非洲马瘟病毒VP7蛋白研究进展

非洲马瘟(African horse sickness, AHS)是由非洲马瘟病毒(African horse sickness virus, AHSV)引起的一种通过库蜢等昆虫传播的、主要感染马科动物的传染病。我国是世界动物卫生组织认可的非洲马瘟无疫国,随着AHS疫情在东南亚的传播,增大了疫情传入我国的风险。AHSV编码了7种结构蛋白(VP1～VP7),其中VP7是病毒内衣壳蛋白的主要组成部分,在AHSV 9个血清型中高度保守,常作检测的靶标。此外,VP7蛋白的自组装特点对于AHSV亚单位疫苗和病毒样颗粒疫苗(VLP)的研究有基础性作用。对当前AHSV VP7蛋白相关研究进展进行了综述,以期为AHSV检测方法及疫苗等研究提供参考。     

African horse sickness in naturally infected,immunised horses

To determine whether subclinical cases, together with clinical cases, of African horse sickness (AHS) occur in immunised horses in field conditions, whole blood samples were collected and rectal temperatures recorded weekly from 50 Nooitgedacht ponies resident in open camps at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort, during 2008–2010. The samples were tested for the presence of African horse sickness virus (AHSV) RNA by a recently developed real‐time RT‐PCR. It was shown that 16% of immunised horses in an AHS endemic area were infected with AHSV over a 2 year period, with half of these (8%) being subclinically infected. The potential impact of such cases on the epidemiology of AHS warrants further investigation.     

非洲马瘟病毒、西尼罗病毒和马冠状病毒的基因芯片检测技术研究

为探索建立马病病毒基因芯片检测方法,采用人工拼接的方式拼接了非洲马瘟病毒(ASHV)核酸序列,通过分子克隆技术获得西尼罗病毒(WNV)和马冠状病毒(ECV)的特异基因片段。用芯片点样仪逐点分配到处理过的玻片上,制备成检测芯片。以拼接、克隆的核酸序列为模板通过多重不对称RT-PCR进行特异性扩增和荧光标记后滴加到芯片上进行杂交,对杂交结果进行扫描检测和计算机软件分析。结果显示,制备的基因芯片可同时检测和鉴别上述3种病毒,ECV质粒样品、WNV质粒样品检测灵敏度为10²拷贝;AHSV质粒样品检测灵敏度为10⁴拷贝。其他病毒材料未出现荧光信号,验证了本方法的特异性。证明基因芯片技术可快速、准确和灵敏地同时进行多种病毒的检测。     

非洲马瘟病毒VP7蛋白原核表达及间接ELISA抗体检测方法的建立

为建立非洲马瘟病毒(African Horse Sickness Virus,AHSV)的血清学诊断方法,本研究构建了非洲马瘟病毒VP7蛋白的原核表达质粒pET-32a-VP7,诱导表达后使用Ni-NTA对可溶性VP7蛋白亲和纯化,以重组VP7蛋白作为包被抗原,优化建立检测血清抗体的间接ELISA方法,并对该方法进行敏感性、特异型和重复性验证,表明该方法的检测效果良好。本研究为非洲马瘟的血清检测试剂盒的开发提供的相关参考。     

非洲马瘟间接ELISA方法的建立及初步应用

为建立检测非洲马瘟间接酶联免疫吸附试验方法,利用重组杆状病毒感染昆虫细胞表达出具有良好抗原性的VP7蛋白,将感染VP7重组杆状病毒的昆虫细胞裂解液作为包被抗原,通过优化包被抗原浓度、二抗稀释度等,建立了检测非洲马瘟的间接ELISA方法,并进行了初步运用。结果表明,直接利用真核细胞表达VP7蛋白的昆虫细胞裂解液作为包被液可成功建立检测非洲马瘟的间接ELISA方法。     

Laboratory confirmation of African horsesickness in the western Cape: application of a F(ab')2-based indirect ELISA

