Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.     

Production and characterization of monoclonal antibodies against excretory-secretory products of Fasciola hepatica.

Four monoclonal antibodies (mAbs) (9.49, 24.27, 46.71 and 179.57) were produced against Fasciola hepatica excretory-secretory products. Isotype analysis revelead the antibodies to be IgM, IgG₃, IgG₁, and IgM. In immunoblot assays, the mAbs recognized different antigenic polypeptides migrating between 29 and 180 kDa. Specificity of the mAbs was evaluated by ELISA against antigens of Fascioloides magna, Anoplocephala magna, Stichorchis subtriquetrus, Haemonchus contortus, sheep liver extract (SLE), bovine liver extract (BLE), bovine serum albumin (BSA), bovine viral diarrhea virus (BVDV), and Madin-Darby bovine kidney (MDBK) cells. Monoclonals 9.49 and 24.27 were specific, and reacted only with Fasciola hepatica antigens. However, mAb 46.71 cross-reacted with antigens of Fascioloides magna, A. magna, Stichorchis subtriquetrus, and H. contortus but not with SLE, BLE, BSA, BVDV or MDBK cells. Monoclonal antibody 179.57 cross-reacted with Fascioloides magna, A. magna, S. subtriquetrus, H. contortus, SLE, and BLE, but not with BSA, BVDV, or MDBK cells.     

Mapping of two antigenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus

The immunodominant NS3 (p80) protein of the pestivirus bovine viral diarrhea virus (BVDV) functions as a serine protease and a RNA helicase. To identify antigenic domains of the BVDV NS3, a panel of monoclonal antibodies (mAbs) was tested against fragments of the protein expressed in E. coli. Two large overlapping NS3 fragments, A (amino acids [aa] 1-434) and B (aa 368-683) which together contain all NS3 sequences, were used to screen mAbs for reactivity. Two mAbs, 21.5.8 and 1.11.3, were reactive to fragment A (in ELISA only) and one mAb, 20.10.6, was reactive to fragment B (in ELISA and Western blotting). Further mapping demonstrated that the smallest fragment mAbs 21.5.8 and 1.11.3 bound to was comprised of aa 205-369 (domain A). In Western blotting, the smallest fragment reactive with mAb 20.10.6 was comprised of aa 368-549 (domain B). However, in indirect ELISA, mAb 20.10.6 also demonstrated high reactivity to a smaller fragment comprising aa 368-512 (domain B'). This indicated that the epitope of mAb 20.10.6 was conformational and not linear. Blocking ELISAs using these mAbs and type 1 and type 2 BVDV antisera demonstrated that an immunodominant region of the NS3 protein in cattle is defined by aa 205-549.     

Study of cattle persistently infected with bovine viral diarrhea virus that lack detectable virus in serum

OBJECTIVE: To determine whether cattle persistently infected with bovine viral diarrhea virus (BVDV) that lack virus detectable in serum by use of the immunoperoxidase microtiter assay (IPMA) can transmit the virus to susceptible herdmates and determine prevalence of these cattle. DESIGN: Clinical trial and serologic survey. SAMPLE POPULATION: 2 cattle and 1,952 blood samples. PROCEDURE: A persistently infected cow in which virus could not be detected in serum was housed with a BVDV-seronegative steer. Blood and nasal swab specimens were tested via virus isolation and serum virus neutralization. Parallel WBC preparations and sera from blood samples of 1,952 adult cows were screened for BVDV by use of IPMA. RESULTS: The steer seroconverted to BVDV within 4 weeks of contact with the cow. Virus was detected in sera and WBC of 5 adult cows that were verified as persistently infected by retest 3 weeks later. Cattle persistently infected with BVDV in which virus could not be detected in both serum and WBC by use of IPMA were not found. CONCLUSION AND CLINICAL RELEVANCE: Cattle persistently infected with BVDV in which virus cannot be detected in serum by use of IPMA may serve as virus reservoirs for infecting susceptible cattle. Persistent infection was detected at a prevalence of 0.26%. Screening adult cattle by use of IPMA on serum samples appears to be a reliable means of detecting persistent infection with BVDV. Prevalence of cattle persistently infected with BVDV that have negative results of IPMA on serum is extremely low.     

