Differentiation of two bovine lentiviruses by a monoclonal antibody on the basis of epitope specificity.

Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

Cloning of the bovine immunodeficiency virus gag gene and development of a recombinant-protein-based enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.

Semiliki forest virus vector carrying the bovine viral diarrhea virus NS3 (p80) cDNA induced immune responses in mice and expressed BVDV protein in mammalian cells.

Bovine viral diarrhea virus (BVDV) is a primary pathogen responsible for bovine enteric, respiratory and reproductive failure. A genetic region is encoding the p80 (NS3) of BVDV as the most conserved protein among Pestiviruses. BVDV infection in cattle induces NS3 specific lymphocyte proliferation and humoral responses. To generate a DNA vaccine against BVDV, the gene for BVDV-NADL NS3 was cloned into an eukaryotic expression vector of Semiliki Forest virus (pSFV-1). Quadriceps muscles of BALB/c mice were injected with recombinant DNA generated statistically significant cytotoxic T-lymphocyte activity (CTL) and cell mediated immune (CMI) responses against cytopathic and noncytopathic BVDV. Whereas, the BVDV-NS3 did not generate neutralizing antibodies against BVDVin mice. pSFV-1-NS3 DNA was subjected to in vitro transcription into mRNA. The mRNA was transfected into baby hamster kidney cells (BHK-21) and Madin-Darby bovine kidney cells (MDBK). The recombinant cells were used in the detection of DNA antigen responses by immunological assays. This report establishes the ability of BVDV-NS3 DNA inoculation to induce a strong cellular immune responses in mice.

Evaluation of baculovirus-expressed bovine herpesvirus-1 (BHV-1) glycoproteins for detection and analysis of BHV-1-specific antibody responses.

Baculovirus (Autographa californica nuclear polyhedrosis)-expressed bovine herpesvirus-1 (BHV-1) glycoproteins B (gB), gC, and gD were developed and characterized. The recombinant proteins retained their antigenic properties as determined by immunoblotting against monoclonal antibodies. The proteins were examined as antigens for detection of BHV-1 infection and for the analysis of antibody responses to the individual viral proteins. A total of 115 bovine serum samples were tested for their reactivity with individual recombinant proteins from baculovirus-infected Spodoptera frugiperda (SF-9) cell lysates by enzyme-linked immunosorbent assay (ELISA), western blotting, and dot blotting assays. These serum samples were previously tested for BHV-1-specific antibodies by virus neutralization (VN) at the veterinary diagnostic laboratory. All 90 serum samples tested positive with VN were positive by ELISA against gC and gD, separately. However, reactivities of sera against gB were generally low and inconsistent. On the other hand, out of 25 sera that were negative with VN, 22 sera were consistent and gave negative results against gC or gD by ELISA, whereas reactivities with gB were inconsistent. Similar results were obtained when the sera were tested by western blotting and dot blotting. The positive sera consistently reacted strongly against gC and to a lesser extent gD. These results suggest that baculovirus expressed gC from infected cell lysate can be used as a potential diagnostic antigen for detection of anti-gC-specific antibody responses in BHV-1 infected cattle.

Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demonstrating the strong expression of bovine viral diarrhea virus receptor in the tissues. Weak staining was found in cerebellum, thymus, spleen, liver, cerebrum, and lung tissues; however, heart tissues were negative. Immunoblotting results correlated with the immunoperoxidase staining assays. Thus, the expression levels of the receptor are variable in different tissues. This pattern of expression may provide clues to the pathogenic potential of bovine viral diarrhea virus in the bovine fetus.

The effect of different bovine viral diarrhea virus genotypes and biotypes on the metabolic activity and activation status of bovine peripheral blood mononuclear cells.

The effects of cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) on the cellular metabolic activity and activation status of bovine peripheral blood mononuclear cells (PBMC) were investigated. Cellular DNA and protein synthesis was determined by [3H]thymidine and [3H]valine incorporation, respectively, in phytohemagglutinin (PHA)-stimulated PBMC. All cp strains and most ncp BVDV strains significantly inhibited DNA synthesis in PHA-stimulated PBMC; however, only cp BVDV strains inhibited protein synthesis. A plaque assay and immunofluorescence test confirmed productive BVDV infection of PBMC. In addition, viral RNA synthesis was demonstrated in BVDV-infected PBMC by RT-PCR. The interleukin-2 receptor (IL-2R) was used as a marker for the activation status of BVDV-infected PBMC. The expression of IL-2R was preserved in virus-infected cells, even though DNA and protein synthesis was suppressed. These findings suggest a novel mechanism of virus-induced immune suppression in which BVDV inhibits basic metabolic activities of bovine PBMC. The activation signals, however, are maintained.

Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization.

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCR in situ hybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conserved pol gene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIV pol gene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed by in situ amplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected to in situ hybridization with an internal biotinylated probe and detected with streptavidin-gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissues in vivo and has a broad cell tropism.

Isolation and characterization of a coronavirus from elk calves with diarrhea.

This is the first report of the isolation of a coronavirus from elk calves. Two fecal samples from elk calves with diarrhea were shown to be positive for coronavirus-like particles by electron microscopy, and the particles were propagated in the human rectal tumor-18 cell line. After 24 h, syncytia were observed, and cell culture supernatants from both samples showed hemagglutinating activity with mouse erythrocytes. Cells infected with both elk coronavirus (ECV) isolates reacted with Z3A5, a monoclonal antibody against the spike protein of bovine coronavirus (BCV), on an indirect fluorescent antibody test. The protein profiles of both ECV isolates were similar to that of BCV as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. On Northern blot analysis, the transcriptional pattern of ECV was typical of coronaviruses, with a nested set of transcripts with common 3' end sequences. Based on a published nucleoprotein gene sequence for BCV (Mebus isolate), we arbitrarily designed two primers for amplification by PCR. After cloning, the nucleoprotein was sequenced and a high degree of homology (99%) between the nucleoprotein gene sequences of ECV and BCV was observed. Thus, ECV is closely related genetically and antigenically to BCV and will be a new member of antigenic group 2 of the mammalian coronaviruses, which possess hemagglutinin-esterase protein.

Application of immunohistochemistry and in situ hybridization for detection of bovine coronavirus in paraffin-embedded, formalin-fixed intestines.

A monoclonal antibody (MAb) (Z3A5) against spike protein subunit of bovine coronavirus (BCV) reacted with the virus in formalin-fixed intestines in an immunoperoxidase test. We found an 88% correlation between immunohistochemistry with Z3A5 and in situ hybridization with a BCV nucleoprotein cDNA probe. MAb Z3A5 reacted with 90 BCV isolates from the United States and was an effective reagent for the diagnosis of BCV.

Characterization of a putative receptor protein for bovine viral diarrhea virus.

In a previous communication, we reported a 50-kDa cell surface protein from Madin-Darby bovine kidney (MDBK) cells as a putative receptor for bovine viral diarrhea virus (BVDV). The present study delineates further characterization of the receptor protein. Protease treatment of cultured MDBK cells adversely affected the receptor, thus abolishing the binding of anti-D89 (BVDV anti-idiotypes) to the cells. However, pretreatment of the cells with either phospholipases or glycosidases did not significantly change the anti-D89 binding to the cells. Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells. These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation may not play a direct role in BVDV attachment to cells. The BVDV receptor gradually regenerated on the cell surface after the protease-treated cells were cultured in normal growth medium. Regeneration of the BVDV receptor to a normal level took about 4 h as indicated by flow cytometric analysis and this process was inhibited in the presence of cycloheximide, a protein synthesis inhibitor. The 50-kDa receptor protein purified by electro-elution inhibited BVDV infection in a plaque reduction assay. It also inhibited anti-D89 binding to cells as analyzed by flow cytometry. These data demonstrated the nature of the 50-kDa protein as a specific receptor for BVDV.

Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle.

The National Animal Disease Laboratory (NADL) vaccine strain of bovine viral diarrhea virus (BVDV) genes for gp48 and p80 were expressed in Escherichia coli. The BVDV-NADL gene for gp62 was integrated into a baculovirus genome for expression in Spodoptera frugiperda (Sf-9) insect ovarian cells. The antigenicity of baculovirus expressed BVDV protein was detected by anti-BVDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent assay (IFA) and radio-immunoprecipitation (RIP). The recombinant proteins isolated from bacteria showed antigenic properties when analyzed by ELISA and immunoblotting using BVDV antibodies. The recombinant proteins were then used in ELISA or IFA to detect BVDV infection by testing 54 independent bovine serum samples. The baculovirus-expressed BVDV protein was used as an ELISA and IFA antigen, and the bacteria-expressed proteins were used as ELISA antigens. BVDV-NADL-infected Madin-Darby bovine kidney (MDBK) cell monolayers served as a control antigen. Statistical analysis showed a high degree of correlation between the reactivity of recombinants and natural antigens in ELISA using bovine sera. The results of ELISA or IFA proved there is a high degree of correlation with the virus neutralization. In the comparative ELISA assays, the insect-cell-mediated expression revealed greater specificity and sensitivity than the bacterial expression or the natural BVDV antigens produced by cell cultures.