Recently a suspected outbreak of African horsesickness in the Western Cape Province resulted in the deaths of four foals and one adult horse. Spleen samples from these animals were subjected to analysis by an enzyme-linked immunosorbent assay (ELISA) which uses F(ab')2 fragments of immunoglobulins to detect African horse sickness virus (AHSV) antigens. The results of the immunoassay were compared with those obtained by isolation followed by serotyping as is currently applied by the Reference Centre at the Veterinary Research Institute, Onderstepoort. Samples of spleen tissue from the four foals contained sufficient antigen to be readily detectable by ELISA. A marginally positive signal was obtained with the tissue from the adult horse. This sample was inoculated onto VERO cells and four days were allowed for viral multiplication. Subsequently, when the cell culture was assayed by F(ab')2-ELISA, a much higher absorbance value than that obtained with the original spleen sample resulted, thus confirming the presence of AHSV in the initial specimen. The F(ab')2-ELISA has potential to be used as an initial diagnostic test to screen for AHSV.     

Evidence for a new field Culicoides vector of African horse sickness in South Africa

Between February and May 1998, approximately 100 horses died of African horse sickness (AHS) in the cooler, mountainous, central region of South Africa. On 14 affected farms, 156,875 Culicoides of 27 species were captured. C. imicola Kieffer, hitherto considered the only field vector for AHS virus (AHSV), constituted <1% of the total Culicoides captured, and was not found on 29% of the farms. In contrast, 65% of the Culicoides were C. bolitinos Meiswinkel, and was found on all farms. Five isolations of AHSV were made from C. bolitinos, and none from 18 other species of Culicoides (including C. imicola).     

A serological survey of African horse sickness in Botswana

A retrospective serological survey of African horse sickness (AHS) in Botswana covering a 10-year period (1995-2004) is reported. The survey involved horses showing clinical symptoms of the disease; the horses had not been vaccinated against AHS. Over the period surveyed, serological evidence suggestive of infection with AHS virus (AHSV) was found in 99 clinical cases out of which 41.4% (41/99) cases were found during the 1st half (1995-1999) and 58.6 % (58/99) cases were found in the 2nd half of the survey period (2000-2004). These serological findings are discussed in relation to AHSV serotypes isolated from diseased horses in Botswana before and during the period of this serological survey.     

Laboratory diagnosis of African horse sickness: comparison of serological techniques and evaluation of storage methods of samples for virus isolation

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at -70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses

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.     

Adaptive strategies of African horse sickness virus to facilitate vector transmission

African horse sickness virus (AHSV) is an orbivirus that is usually transmitted between its equid hosts by adult Culicoides midges. In this article, we review the ways in which AHSV may have adapted to this mode of transmission. The AHSV particle can be modified by the pH or proteolytic enzymes of its immediate environment, altering its ability to infect different cell types. The degree of pathogenesis in the host and vector may also represent adaptations maximising the likelihood of successful vectorial transmission. However, speculation upon several adaptations for vectorial transmission is based upon research on related viruses such as bluetongue virus (BTV), and further direct studies of AHSV are required in order to improve our understanding of this important virus.     

基于E184L基因的非洲猪瘟病毒实时荧光定量PCR检测方法的建立

非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)感染引起的一种高度接触性传染病。由于ASFV的感染机制极为复杂,基因型多,至今尚无有效疫苗用于防控,防止该病暴发主要依赖于早期快速诊断和控制。为建立一种高效快速、特异的ASFV检测方法,根据ASFV的E184L基因序列,设计了TaqMan荧光定量PCR引物及探针,建立了检测ASFV的TaqMan荧光定量PCR方法。结果表明,该方法设计的引物具有高度特异性,以构建的重组质粒为标准品建立的TaqMan荧光定量PCR方法的标准曲线具有良好的线性关系,线性相关系数为0.992,对ASFV核酸最低检测限为1.51拷贝,且与伪狂犬病病毒、猪细小病毒、猪圆环病毒2型等不存在交叉反应。建立的基于ASFV E184L基因实时荧光定量PCR检测方法能够快速、准确、特异地对ASFV核酸进行定量分析,丰富了ASFV的检测方法。     

