An enzyme-linked immunosorbent assay for the detection of bovine antibodies to vesicular stomatitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibody to vesicular stomatitis virus (VSV). Serum samples from cows experimentally infected with the New Jersey serotype of VSV (VSV-NJ) were assayed by the ELISA and serum-neutralization (SN) assay. The ELISA was as sensitive as the SN assay in detecting bovine antibody to VSV. The correlation between SN titers and ELISA values at absorbance at 405 nm was statistically significant. The ELISA was not specific for VSV-NJ, however, and could detect serum samples positive to the Indiana serotype of VSV that had SN titers of greater than or equal to 480. Nonspecific reactions were due to cross-reactive group-specific viral proteins that are shared by both serotypes. The cross-reactivity allows the use of a single rapid test in identifying both serotypes of VSV from the other exotic vesicular diseases, especially foot-and-mouth disease. The ELISA titers of serum samples positive for VSV-NJ were comparable with the corresponding SN titers of each sample. The sensitivity, rapidity, and ease of the ELISA system and the use of a single test in identifying both serotypes of VSV from the other exotic vesicular diseases make this ELISA suitable as a rapid diagnostic assay for VS.     

TaqMan~T-PCR对水疱性口炎病毒的鉴定检测

水疱性口炎分为印第安那型 ( Indiana)和新泽西型 ( New Jersey)。这两种血清型病毒的快速和可靠的鉴别对该病的诊断、检疫、分子流行病学调查和监测至关重要。文章按照 VSV核蛋白基因序列 ,设计了一对两型通用引物和两型各自特异性探针。研究建立了 VSV实时荧光定量 PCR检测方法 ,对 VSV细胞培养物、人工感染实验动物组织、血清样品 ,以及系列稀释的不同 TCID50 样品、其它相关或相似病毒进行鉴定 ,同时与常规 PCR、病毒分离试验作了比较。Taq Man○R RT-PCR的特异性和敏感性相当于或优于对照方法。重复性和稳定性试验证实 ,该方法可靠。每个试验中设立阳性、阴性对照和标准稀释度对照 ,使试验结果可对病毒 RNA作准确定量 ,并可在 4h内获得结果。研究结果表明 ,Taq Man○R RT-PCR方法是一种特异性强、敏感性高、快速安全的定量检测方法。因此 ,可以作为 VSV的快速检测和定型。     

水疱性口炎研究进展

水疱性口炎(VS)是由水疱性口炎病毒(VSV)引起的人畜共患的重大动物疫病.VS病毒生态学复杂,易感动物较多,传播媒介种类广,病毒可在一定区域内长期存在.VSV主要呈嗜上皮性,大量流涎是家畜感染最重要的临床症状,其特征为口腔黏膜、乳房皮肤及蹄冠部皮肤出现水泡及糜烂,人感染后出现类似流感的症状.由于其临床症状与口蹄疫不易区别,发病时容易引起国际贸易恐慌,因此,对VS诊断防治的研究有着重要的社会经济和公共卫生意义.文章从分子生物学特征、流行病学、诊断和防控4个方面对水疱性口炎的研究进行了综述.     

Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for na?ve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificity.     

猪水疱性口炎病毒基因及其疫苗的研究进展

猪水疱性口炎是由水疱性口炎病毒引起的高度接触传染性的病毒性疾病。其临床特征为猪的唇部,鼻及口腔等处发生水疱,并从口中不断向外流涎,有时常常还发生在蹄冠和趾间皮肤上,其症状主要以水疱为主。该病在全球许多地区造成广泛流行。近年来,由于产品贸易量的增加,猪水疱性口炎病毒也陆续的传入我国。由于该病与猪水疱病、猪口蹄疫和猪水疱性疹等病毒性疾病容易混淆,因此对该病做出准确地诊断与防制显得尤为重要。在VSV疫苗的研究方面,主要是灭活疫苗和弱毒疫苗的研究,而在新型疫苗的研究方面很少。本文主要综述了猪水疱性口炎病毒的基因及其疫苗的研究进展,为进一步了解和预防该病提供参考依据。     

多重RT-PCR检测猪口蹄疫病毒、猪水泡病病毒和猪水疱性口炎病毒的研究

建立了一种同时检测猪口蹄疫病毒(FMDV)、猪水泡病病毒(SVDV)和猪水疱性口炎病毒(VSV)三种病原体的多重RT-PCR方法。参照文献报道的基因序列,设计合成了三对特异性引物;PCR扩增条件进行优化后,用这三对引物对同一样品中的FMDV、SVDV、VSVRNA模板进行扩增,结果同时得到了三条特异性条带,大小与试验设计相符:FMDV(208bp)、SVDV(862bp)、VSV(638bp),且对猪瘟病毒(CSFV)、猪繁殖与呼吸综合症病毒(PRRSV)和猪传染性胃肠炎病毒(TGEV)核酸扩增结果为阴性;三种病毒RNA模板检出的最小量均为10fg。试验证明,此方法经济、快速、敏感、特异,可用于FMDV、SVDV和VSV这三种猪水泡性疾病的鉴别诊断及流行病学调查。     

