ABSTRACT
A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV.
Subject(s)
Adenoviruses, Human/genetics , Antibodies, Viral/blood , Diarrhea Viruses, Bovine Viral/immunology , Immunity, Cellular , Nucleocapsid Proteins/immunology , Vaccines, Synthetic/immunology , Adenoviruses, Human/immunology , Animals , Cattle , Diarrhea Viruses, Bovine Viral/chemistry , Genetic Vectors , Interferon-gamma/biosynthesis , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Nucleocapsid Proteins/genetics , Promoter Regions, Genetic , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/immunology , Tetracycline/pharmacology , Vaccination , Viral Vaccines/immunologyABSTRACT
The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.
Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/immunology , Diarrhea Viruses, Bovine Viral/immunology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Adenoviridae , Animals , Antibodies, Viral/blood , Antibody Formation , Cattle , Cell Line , Diarrhea Viruses, Bovine Viral/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Viral , Humans , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Neutralization Tests , Promoter Regions, Genetic , Recombinant Proteins/immunology , Time Factors , TransfectionABSTRACT
Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.
Subject(s)
Diarrhea Viruses, Bovine Viral/immunology , Immunity, Cellular , Peptide Hydrolases , RNA Helicases , Viral Nonstructural Proteins/immunology , Adenoviridae , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Cattle , Cell Line , DNA, Recombinant , Gene Expression , Genetic Vectors , Humans , Mice , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Viral Nonstructural Proteins/geneticsABSTRACT
A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.
Subject(s)
Diarrhea Viruses, Bovine Viral/genetics , Diarrhea Viruses, Bovine Viral/immunology , Fowlpox virus/genetics , Fowlpox virus/immunology , Viral Envelope Proteins/genetics , Animals , Antibodies, Monoclonal , Antibodies, Viral/blood , B-Lymphocytes/immunology , B-Lymphocytes/virology , Cells, Cultured , Chick Embryo , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Viral , Immunization , Interferon-gamma/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Mice , Mice, Inbred BALB C , Plasmids , Recombinant Fusion Proteins/immunology , Viral Envelope Proteins/immunologyABSTRACT
Bovine viral diarrhea virus (BVDV) is a worldwide pathogen in cattle which has not been controlled by classical vaccination. The region encoding the BVDV major glycoprotein gp53 (E2) known to possess virus-neutralizing activity was cloned into a mammalian expression vector under the human cytomegalovirus (CMV) intermediate early promoter. Intramuscular and intradermal administration of the recombinant plasmid DNA into BALB/c mice induced BVDV gp53-specific antibody responses to both biotypes (cytopathic and noncytopathic) of BVDV genotype 1, and to cytopathic BVDV genotype 2. BVDV-neutralizing antibodies were generated against BVDV genotype 1 strains and they also persisted 6 months after the last injection.
Subject(s)
Bovine Virus Diarrhea-Mucosal Disease/prevention & control , DNA, Viral/immunology , Vaccination , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/immunology , Bovine Virus Diarrhea-Mucosal Disease/immunology , COS Cells/microbiology , Cattle , Cloning, Molecular , DNA, Complementary , DNA, Viral/pharmacology , Female , Gene Expression Regulation, Viral/immunology , Mice , Mice, Inbred BALB C , Microinjections , Neutralization Tests , Plasmids , Transfection , Viral Envelope Proteins/geneticsABSTRACT
Antigenic variation among 13 Quebec isolates of bovine viral diarrhea virus (BVDV), 4 reference strains and 2 American isolates were studied by peroxidase-linked antibody assay (PLA assay) and neutralization test (NT). The Quebec strains consisted of 3 isolates before 1993 and 10 isolates from 1993. In the PLA assay, we compared 2 different fixatives, acetone and formalin. Acetone-fixation allowed us to identify 6 groups from amongst the viruses tested. All the Quebec isolates were different from the reference strains. In addition, antigenic variation was detected between Quebec isolates obtained before and during 1993. However, PLA assays performed after formalin fixation did not detect these antigenic variations. Neutralization tests were carried out with 2 polyclonal antibodies (PAb) and 6 monoclonal antibodies (MAb). They were used to classify BVDV strains and isolates into 4 groups and 7 subgroups respectively. In conclusion, we demonstrated that the BVDV isolates from the 1993 outbreak in Quebec are antigenically different from reference strains and from isolates existing in Quebec before 1993. In addition, we have shown that 2 internationally used fixation-methods in PLA assay give different results. The usefulness of each method is discussed.
Subject(s)
Antigenic Variation , Bovine Virus Diarrhea-Mucosal Disease/virology , Diarrhea Viruses, Bovine Viral/genetics , Animals , Antibodies, Monoclonal , Antibodies, Viral/analysis , Antibodies, Viral/genetics , Antibody Specificity , Cattle , Diarrhea Viruses, Bovine Viral/immunology , Diarrhea Viruses, Bovine Viral/isolation & purification , Immunoenzyme Techniques , Immunoglobulin G , Neutralization Tests , Quebec , Sensitivity and SpecificityABSTRACT
The 5' untranslated region (UTR) of several bovine viral diarrhea virus (BVDV) isolates from the severe Quebec outbreak was amplified by polymerase chain reaction (PCR) and sequenced. Sequences revealed the loss, for the BVDV type II isolates, of an internal PstI restriction site, which is present in all known BVDV type 1 5' UTR sequences. A single restriction enzyme digestion (PstI) of an aliquot of PCR product allowed us to differentiate BVDV type I and BVDV type II.
