RESUMO
Based on the DNA sequence and known mRNA structures, the early region 3 (E3) of bovine adenovirus (BAV)-3 has the potential to encode four proteins. One of them (121R) is produced as a 14.5-kD protein throughout infection. Analysis of the 121R protein showed limited homology to a 14.7K protein of human adenovirus (HAV)-5. Interestingly, both anti-14.7K and anti-121R sera immunoprecipitated a 14.5-kD protein from cells infected with BAV-3. To determine if 121R is functionally related to 14.7K, we constructed a recombinant E3-deleted HAV-5 (AdKV121) expressing the BAV-3 E3 121R protein. Mouse C3HA cells infected with HAV-5 mutant dl 758 (deletion of 14.7K) were sensitive to TNF lysis. However, wild-type HAV-5- or recombinant AdKV121-infected cells were resistant to TNF-induced cytolysis. Our result show that the BAV-3 E3 121R protein is serologically and functionally related to the 14.7K protein encoded by the E3 region of HAV-5.
Assuntos
Adenoviridae/química , Proteínas E3 de Adenovirus/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adenoviridae/fisiologia , Proteínas E3 de Adenovirus/química , Proteínas E3 de Adenovirus/genética , Sequência de Aminoácidos , Animais , Bovinos , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Deleção de Genes , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Alinhamento de Sequência , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.
Assuntos
Doenças dos Bovinos/imunologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Vacinação/veterinária , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Bovinos , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Vetores Genéticos/imunologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/genética , Testes de Neutralização , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Vacinas Virais/genética , Vacinas Virais/normasRESUMO
To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.
Assuntos
Proteínas E3 de Adenovirus/genética , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/imunologia , Mastadenovirus/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Proteínas E3 de Adenovirus/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Vetores Genéticos , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Imunidade nas Mucosas , Imunização , Mastadenovirus/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagemRESUMO
A ribozyme gene directed at a specific cleavage of mRNA coding for PB1 protein, a component of RNA-dependent RNA-polymerase of influenza A virus, was constructed. The avian adenovirus CELO virus-associated RNA (VA RNA CELO) promoter and human cytomegalovirus (CMV) promoter were used for the permanent expression of the ribozyme in cell lines. The cells were infected with influenza A virus strains A/Singapore/1/57 and A/WSN/33, and the suppression of the virus reproduction and virus-specific protein synthesis was measured. The maximal level of the inhibition of virus reproduction as compared to the reproduction in non-transformed cells was 93.5%. Defective recombinant adenoviruses were constructed carrying the genes of functional and non-functional ribozymes under the control of human cytomegalovirus (CMV) promoter. The reproduction of A/WSN/33 virus in CV-1 cells preinfected with recombinant adenoviruses was shown to be suppressed.
Assuntos
Vírus da Influenza A/fisiologia , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Adenoviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Citomegalovirus/genética , Humanos , Vírus da Influenza A/genética , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas Virais/biossíntese , Replicação Viral/fisiologiaRESUMO
Using the homologous recombination machinery of E. coli, a 1.245-kb deletion was introduced in the E3 region of bovine adenovirus 3 (BAV3) genomic DNA cloned in a plasmid. Transfection of the restriction enzyme-excised, linear E3-deleted BAV3 genomic DNA into primary fetal bovine retina cells produced infectious virus (BAV3. E3d), suggesting that all the E3-specific open reading frames are nonessential for virus replication in vitro. Using a similar approach, we constructed replication-competent (BAV3.E3gD and BAV3. E3gDt) BAV3 recombinant expressing full-length (gD) or truncated (gDt) glycoprotein of bovine herpes virus 1. Recombinant gD and gDt proteins expressed by BAV3.E3gD and BAV3.E3gDt, respectively, were recognized by gD-specific monoclonal antibodies directed against conformational epitopes, suggesting that antigenicity of recombinant gD and gDt was similar to that of the native gD expressed in bovine herpes virus 1-infected cells. Intranasal immunization of cotton rats induced strong gD- and BAV3-specific IgA and IgG immune responses. These results suggest that replication-competent bovine adenovirus 3-based vectors have potential for the delivery of vaccine antigens to the mucosal surfaces of animals.
