Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

The isolation and preliminary characterization of a rhabdovirus in Australia related to bovine ephemeral fever virus

CSIRO 368 virus was isolated from blood collected in the Northern Territory from a healthy cow and electron microscope studies showed that the isolate had rhabdovirus morphology. Fluorescent antibody studies and complement fixation tests related the virus to bovine ephemeral fever (BEF) virus. Neutralization tests in both suckling mice and Vero cells showed that the virus was not BEF virus. Antibodies to CSIRO 368 virus were found in cattle sera from northern and eastern Australia and Papua New Guinea. Antibodies were found in 16 out of 45 buffalo, some of which also had antibodies to BEF virus. In contrast, none of the 419 deer tested had antibodies to CSIRO 368 virus, although 142 of the same deer had antibodies to BEF virus. No antibodies to CSIRO 368 virus were detected in 16 goats, 54 horses, 10 pigs, 31 sheep, 25 kangaroos, or 14 human beings. Both CSIRO 368 and BEF viruses were found to be sensitive to ether and chloroform, but were not affected by the DNA inhibitor 5-bromo-2'-deoxyuridine, showing that they probably had the same type of nucleic acid--namely RNA. CSIRO 368 was also shown to grow to higher titres in BHK21 cells than in Vero cells. Temperature sensitivity studies at -20, 4 and 37 degrees C showed that the presence of foetal calf serum increased the survival time markedly at -20 degrees C, but only slightly at 4 and 37 degrees C. The virus survived the longest at -20 degrees C in the presence of foetal calf serum.     

Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea

Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.     

An experimental inactivated virus vaccine against bovine ephemeral fever 2. Do neutralizing antibodies protect against infection?

In order to develop a safe vaccine against bovine ephemeral fever (BEF) which could be used in areas normally free of the disease, studies were carried out on inactivated virus vaccines. Initial experiments were carried out in cattle using virus vaccines that had been inactivated with β-propiolactone or formalin and then made-up in aluminium phosphate gel or Freund's incomplete adjuvant. A minimum inactivated virus dose of 10⁶ PFU was necessary to stimulate a serum neutralizing antibody response in cattle. β-propiolactone inactivated BEF virus vaccines in Freund's incomplete adjuvant gave the best serum neutralizing antibody responses, producing high levels of neutralizing antibody with both high and low passage level virus. However, the magnitude of the antibody response bore little relationship to resistance of vaccinated animals to challenge with virulent BEF virus. A number of animals with high neutralizing antibody titres to BEF virus did not resist challenge. Using 500-fold less live virus at equivalent passage level to the low passage inactivated vaccine, similar or slightly lower antibody levels were attained, but most of the animals resisted challenge. It is suggested that the nature of the immune response and resistance to BEF infection may be complex and that reliance on serum neutralizing antibody as an indicator of resistance may give misleading results.     

Transmission of virus from serosal fluids and demonstration of antigen in neutrophils and mesothelial cells of cattle infected with bovine ephemeral fever virus

Following intravenous injection of bovine ephemeral fever (BEF) virus 6 cattle were autopsied after clinical disease became evident. Fluid from serosal cavities with serofibrinous inflammatory changes showed large increases in neutrophil numbers. BEF virus was detected for the first time in pericardial, thoracic and abdominal fluids. Virus was also detected in synovial fluids, confirming an earlier report of transmission with a synovial fluid sample. Using a direct fluorescent antibody technique, BEF virus antigen was identified for the first time in synovial, pericardial, thoracic and abdominal fluids, in synovial membranes and epicardium. In synovial membranes and epicardium, specific fluorescence was observed in two cell types, mesothelial cells and neutrophils. In the fluids, fluorescence was restricted to neutrophils, the predominant cell type. Specific fluorescence was observed in blood smears from only one animal although blood samples collected at autopsy from all animals contained infective virus.     

Comparative studies on the growth of Australian bluetongue virus serotypes in continuous cell lines and embryonated chicken eggs

The replication of cell culture passaged Australian bluetongue virus (BTV) isolates, serotypes 20 (CSIRO19) and 1 (CSIRO156), and an untyped BTV (CSIRO154) was assessed in eight continuous cell lines (one derived from baby hamster kidney cells, BHK-21; three derived from monkey kidney cells, Vero, LLC-MK2 and CV-1P; a foetal ovine lung and a mouse fibroblast cell line, CSL503 and L929, respectively, a Super-Vero-Porcine stable cell line, SVP; and a mosquito cell line, Aedes albopictus cells) and in 11-day-old embryonated chicken eggs (ECE) at different multiplicities of infection. All three viruses replicated in the cell lines tested, maximum extracellular virus yields being attained from BHK-21 cells at high multiplicities of infection (approximately 10 PFU per cell). Also BHK-21 cells produced much higher yields of virus than the other cell lines tested when low multiplicities of infection were used (approximately 10(-4) PFU per cell). All BTV serotypes multiplied in Singh's Aedes albopictus cells with no cytopathogenic effects over the 4 day period tested. The viruses also replicated in 11-day-old ECE; however, the sensitivity of ECE for growth of the Australian serotypes was not as high as has been reported for BTV isolates in other countries. In all cell culture systems and in ECE, BTV1 and BTV20 replicated more efficiently than did CSIRO154 virus.     

