Development and evaluation of an enzyme-linked immunosorbent assay for detection, typing, and subtyping of vesicular stomatitis virus.

An indirect sandwich enzyme-linked immunosorbent assay (ELISA) has been used for vesicular stomatitis virus (VSV) typing using sets of monovalent and polyvalent rabbit/guinea pig antisera for identification of VSV types New Jersey (VNJ) and Indiana (VIND). The VIND polyvalent antiserum (VIND-P) detects any strain of the 3 subtypes of the VIND type (VIND-1, VIND-2, and VIND-3) with the same strong reactivity. It is also possible to subtype the VIND strains using VIND-P rabbit antiserum as capture antibody and monovalent VIND-1, VIND-2, or VIND-3 guinea pig antisera as detector. The ELISA proposed has about 10 times more sensitivity and provides 10% more positive results than does the complement fixation 50% (CF50) test when epithelial samples are tested.     

Comparison of the serum neutralization test and a competitive enzyme-linked immunosorbent assay for the detection of antibodies to vesicular stomatitis virus New Jersey and vesicular stomatitis virus Indiana.

A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587-0.9155) and 0.6912 (95% CI: 0.6246-0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.     

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.     

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

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to vesicular stomatitis virus in cattle in an enzootic region of Mexico.

An ELISA was compared with the plaque-reduction serum neutralization (PRSN) test, for detection of vesicular stomatitis virus (VSV) antibodies in cattle in a vesicular stomatitis enzootic region of Mexico. A total of 325 bovine serum samples were screened for VSV antibodies. The PRSN test was performed, using Vero cells. The ELISA contained gradient-purified VSV Indiana (Lab strain) and VSV New Jersey (Hazelhurst) as the antigens. Regression analysis and weighted kappa statistic were used to estimate measures of agreement between the 2 assays for detection of VSV antibodies. The ELISA method proved useful for serodiagnosis of vesicular stomatitis. The ELISA and PRSN test results were highly correlated for detection of VSV antibodies.     

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。     

Neutralizing antibodies against vesicular stomatitis viruses (serotypes New Jersey and Indiana) in horses in Costa Rica.

Serum samples were collected from domestic horses in 4 different regions of Costa Rica to detect antibodies against vesicular stomatitis viruses, serotypes New Jersey (VSV-NJ) and Indiana (VSV-IN). A total of 214 samples were tested by the virus neutralization test. The sampling regions were identified as low North Pacific dry area (1), low Middle Atlantic humid area (2), low South Pacific humid area (3), and the highlands (4). In region 1, 97.1% of horses were positive for VSV-NJ and 16.5% were positive for VSV-IN. The mean antibody titer and its standard deviation after logarithmic transformation were 5.86 +/- 0.9 for VSV-NJ and 3.55 +/- 1.66 for VSV-IN for region 1. In region 2, 40.7% of horses were positive for VSV-NJ and 32.2% were positive for VSV-IN. The mean antibody titer in region 2 was 4.33 +/- 1.82 for VSV-NJ and 3.47 +/- 1.73 for VSV-IN. In region 3, 20.79% of horses were positive for VSV-NJ and 27.6% were positive for VSV-IN. The mean antibody titer in region 3 was 4.39 +/- 1.89 for VSV-NJ and 3.47 +/- 1.82 for VSV-IN. In region 4, 91.3% of horses were positive for VSV-NJ and 73.9% were positive for VSV-IN. The mean antibody titer in region 4 was 5.77 +/- 1.10 for VSV-NJ and 4.85 +/- 1.63 for VSV-IN. This is the first published report of the detection of virus-neutralizing antibodies against VSV-NJ and VSV-IN in horses in Costa Rica.     

Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies.

A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated greater than 99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antigen

An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for the laboratory diagnosis of vesicular stomatitis (VS). The assay which uses rabbit and guinea-pig antisera to the purified glycoprotein antigens of Serotypes New Jersey (VSV-NJ) and Indiana (VSV-IND-1), has both high sensitivity and specificity, but has lower reactivity for the two other Indiana subtypes VSV-IND-2 and VSV-IND-3.     

茨城病的琼脂免疫扩散试验诊断

茨城病 ( Ibaraki disease ID)又称类蓝舌病 ,是由茨城病病毒引起的虫媒性传染病 ,该病毒属呼肠孤病毒科 ( Reovividae)环状病毒属 ( Orbivirus)的成员。其特征为突发高热、精神沉郁、口鼻黏膜溃疡、吞咽困难、蹄冠肿胀。该病曾在日本多次流行 ,当时误认为流行性感冒。1 961年 Omori首先从茨城的病牛中分离到病毒 ,并命名为茨城病毒。在 1 991年的国际病毒分类委员会会议上才予以确认。该病在日本的南九州地区、关东地区的茨城县、爱知县和歧阜县等地多次发生。除日本外尚有韩国、澳大利亚、加拿大、印度尼西亚和菲律宾等国有发生本病的…     

口蹄疫病毒和水泡性口炎病毒二重RT-LAMP鉴别检测方法的建立

LAMP技术是一种快速新型的核酸检测技术,该技术利用4条引物在恒温下扩增目的 DNA,特异性好敏感性高。本试验旨在建立一种能同时鉴别诊断FMDV和VSV的二重RT-LAMP检测方法。根据口蹄疫病毒(FMDV)3D基因和水泡性口炎病毒(VSV)N基因的保守序列,设计了2套特异性引物,在每套引物的内引物中插入酶切位置EcoRⅠ,对反应条件进行了优化,建立了恒温快速的检测方法。结果显示:该方法特异性好,能检测到口蹄疫病毒的A,O,Asia1亚型和水泡性口炎的NJ和IND亚型,并与其他对照牛病原体不发生交叉反应;敏感性高,最低能够检测个100个FMDV病毒RNA和100个VSV病毒RNA;干扰性小,能同时检测两个模板的不同浓度组合。本试验建立的口蹄疫和水泡性口炎二重RT-LAMP方法具有简便、快速、特异、敏感等优点,可用于FMDV和VSV的临床检测和流行病学调查。