水疱性口炎病毒RT-LAMP快速检测方法的研究

根据逆转录环介导等温扩增(RT-LAMP)原理,针对水疱性口炎病毒(VSV)糖蛋白G基因序列中的6个区域设计内外各1对特异引物,建立扩增VSV糖蛋白(G)基因的RT-LAMP方法。扩增产物电泳呈特异的阶梯状条带分布,扩增产物加SYBR GREEN I染色呈特征性的黄绿色,肉眼可直接观察判定。同时,还建立了可以进行定量检测的实时RT-LAMP方法。特异性试验显示,本方法可快速检验鉴别VSV与口蹄疫病毒(FMDV)和猪水泡病病毒(SVDV)。敏感性试验显示,建立的实时RT-LAMP方法检测VSVRNA的最低检测量为0.01 PFU,比实时荧光RT-PCR显著提高。建立的LAMP方法可检测p-VSVNJ质粒DNA的最低量为6.36×10-3pg/μL(1.4×103copies/μL),比PCR也显著提高。综合表明,本研究建立RT-LAMP检测VSV的方法具有特异、敏感、快速、简便的特点,具有开发应用前景。     

O、A、Asia 1型口蹄疫病毒胶体金定量检测免疫层析方法的建立

旨在建立口蹄疫病毒(foot-and-mouth disease virus, FMDV)抗原血清型的快速分型和定量的检测方法,利用双抗体夹心法,将口蹄疫病毒的兔抗及豚鼠抗体作为标记胶体金与NC膜检测带的原料,分别制备出检测O、A、Asia 1血清型的3种层析试纸卡。通过对标定的抗原标准品146S检测,拟合出定量标准曲线。免疫层析方法的质量验证通过特异性、敏感性、重复性及与蔗糖密度梯度法(sucrose density gradient, SDG)的相关性进行评价。结果显示:建立的快速定量检测方法,3种血清型口蹄疫病毒间无交叉反应,同时与其他非口蹄疫病毒,如塞内卡病毒A型(SVA)、猪水疱病病毒(SVDV)、猪水疱性口炎病毒(VSV)无非特异反应;敏感性研究,对O、A、Asia 1型病毒的最低检出量分别为0.567、0.693、0.219μg·mL^-1,拟合的3条标准曲线的线性相关系数R~2>0.97,新建立方法与蔗糖密度梯度法检测结果的相关系数均>0.9, 3种层析试纸卡的变异系数均小于10%。综上表明,建立的口蹄疫O、A、Asia1型病毒胶体金免疫层析定量检测试纸卡方法,可以用于口蹄疫病毒抗原快速鉴别、血清分型与146S定量检测。     

A prospective study of vesicular stomatitis in cattle in an enzootic region of Mexico

A prospective study of vesicular stomatitis was conducted in two bovine herds in southeastern Mexico. In July 1987, an initial serological screening showed that 64% and 87% of the 654 cattle tested negatively to vesicular stomatitis New Jersey and Indiana antibodies, respectively, using the plaque-reduction serum neutralization test. Most seropositive animals were at least 24 months of age. Based on the initial serological screening, cohorts of seronegative and seropositive cattle were monitered (January–December 1988) for the prevalence of vesicular stomatitis virus (VSV) antibodies using an enzyme-linked immunosorbent assay (ELISA). The serological results, using ELISA, indicated that no VSV activity occured in the two study herds. The seronegative cohort of cattle did not yield a positive seroconversion pattern to either VSV Indiana or New Jersey. The seropositive cohort showed a variable antibody response pattern against the VSV. There were no clinical cases of vesicular stomatitis (VS) in the two herds. The data from the national surveillance program for vesicular diseases suggested that 1988 was a year of low VSV infection incidence in southeastern Mexico.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

Immunoelectron microscopic comparisons of caliciviruses

Using immunoelectron microscopy, 9 serotypes of vesicular exanthema of swine virus (VESV) were compared with 5 serotypes of San Miguel sea lion virus and 7 additional calicivirus isolates from marine animals. In addition, swine caliciviruses and marine caliciviruses were compared with the vaccinal strain of feline calicivirus (FCV) F-9. Of 9 VESV types, 8 showed common antigenicity with San Miguel sea lion virus. Of 9 VESV types, 2 showed common antigenicity with FCV F-9. All 12 marine caliciviruses showed common antigenicity with VESV, but not with FCV F-9.     