Detection of bovine immunodeficiency virus antibodies in cattle by western blot assay with recombinant gag protein.

A western blot assay using purified recombinant bovine immunodeficiency virus gag protein has been developed for detection of bovine immunodeficiency virus antibodies in bovine serum samples. The test was standardized with known bovine immunodeficiency virus positive and negative bovine serum samples and the monoclonal antibody to gag protein. Both naturally and experimentally infected cattle sera demonstrated positive test results. The result of western blot assay was compared with polymerase chain reaction test results in 134 blood samples collected from Kansas. Twenty-six samples tested positive for bovine immunodeficiency virus DNA with polymerase chain reaction (18.7%) and 25 were positive for the antibody to gag protein by western blot analysis (17.9%). Of 26 cattle testing positive using the polymerase chain reaction assay, 24 were antibody-positive by western blot assay, thus establishing a strong correlation between the two tests. The sensitivity and specificity of western blot relative to polymerase chain reaction are 0.92 and 0.99, respectively. The western blot assay proved to be a specific and sensitive test.

Immune suppression in calves with bovine immunodeficiency virus.

The present study was designed to evaluate the effect of bovine immunodeficiency virus (BIV) infection on immune functions and possible interactions between BIV and other bovine viruses in calves. Ten calves were inoculated intravenously with BIV, and five served as controls. An increased lymphocyte proliferation to BIV gag protein was demonstrated 2 to 6 weeks after BIV inoculation (P < 0.05). Lymphocyte subset differentiation revealed a decreased CD4/CD8 ratio (P < 0.05) during weeks 2 to 7, suggesting a possible immune dysfunction in BIV-infected calves. When the calves were inoculated with bovine herpesvirus type 1 (BHV-1), the antibody response to BHV-1 in BIV-infected calves was delayed and the antibody titers were significantly lower (P < 0.05). Injection of bovine viral diarrhea virus vaccine also elicited a lower neutralizing antibody response in BIV-infected calves. The results indicated that immune suppression occurred in BIV-infected calves.

A 50 kDa membrane protein from bovine kidney cells is a putative receptor for bovine viral diarrhea virus (BVDV).

A 50 kDa cell surface protein from MDBK cells has been identified as a putative receptor for bovine viral diarrhea virus (BVDV) by using a BVDV specific anti-idiotypic antibody (Anti-D89). This study delineates further characterization of the receptor protein. Protease treatment of cultured MDBK cells adversely affected the receptor thus abolishing the binding of anti-D89 to the cells. However, pretreatment of the cells with either phospholipases or glycosidases did not signficantly alter the extent of anti-D89 binding. Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells. These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation of the receptor protein may not play a direct role in BVDV attachment to cells. The BVDV receptor protein gradually regenerated on cells when they were maintained in culture following protease treatment. The purified 50 kDa receptor protein also significantly inhibited BVDV infection in a plaque reduction assay.

Identification of a 56 kDa putative bovine herpesvirus 1 cellular receptor by anti-idiotype antibodies.

Polyclonal anti-idiotype antibodies (anti-ids) to anti-bovine herpesvirus 1 (BHV-1) MAbs blocked virus infection in cell cultures, indicating that they contain internal images of a viral attachment protein(s). In the present study anti-id (anti-83) of BHV-1 gB was used as a probe to detect the cellular receptor. Anti-id specifically identified a 56 kDa protein in radioimmunoprecipitation analysis (RIPA) of Madin-Darby bovine kidney (MDBK) cell membranes suggesting the involvement of this cell surface component in BHV-1 binding. Anti-83 pretreated with MAb 83 failed to identify the 56 kDa cellular component proving its specificity for the reacting epitope of MAb 83. The recognition of 56 kDa protein by anti-id was inhibited by prior incubation of radiolabelled membrane proteins with BHV-1 suggesting that the ligands competed for the same binding sites on the cells. 35S-Radiolabelled BHV-1 virions also bound a 56 kDa protein from purified MDBK cell membrane proteins in a virus overlay protein blot assay. RIPA using anti-id as probe detected the 56 kDa protein in permissive MDBK cells but not in non-permissive bat lung cells. The protein nature of the 56 kDa component was confirmed by protease treatment of membranes which resulted in abolition of the 56 kDa signal in RIPA. In addition, purified 56 kDa protein inhibited biotinylated BHV-1 attachment in flow cytometry. These findings indicate that the 56 kDa protein identified by anti-id is a putative receptor for BHV-1.