牛传染性鼻气管炎病毒LUXTM新型荧光PCR检测方法的建立

本研究针对牛传染性鼻气管炎病毒（Infectious bovine rhinotracheitis virus，IBRV）高度保守的gC基因设计单标记并具有自身荧光淬灭功能的LUX^TM引物，建立L刚新型实时荧光PCR方法用于快速检测IBRV。该方法对四株IBRV细胞培养物的检测均呈典型阳性反应，而对其它动物疱疹病毒以及健康牛组织DNA和细胞对照的检测结果为阴性，检测时间包括核酸提取仅需1h～2h。试验表明，LUX^TM荧光PCR法对IBRV细胞增殖病毒液的检测敏感性可达0．04TCID50，比病毒分离敏感性至少提高10倍；对10倍系列稀释的纯化IBRV核酸样品，L刚荧光PCR的检测敏感性比常规PCR可提高10^3倍。将病毒液添加到健康牛精液和血液样品中，该荧光PCR可检测到牛冻存精液中40TCID50牛抗凝全血、血清和临床精液中0．04TCID50的病毒，说明对临床样品的检测有效。本研究所建立的LUXTM荧光PCR方法快速敏感，适合应用于活牛及其遗传物质的进出口检疫、养牛业疾病防控等领域对IBRV的快速检测。     

An investigation into causes of resistance of a cloned line of BHK cells to a strain of foot-and-mouth disease virus

The reduced ability of foot-and-mouth disease virus (FMDV) strain Asia 1 Iran 1/73 to replicate in the cloned BHK cell line AA7 was not due to lack of virus attachment at the cell surface. Instead, the main restriction in the viral growth cycle occurred during synthesis and processing of viral macromolecules, and/or during the earliest stages of their assembly. Reduced efficiency of penetration and uncoating of virus attached to the cells may also have contributed to inhibition of virus replication. Viral components or subviral particles did not accumulate and defective interfering particles were not detected. The reduced number of infective virions produced was released from infected cells at the normal rate. No interferon production could be demonstrated.     

Fluorogenic RT-PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus

A single tube fluorogenic RT-PCR-based 'TaqMan' assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan-PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5' untranslated region (5' UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT-PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10-100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT-PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values (C(T)) versus copy number. Single tube TaqMan-PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.     

Homologous and heterologous antibody reactions in sera from cattle naturally infected with bovine ephemeral fever group viruses

Four viruses belonging to the bovine ephemeral fever (BEF) group have been isolated from bovine blood. Infection of cattle with BEF virus was associated with neutralizing antibody responses to BEF, Kimberley (KIM), Berrimah (BRH) and Adelaide River (ADE) viruses, with highest antibody titres to BEF and KIM viruses. Infection of one cow with KIM virus was associated with a homologous neutralizing antibody response and nil or minimal responses to the other three viruses. Infection of a steer with ADE virus was associated with a rise in neutralizing antibody levels to ADE virus and to KIM virus, but not to BEF or BRH viruses. Infection of a steer with BRH virus was associated with marked neutralizing antibody rises to BRH and BEF viruses and small rises to KIM and ADE viruses. An antibody rise to BEF virus did not necessarily indicate recent BEF virus infection, and should be considered of diagnostic value only when taken in conjunction with clinical signs of disease.     

利用RNAi抑制口蹄疫病毒的复制

根据口蹄疫病毒 IRES和 L 串联序列两侧的保守区域设计了 2个引物 ,利用 RT- PCR和 PCR方法扩增出该串联序列 ,并进行了测序。测序结果表明 ,扩增产物与 Gen Bank上相应的序列具有很高的序列同源性 (大于 99% )。在测序的基础上 ,选择了 L 基因上的 1个靶位点 (位于启始密码子下游第 2 2 9nt后 2 1 nt长的序列 ) ,合成了 si RNA表达盒SEC- L2 2 9。细胞单层长成 5 0 %～ 70 %时 ,将纯化的 SEC- L2 2 9转染到 BHK细胞中 ,转染 4 h后用高感染复数 FMDV接种 ,2 4 h后用间接免疫荧光方法对口蹄疫病毒在 BHK细胞中的复制进行检测。研究结果表明 ,SEC- L 2 2 9极大地抑制了口蹄疫病毒在 BHK细胞中的复制 ,且该抑制作用具有序列特异性 ,并降低了 BHK细胞的死亡率。另外 ,2 5 ng和5 0 ng SEC- L2 2 9处理组间对病毒复制的抑制作用差异不明显 ,可能是病毒基因组发生了突变。本试验表明 ,利用 PCR方法合成的 SEC在 BHK细胞中能特异性地抑制 FMDV的复制 ,RNAi技术可能为防治口蹄疫提供一个新的途径     