Identifying equine premises at high risk of introduction of vector-borne diseases using geo-statistical and space-time analyses

The identification of premises that may play an important role in the introduction or spread of animal diseases is fundamental to the development of risk-based surveillance and control programs. A combination of geo-statistical and cluster analysis methods was used to identify geographical areas and periods of time at highest risk for introduction of the African horse sickness virus (AHSV) into the Castile and Leon (CyL) region of Spain. Risk was estimated based on the predicted premises-specific abundance of Culicoides spp., a vector for AHSV, and on the frequency of equine introductions from outside regions. The largest abundance of Culicoides spp. was observed between May and September in the northern region of CyL. Six significant (P-value <0.01) space-time clusters of equine premises were found, at which presence of Culicoides spp. was predicted and live equidae were introduced from outside CyL. The clusters included 37 equine premises and took place between April and December. These results will contribute to updating plans for prevention of AHSV introduction and spread in Spain. The methodological approach developed here may be adapted and applied to design and establish risk-based surveillance and control programs for Spain and other European countries.     

Production and properties of monoclonal antibodies against African horsesickness virus, serotype 3.

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.     

Re-emergence of bluetongue, African horse sickness, and other Orbivirus diseases

Arthropod-transmitted viruses (Arboviruses) are important causes of disease in humans and animals, and it is proposed that climate change will increase the distribution and severity of arboviral diseases. Orbiviruses are the cause of important and apparently emerging arboviral diseases of livestock, including bluetongue virus (BTV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and epizootic hemorrhagic disease virus (EHDV) that are all transmitted by haematophagous Culicoides insects. Recent changes in the global distribution and nature of BTV infection have been especially dramatic, with spread of multiple serotypes of the virus throughout extensive portions of Europe and invasion of the south-eastern USA with previously exotic virus serotypes. Although climate change has been incriminated in the emergence of BTV infection of ungulates, the precise role of anthropogenic factors and the like is less certain. Similarly, although there have been somewhat less dramatic recent alterations in the distribution of EHDV, AHSV, and EEV, it is not yet clear what the future holds in terms of these diseases, nor of other potentially important but poorly characterized Orbiviruses such as Peruvian horse sickness virus.     

Serological survey of African horse sickness in selected districts of Jimma zone,Southwestern Ethiopia

A cross-sectional serological survey was undertaken in selected districts of different agro-ecology of Jimma zone (Dedo, Yebu, Seka, Serbo, and Jimma town) from November 2009 to February 2010 to determine the seroprevalence of African horse sickness virus and associated risk factors of the disease. Two hundred seventy-four equids (189 horses, 43 mules, and 47 donkeys) with a history of non-vaccination for at least 2 years were selected randomly from the above areas. Sera samples were collected and assayed for the presence of specific antibody against African horse sickness virus using blocking ELISA. An overall seroprevalence of 89 (32.5%) was found and it was 24 (51.1%) for donkeys, 13 (30.2%) for mules, and 52(28.3%) for horses. Seroprevalence was significantly (X ² = 11.05, P < 0.05) different among the different species of equids. Seroprevalence was also significantly (X ² = 11.43, P < 0.05) different among the different agro-ecological areas being higher in highlands 47 (40.5%) followed by midland 30 (34.5%) and lowland 12 (16.9%). Age and sex were not significantly (X ² = 3.15, P > 0.05 and X ² = 3.38, P > 0.05, respectively) associated with seroprevalence of AHSV. The present study showed that African horse sickness (AHS) is highly prevalent disease for the horses followed by mules and then donkeys in Jimma zone explained by lower seroconversion rate. Therefore, control strategy against AHS should target at high risk species of all age and sex in their locality in the initial stage for better containment of the disease.