Production and characterization of monoclonal antibodies against excretory-secretory products of Fasciola hepatica

Four monoclonal antibodies (mAbs) (9.49, 24.27, 46.71 and 179.57) were produced against Fasciola hepatica excretory-secretory products. Isotype analysis revelead the antibodies to be IgM, IgG₃, IgG₁, and IgM. In immunoblot assays, the mAbs recognized different antigenic polypeptides migrating between 29 and 180 kDa. Specificity of the mAbs was evaluated by ELISA against antigens of Fascioloides magna, Anoplocephala magna, Stichorchis subtriquetrus, Haemonchus contortus, sheep liver extract (SLE), bovine liver extract (BLE), bovine serum albumin (BSA), bovine viral diarrhea virus (BVDV), and Madin-Darby bovine kidney (MDBK) cells. Monoclonals 9.49 and 24.27 were specific, and reacted only with Fasciola hepatica antigens. However, mAb 46.71 cross-reacted with antigens of Fascioloides magna, A. magna, Stichorchis subtriquetrus, and H. contortus but not with SLE, BLE, BSA, BVDV or MDBK cells. Monoclonal antibody 179.57 cross-reacted with Fascioloides magna, A. magna, S. subtriquetrus, H. contortus, SLE, and BLE, but not with BSA, BVDV, or MDBK cells.     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.     

Competitive binding with putative Bo5 (CD5) cluster of monoclonal antibodies.

The relationship of seven monoclonal antibodies, putatively to the Bo5 (CD5) antigen, was tested. Five of the mAbs were confirmed to be directed against the Bo5 antigen. Three mAbs, CC29, BLT-1 and 8C11, effectively blocked binding to bovine PBM of mAb CC17, previously reported to be directed against this antigen. MAb 8-3F4 also blocked binding of mAb CC17, but less effectively than the others. MAbs IL-A67 and 79-5 did not inhibit binding of mAb CC17 because of antibody allelic specificity or technical reasons.     

Protection against fetal infection with either bovine viral diarrhea virus type 1 or type 2 using a noncytopathic type 1 modified-live virus vaccine

Two bovine viral diarrhea virus (BVDV) fetal protection studies were done using a monovalent noncytopathic (NCP) BVDV vaccine containing type 1 BVDV. In study 1, thirty-two fetuses (23 vaccinates and nine controls) were recovered following fetal challenge with the type 1a BJ strain. Twenty of twenty-three fetuses from the vaccinates were negative for BVDV type 1 while all of the controls (nine of nine) were infected. In study 2, twenty-two animals (14 vaccinates and eight controls) were challenged with the type 2 PA131 strain. Thirteen of the fourteen fetuses from the vaccinates were negative for BVDV type 2 while all of the nonvaccinated controls (eight of eight) were infected. These results indicate the efficacy of a monovalent NCP BVDV vaccine in providing excellent protection against either BVDV type 1 or 2 fetal infection.     

抗CP型、NCP型牛病毒性腹泻病毒高免卵黄抗体的制备

本研究采用致细胞病变（cytopathic,CP）型牛病毒性腹泻病毒（bovine viral diarrhea virus,BVDV）标准毒和非致细胞病变（noncytopathic,NCP）型新疆优势毒株免疫产蛋鸡,用改良PEG法提取卵黄抗体(IgY),并对提取的IgY采用SDS-PAGE检测纯度,间接ELISA检测免疫后每隔7 d的抗体效价,并测定所得抗体对NCP型BVDV的中和效价。结果表明,用该法提取的IgY纯度较高;间接ELISA结果证明,经过4次免疫后,抗CP型BVDV的效价达到1∶32000,抗NCP型BVDV的效价达到1∶40000,3个月后再次检测,卵黄抗体效价未见明显下降。最后一次免疫14 d的抗体对NCP型BVDV的中和效价达到1×10_^-3。     

Monoclonal antibodies to bovine viral diarrhea virus: cross-reactivities to field isolates and hog cholera virus strains.

Monoclonal antibodies to bovine viral diarrhea virus (BVDV) were examined for binding with a large number of North American BVDV isolates and eight strains of the serologically related pestivirus, hog cholera virus (HCV). No single BVDV monoclonal antibody reacted with all BVDV isolates. The most cross-reactive monoclonal antibody was an anti-p80/p125 antibody which showed a positive reaction with 173 of 180 (96%) North American isolates. From a fewer number of isolates tested, one anti-gp53 monoclonal antibody also showed a high cross-reactivity (94%). All BVDV isolates showed a positive reaction with at least one of the seven monoclonal antibodies in the panel. Thus, the results indicated that a pool of these monoclonal antibodies may be used in place of polyclonal antisera for the detection of BVDV contamination of cell lines or for virus isolation. For HCV, all three anti-p80/p125 monoclonal antibodies reacted positively with all eight virus strains. In contrast, none of the anti-gp53 monoclonal antibodies were reactive to HCV strains. Thus, the anti-gp53 monoclonal antibodies may be useful for distinguishing between usually innocuous BVDV infections and the highly significant HCV infections in swine for foreign animal disease surveillance.     

Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis

Thirteen hybridoma cell lines which secrete monoclonal antibodies (MCAs) against swine fever virus (SFV) strain Brescia were produced. The hybrid cells resulted from fusion of P3X63-Ag8.653 myeloma cells with splenocytes of Balb/c mice which had been immunized with purified SFV. Screening of supernatant fluids was performed by an indirect enzyme-linked immunosorbent assay (ELISA) and an immunoperoxidase monolayer assay (IPMA). The IPMA, and an immunoperoxidase test (IPT) performed on cryostat sections, were used to characterize these MCAs on several pestivirus strains. All MCAs reacted to a varying degree with all but one of the SFV strains tested. None of the MCAs reacted with the bovine viral diarrhoea virus (BVDV) strains. Two MCAs are now used routinely in the differential diagnosis between infections with field strains of SFV, and the Chinese vaccine strain and BVDV strains.     

Response of cattle persistently infected with bovine virus diarrhoea virus to bovine leukosis virus

Six cattle persistently infected with bovine virus diarrhoea virus (BVDV) and seronegative, and two control, virus negative seropositive cattle were inoculated with lymphocytes infected with bovine leukosis virus (BLV). The two controls produced a normal immune response to BLV, developing antibodies at four and five weeks after inoculation. Two of the six cattle persistently infected with BVDV developed a strong antibody response by six weeks after inoculation with BLV. Four developed a depressed response to BLV, characterised in three by a 'hooking' reaction in the immunodiffusion test which persisted in successive bleedings but was interspersed occasionally by a weak positive reaction. In one of these animals, a series of 'hooking' reactions was followed by a number of negative results. The fourth animal remained serologically negative until 16 weeks after inoculation when a 'hooking' reaction was observed followed by a series of negative results. BLV was isolated from all the cattle persistently infected with BVDV at 42 or 58 weeks after inoculation regardless of whether the serum samples gave negative, 'hooking', weak positive or positive reactions in the immunodiffusion test. BLV was consistently isolated from the nasal secretions of a steer which was BVDV negative but seropositive. The possibility of decreased immune responsiveness to BLV in animals persistently infected with BVDV should be considered when formulating regulations governing the testing of animals for freedom from BLV.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Development and evaluation of a MAb based competitive-ELISA using helicase domain of NS3 protein for sero-diagnosis of bovine viral diarrhea in cattle and buffaloes

The aim of this study was to develop a competitive inhibition ELISA (CI-ELISA) for detection of antibodies to bovine viral diarrhea virus (BVDV) using the helicase domain of NS3 (non-structural) protein and monoclonal antibody (MAb) against it and to estimate its sensitivity and specificity using two commercial ELISA kits as independent references. The 45-kDa helicase domain of NS3 protein of BVDV was expressed in Escherichia coli and 18MAbs were developed against it. MAb-11G8 was selected for use in CI-ELISA on the basis of maximum inhibition (90%) obtained with BVDV type 1 infected calf serum. Based on the distribution of percent inhibition of known negative sera (n=166), a cut-off value was set at 40% inhibition. In testing 914 field serum samples of cattle (810) and buffaloes (104), the CI-ELISA showed a relative specificity of 95.75% and 97.38% and sensitivity of 96% and 94.43% with Ingenesa kit and Institut Pourquier kit, respectively. This study proved that the use of helicase domain of NS3 (45-kDa) is equally good as the whole NS3 protein (80-kDa) used in commercial kits for detection of BVDV antibodies in cattle and buffaloes.     

Seroprevalence of infectious bovine rhinotracheitis and bovine viral diarrhea virus type 1 and type 2 in non-vaccinated cattle herds in the Pacific Region of Central Costa Rica

The objectives of this cross-sectional study were to estimate the seroprevalence of infectious bovine rhinotracheitis (IBR, BHV-1) and bovine viral diarrhea virus (BVDV) in a population of non-vaccinated, double purpose, dairy and beef herds in the Pacific Region of Central Costa Rica. Blood samples were collected from a total of 496 animals from 35 herds. Sera were tested for antibodies against BHV-1(IBR) and BVDV types 1 and 2 using serum neutralization test. The average number of animals tested in each herd for each of the viruses was 14. Overall individual seroprevalence was 48%, 27%, and 19% for IBR, BVDV type 1, and BVDV type 2, respectively. Median within-herd seroprevalence for IBR, BVDV type 1 and type 2 were 43%, 27%, and 24%, respectively.     

Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.     

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.