Retrospective epidemiological investigation of an outbreak of bovine ephemeral fever in 1991 affecting dairy cattle herds on the Mediterranean coastal plain

From August to October 1991 bovine ephemeral fever (BEF) occurred sporadically in two localities in Israel. The morbidity and mortality rates reached 2.6% and 0.1%, respectively. Only 12/50 dairy cattle herds were clinically infected with BEF in the dairy community. The total morbidity rate reached 0.8%. The lowest morbidity rate was recorded in young heifers (5.5%) and the highest in adult cows (75%). Only heifers over the age of three months were clinically affected. The spread of the disease apparently followed the local prevailing night winds, which blow from east to west, i.e., from the land toward the sea. The morbidity period lasted 61 days. The low incidence and morbidity rates were possibly due to the low virulence of the virus strain involved in the 1991 epidemic. Retrospective analysis indicates that vectors - apparently mosquitoes - infected with BEF virus could have been overwintering.     

The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.     

Testing the susceptibility of cultured cells to infection with bovine leukemia virus

Different cell cultures were studied for their susceptibility to bovine leucosis virus infection. Syncytial assay was used for this study. The FLS/BLV+ cell line served as virus source. Cell lines BHK-21 and ZP-1/58 were found to be susceptible to syncytium formation. Large cells with one to three large nuclei, and loose nuclei reaching the size of syncytium were observed to occur in the BHK-21 and ZP-1/58 cell lines, apart from the syncytial formations. The virus specificity of the syncytia arising in these two cell lines was confirmed by the immunofluorescence assay. In the case of the immunoperoxidase assay, a positive result was obtained only in the BHK-21 cell line. The occurrence of syncytia and large nuclei was observed even in the cases when the BHK-21 cells were infected with the lymphocytes of leucotic cows.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine virus diarrhea virus in sera from border disease virus-infected sheep.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific antibodies against the causative agent of border disease in ovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified bovine virus diarrhea virus was used as test antigen. The optimal amount of antigen was 0.5 microgram/well, and the optimal concentration of conjugate was at 1/4,000 dilution. A total of 20 ovine serum samples, which had been collected from animals with or without border disease, were compared by ELISA and serum neutralization test for the detection of border disease-specific antibodies. ELISA was shown to be equally specific but less time-consuming and easier to perform than serum neutralization test. A positive correlation (r = 0.60) between the two tests was found.     

THE SUSCEPTIBILITY OF YOUNG AND NEWBORN CALVES TO BOVINE EPHEMERAL FEVER VIRUS

The pathogenicity of a field strain, 417, of bovine ephemeral fever (BEF) virus for newborn and young calves was investigated. Three colostrum-deprived newborn calves inoculated intravenously developed severe clinical disease and viraemia, and produced long-lasting neutralising antibody. The incubation period in these animals was 10 and 11 days, compared with 5 to 7 days for older calves. Two newborn calves which received colostrum from immune dams and 2 which received colostrum from non-immune dams failed to respond clinically to intravenous inoculation with strain 417. The neutralising antibody response of these calves was of short duration. Four calves, 7 to 8 weeks old and lacking detectable neutralising antibody to BEF virus, or having low levels of antibody, did not develop clinical disease when inoculated intravenously. Four calves 12 to 14 weeks of age and free of detectable neutralising antibody to BEF virus developed clinical disease when inoculated with strain 417.     

Evaluation of the critical parameters of a sensitive ELISA test using purified infectious bovine rhinotracheitis virus antigens

An enzyme-linked immunosorbent assay (ELISA) for the survey or titration of bovine sera for the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR) virus was developed. The optimal conditions of serum dilution, antigen concentration, conjugate dilution, substrate concentrations, and reaction time were established using the signal/ noise (S/N) ratio as the determining criterion. Equilibrium density gradient purified IBR virus was used as antigen at an optimal concentration of 0.60 μg/cuvette. The use of purified antigen allowed the testing of sera at a 1 : 10 dilution without nonspecific reaction.The conditions of conjugate dilution, substrate concentration and reaction time were shown to have significant effects on the ELISA test. Results from 35 sera showed this optimized ELISA procedure to be as much as 1000-fold more sensitive than the serum neutralization plaque reduction assay. Numerous sera showing no neutralizing titer to IBR virus were found to be positive when examined by this ELISA method.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

Production and characterization of monoclonal antibodies to psittacine beak and feather disease virus.

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.     

Interactions of porcine alveolar macrophages and bone marrow cells with African swine fever virus and virus-infected cells

Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC.Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.