RESUMO
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.
Assuntos
Adenovírus Humanos/genética , Anticorpos Antivirais/sangue , Vírus da Diarreia Viral Bovina/imunologia , Imunidade Celular , Proteínas do Nucleocapsídeo/imunologia , Vacinas Sintéticas/imunologia , Adenovírus Humanos/imunologia , Animais , Bovinos , Vírus da Diarreia Viral Bovina/química , Vetores Genéticos , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Nucleocapsídeo/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Tetraciclina/farmacologia , Vacinação , Vacinas Virais/imunologiaRESUMO
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.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Adenoviridae , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/genética , Ensaio de Imunoadsorção Enzimática , Regulação Viral da Expressão Gênica , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Regiões Promotoras Genéticas , Proteínas Recombinantes/imunologia , Fatores de Tempo , TransfecçãoRESUMO
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.
Assuntos
Vírus da Diarreia Viral Bovina/imunologia , Imunidade Celular , Peptídeo Hidrolases , RNA Helicases , Proteínas não Estruturais Virais/imunologia , Adenoviridae , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Bovinos , Linhagem Celular , DNA Recombinante , Expressão Gênica , Vetores Genéticos , Humanos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas não Estruturais Virais/genéticaRESUMO
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.
Assuntos
Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/imunologia , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Linfócitos B/virologia , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Regulação Viral da Expressão Gênica , Imunização , Interferon gama/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
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.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , DNA Viral/imunologia , Vacinação , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Células COS/microbiologia , Bovinos , Clonagem Molecular , DNA Complementar , DNA Viral/farmacologia , Feminino , Regulação Viral da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microinjeções , Testes de Neutralização , Plasmídeos , Transfecção , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Variação Antigênica , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/genética , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/análise , Anticorpos Antivirais/genética , Especificidade de Anticorpos , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Técnicas Imunoenzimáticas , Imunoglobulina G , Testes de Neutralização , Quebeque , Sensibilidade e EspecificidadeRESUMO
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.