Assuntos
Vetores Genéticos/genética , Herpesvirus Bovino 1/imunologia , Mastadenovirus/genética , Vacinas de DNA/genética , Proteínas Virais/genética , Vacinas Virais/genética , Proteínas E3 de Adenovirus/genética , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Bovinos , Linhagem Celular , Citarabina/farmacologia , DNA Recombinante , DNA Viral/análise , DNA Viral/biossíntese , Deleção de Genes , Expressão Gênica , Herpesvirus Bovino 1/genética , Pulmão/imunologia , Mastadenovirus/imunologia , Mucosa Nasal/imunologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Sigmodontinae , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
We have identified and sequenced 3614 nucleotides located at the extreme right-end of the bovine adenovirus type 3 (BAV3) genome from map units 89.5-100. Analysis of the sequence revealed an inverted terminal repeat (ITR) of 195 bp, and identified five open reading frames (ORFs) designated ORF1, ORF2, ORF3, ORF4 and ORF5. When compared with known E4 ORFs of other adenoviruses, ORFs 1, 2 and 4, which code for proteins of 143, 69 and 143 amino acids respectively, were found to be unique to BAV3. ORFs 3 and 5, which code for proteins of 268 and 219 amino acids respectively, showed partial homology to the E4 34 kDa protein of human adenovirus 2. Nucleotide sequence analysis also identified two potential TATA boxes upstream of ORF1 and a potential polyadenylation signal downstream of ORF5 suggesting that E4 transcripts may be 3' co-terminal.
Assuntos
Adenoviridae/genética , DNA Viral/genética , Adenoviridae/química , Proteínas E4 de Adenovirus/genética , Animais , Sítios de Ligação/genética , Bovinos , DNA Viral/química , Desoxirribonuclease EcoRI , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Análise de Sequência de DNA , Sequências Repetidas Terminais/genéticaRESUMO
We have determined the nucleotide sequence of a 6999 base pair region of bovine adenovirus-3 covering map units 9.0 to 29.17, which contained the adenovirus homologs of IVa2 protein and the DNA replication proteins, precursor of terminal protein and DNA polymerase proteins. Analysis of the sequence for cis-acting elements suggests that transcripts of DNA polymerase and precursor of terminal protein are 3' co-terminal. In addition, this region also contains major late promoter sequence. The sequence to the left of IVa2 contains the ORF of pIX with a potential TATA box immediately upstream and two polyadenylation consensus signals immediately downstream of the ORF.
Assuntos
Genes pol , Mastadenovirus/genética , Fosfoproteínas/genética , Precursores de Proteínas/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Viral , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The complete DNA sequence of bovine adenovirus type 3 is reported here. The size of the genome is 34,446 bp in length with a G+C content of 54%. All the genes of the early and late regions are present in the expected locations of the genome. However, the late-region genes are organized into seven families, instead of five as they are in human adenovirus type 2. The deduced amino acid sequences of open reading frames (ORFs) in the late regions and early region 2 (E2) and for IVa2 show higher degrees of homology, whereas the predicted amino acid sequences of ORFs in the E1, E3, and E4 regions and the pIX, fiber, and 33,000-molecular-weight nonstructural proteins show little or no homology with the corresponding proteins of other adenoviruses. In addition, the penton base protein lacks the integrin binding motif, RGD, but has an LDV motif instead of an MDV motif. Interestingly, as in other animal adenoviruses, the virus-associated RNA genes appear to be absent from their usual location. Sequence analysis of cDNA clones representing the early- and late-region genes identified splice acceptor and splice donor sites, polyadenylation signals and polyadenylation sites, and tripartite leader sequences.