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.     

Vesicular stomatitis.

Vesicular stomatitis is an infrequent yet important vesicular disease of cattle, horses, and swine. Periodic outbreaks of this disease in the United States have caused economic losses in cattle herds because of decreased production, movement restrictions, and trade embargoes. Vesicular stomatitis causes clinical signs indistinguishable from those of foot-and-mouth disease. It is of utmost importance that appropriate samples are collected from clinical cases of vesicular disease in cattle and swine so a rapid laboratory diagnosis can be made.     

应用RT-PCR方法快速检测水泡性口炎病毒

针对水泡性口炎病毒(VSV)的两种血清型设计了2对引物，建立RT-PCR方法，用于检测VSV。VSV接种细胞出现明显的细胞病变，经RT-PCR检测为阳性，而检测口蹄疫、猪水泡病均为阴性，说明引物具有较好的特异性。     

Serologic survey of viral antibodies in the Peruvian alpaca (Lama pacos)

Sera from more than 100 alpacas (Lama pacos) from the Peruvian southern sierra were examined for antibodies to 8 viruses known to infect other domestic animals. On the basis of these serologic findings and previously published serologic or clinical data, it is now known that the alpaca can be infected with the following viruses: parainfluenza-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bluetongue virus, border disease virus, influenza A virus, rotavirus, rabies virus, vesicular stomatitis virus, foot-and-mouth disease virus, and contagious ecthyma virus.     

Oral vesicular lesions in horses without evidence of vesicular stomatitis virus infection

OBJECTIVE: To report clinical and serologic findings in horses with oral vesicular lesions that were consistent with vesicular stomatitis (VS) but apparently were not associated with VS virus (VSV) infection. DESIGN: Serial case study. ANIMALS: 8 horses. PROCEDURE: Horses were quarantined after appearance of oral lesions typical of VS. Severity of clinical signs was scored every 2 to 5 days for 3 months. Serum samples were tested for antibodies by use of competitive ELISA (cELISA), capture ELISA for IgM, serum neutralization, and complement fixation (CF). Virus isolation was attempted from swab specimens of active lesions. RESULTS: 2 horses with oral vesicular lesions on day 1 had antibodies (cELISA and CF) against VSV; however, results of CF were negative by day 19. Five of the 6 remaining horses were seronegative but developed oral lesions by day 23. Virus isolation was unsuccessful for all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses were quarantined for 75 days in compliance with state and federal regulations. However, evidence suggests that oral lesions were apparently not associated with VSV infection. The occurrence in livestock of a vesicular disease that is not caused by VSV could confound efforts to improve control of VS in the United States and could impact foreign trade. Vesicular stomatitis is of substantial economic and regulatory concern.     

San Miguel sea lion virus fed to mink and pigs.

Mink became infected with San Miguel sea lion virus when fed ground meat from seal carcasses showing vesicular-like lesions in the skin. The mink also contracted the infection when they were fed San Miguel sea lion virus infected pig meat or cell culture propagated virus. San Miguel sea lion virus infection in mink was inapparent but the virus was isolated from blood and rectal swabs. Pigs treated similarly with the same virus preparations given to mink developed a severe vesicular disease syndrome similar to that produced by vesicular exanthema of swine. In a separate trial, pigs fed a large sample of commercial ground seal meat did not develop disease signs or antibodies. Further work is needed to assess the hazard of introducing San Miguel sea lion virus into swine on the same premises when potentially San Miguel sea lion virus infective seal meat is fed to mink.     

水泡性口炎病毒双抗体夹心ELISA检测方法的建立

为建立方便快捷的水泡性口炎病毒(VSV)检测方法,本研究以抗VSV单克隆抗体(MAb)为捕获抗体,兔抗VSV多克隆抗体为检测抗体,建立VSV双抗体夹心ELISA检测方法。结果显示,该方法的最佳工作条件为:抗VSV MAb 1A2的包被浓度为3.09μg/mL,兔抗VSV多克隆抗体和酶标抗体的工作浓度分别为5.16μg/mL和1∶5 000,以OD450nm≥0.231作为阳性判定标准。该ELISA方法对猪水泡病病毒、猪水疱疹病毒及羊传染性脓疱病毒等均无交叉反应;敏感度可达3.125μg/mL(101TCID50);其重复性变异系数小于10%。采用建立的ELISA方法与RT-PCR方法同时检测187份临床样品,符合率达到97.9%,具有良好的相关性。本实验建立的VSV双抗体夹心ELISA检测方法具有特异性好、敏感性高、成本低及方便快捷等优点,可以用于VSV的快速检测。