Identification of bovine viral diarrhea virus receptor in different cell types.

Anti-idiotypic antibodies (anti-ids) have been used successfully in studies on bovine viral diarrhea virus (BVDV) receptor(s) in our laboratory. The anti-ids specifically bound to cultured cells and identified a 50 kDa cellular membrane protein, which is thought to be a specific receptor for BVDV. In this study, flow cytometric analyses demonstrated that the anti-ids also specifically bound to different cell types, namely MDBK, EBK, BT, PK15, MA1O4, and Vero. Experiments on virus attachment and replication showed that BVDV adsorbed to all cells and replicated in them except monkey kidney cells MA 104 and Vero (non-permissive). Results from plaque reduction assays indicated that cellular membrane proteins from all cell lines competitively inhibited BVDV attachment to cultured MDBK cells, suggesting the presence of BVDV receptor on all cells. Immunoblotting of cell membrane proteins with the anti-ids revealed a 50 kDa protein in both permissive and nonpermissive cells. Subcloned or synchronized MDBK cells demonstrated no significant difference of binding with anti-ids as compared to normal cultured cells.

Antigenic differences between a field isolate and vaccine strains of bovine viral diarrhea virus.

Antigenic diversity among the bovine viral diarrhea virus (BVDV) cytopathic strains 87-2552 (field isolate) and NADL and Singer (prototype strains) was demonstrated with monoclonal antibodies (MAbs) in enzyme-linked immunosorbent, immunofluorescence, virus neutralization, and immunoprecipitation assays. Two MAbs against BVDV strain 87-2552, designated D11 and B7, strongly neutralized this field strain and were specific for the 48-kDa glycoprotein of the virus. These two MAbs have different subisotypes, immunoglobulin G1 for D11 and immunoglobulin G3 for B7. MAbs against BVDV strains 87-2552 and NADL were specific for their respective strains in virus neutralization assays. The results indicated significant antigenic differences between BVDV strain 87-2552 and the NADL and Singer strains.

Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD.

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusion proteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52-126 and 165-216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92-106 (3402 epitope) and amino acids 202-213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.

Immune response to bovine viral diarrhea virus induced by anti-idiotypic antibodies.

We have previously prepared rabbit anti-idiotypic antibodies (anti-Ids) against the neutralizing monoclonal antibodies (MAbs) specific for the gp53 of bovine viral diarrhea virus (BVDV). The anti-Ids, purified by sequential immunoaffinity chromatography, inhibited the immunizing MAb from binding to the original antigens and blocked BVDV infection of cell cultures. This study evaluated immune responses in mice to the purified anti-Id reagents. BVDV-specific neutralizing antibodies were induced by the anti-Ids. The antisera (Ab3) induced by the anti-Ids immunoprecipitated gp53 from BVDV-infected Madin-Darby bovine kidney (MDBK) cell lysates. However, lymphocyte-proliferative responses were specific only for the respective immunizing antigens. These results suggest that the anti-Ids may bear an internal image of the gp53 to stimulate production of antibody but not to stimulate a virus-specific cellular immune response in mice.

Immunopotentiation of bovine respiratory disease virus vaccines by interleukin-1 beta and interleukin-2.

Three experiments, using 85 crossbred beef calves, were conducted to evaluate the adjuvanticity of single, multiple, and combined doses of recombinant bovine IL-1 beta (rBoIL-1 beta) and recombinant bovine IL-2 (rBoIL-2), with a modified-live bovine herpesvirus-1/parainfluenza-3 (BHV-1/PI-3) virus vaccine and a killed bovine viral diarrhea (BVD) virus vaccine. Cytokines were administered intramuscularly at vaccination but at different injection sites. All cytokine treatments increased non-major histocompatibility complex (MHC)-restricted cytolytic capability of peripheral blood mononuclear cells (PBMC) against virus-infected target cells and serum neutralizing (SN) antibody titers to BHV-1 and BVD virus. Multiple, consecutive injections of rBoIL-2 generally showed the greatest adjuvant effect, and no additive effect was observed when rBoIL-1 beta and rBoIL-2 were administered together. In a challenge experiment, calves were vaccinated with a modified-live BHV-1/PI-3 vaccine and infected with BHV-1 on Day 21. Cytokine-treated calves had higher SN antibody titers to BHV-1 than did the control calves at the time of challenge. Calves that were administered rBoIL-2 on 5 consecutive days shed less BHV-1 and had the highest SN antibody titer to BHV-1 (Day 28). These data suggest that rBoIL-1 beta and rBoIL-2 may be useful immunoadjuvants for bovine respiratory disease virus vaccines.