-30a—VP7转化BL21（DE3）,经1．0mmol／LIPTG诱导,外源基因以包涵体的形式获得高效表达。通过Dot-EuSA以及ELISA试验证明表达产物具有良好的反应原性。以纯化后表达产物作为诊断抗原包被酶标板建立了检测AHSV抗体的间接ELISA方法。结果表明,抗原的最佳包被浓度为0．25μg／mL,血清的最佳稀释度为1：40,待检血清阳性临界值初步定为0．25。用此方法和商品化ELISA试剂盒检测了184份血清样品,结果完全符合。 相似文献
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African horse sickness virus structure 总被引：4，自引：0，他引：4
Polly Roy Peter P.C. Mertens Ignacio Casal 《Comparative immunology, microbiology and infectious diseases》1994,17(3-4):243-273
African horse sickness virus (AHSV), of which there are nine serotypes (AHSV-1, -2, etc.), is a member of Orbivirus genus within the Reoviridae family. Both in morphology and molecular constituents AHSV particles are comparable to those of bluetongue virus (BTV), the prototype virus of the genus. The two viruses have seven structural proteins (VP1–7) organized in two layered capsid. The outer capsid is composed of VP2 and VP5. The inner capsid, or core, is composed of two major proteins, VP3 and VP7, and three minor proteins, VP1, VP4 and VP6. Within the core is the virus genome. This genome consists of 10 double-stranded (ds)RNA segments of different sizes, three large, designated L1–L3, three medium, M4–M6, and four small, S7–S10. In addition to the seven stuctural proteins that are coded by seven of the RNA species, four non-structural proteins, NS1, NS2, NS3 and NS3A, are coded by three RNA segments, M5, S8 and S10. The two smallest proteins (NS3 and NS3A) are synthesized by the S10 RNA segment, probably from different in-frame translation initiation codons. Nucleotide sequences of eight RNA segments (L2, L3, M4, M5, M6, S7, S8 and S10) and the predicted amino acid sequences of the encoded gene products are also available, mainly representing one serotype, AHSV-4. In this review the properties of the AHSV genes and gene products are discussed. The sequence and hybridization analyses of the different AHSV dsRNA segments indicate that the segments that code for the core proteins, as well as those that code for NS1 and NS2 proteins, are highly conserved between the different virus serotypes. However, the RNA encoding NS3 and NS3A, and the two segments encoding the outer capsid proteins, are more variable between the AHSV serotypes. A close phylogenetic relationship between AHSV, BTV and epizootic haemorrhagic disease virus (EHDV), three Culicoides-transmitted orbiviruses, has been revealed when the equivalent sequences of genes and gene products are compared. Recently, the four major AHSV capsid proteins have been expressed using recombinant baculoviruses. Biochemically and antigenically these proteins are similar to the authentic proteins. Since the AHSV VP7 protein is highly conserved among the different serotypes, it has been utilized as a diagnostic reagent. The expressed VP7 protein has also been purified to homogeneity and crystallized for three-dimensional X-ray analysis. The expressed outer capsid proteins, VP2 and VP5, have been purified and used to raise antisera in rabbits. The VP2 antisera neutralize virus infections in vitro indicating the importance of this protein for vaccine development. 相似文献
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W Van Wyngaardt D H Du Plessis S Van Wyngaardt J A Verschoor 《The Onderstepoort journal of veterinary research》1992,59(2):129-133
Four polyethylene glycol-mediated cell fusions yielded a total of 23 monoclonal antibodies (McAbs) specific for African horsesickness virus (AHSV). Two recognised the major core structural polypeptide, VP7, while one each was specific for the outer capsid proteins, VP2 and VP5. The remainder co-precipitated both VP2 and VP7. An inhibition ELISA and radio-immunoprecipitation revealed two types of co-precipitating McAbs, distinguishable from each other by the different relative amounts of the two proteins they precipitated. Only co-precipitating McAbs reduced the size and number of plaques formed by AHSV on VERO cell monolayers, but even at low dilution did not completely abolish virus infectivity. A McAb specific for VP7 showed potential as a group-reactive diagnostic reagent since guinea pig antisera to all nine serotypes of AHSV, as well as an anti-serotype 4 horse serum and an anti-serotype 3 rabbit serum, inhibited its binding in ELISA to AHSV serotype 3. 相似文献
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H El Garch JE Crafford P Amouyal PY Durand C Edlund Toulemonde L Lemaitre V Cozette A Guthrie JM Minke 《Veterinary immunology and immunopathology》2012,149(1-2):76-85
A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(?)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(?)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(?)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(?)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4. 相似文献