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2.
Virology ; 290(1): 153-63, 2001 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-11883000

RESUMO

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, or Orf4 showed growth characteristics similar to those of the E3-deleted BAV3 (BAV302). DNA accumulation and early/late protein synthesis were also indistinguishable from those of BAV302. However, mutant viruses carrying a deletion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compromised in their ability to grow in bovine cells and express early/late proteins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicated as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-specific IgA and IgG responses were detected in serum and at the mucosal surfaces in cotton rats inoculated with mutant viruses. In vitro and in vivo characterization of these E4 mutants suggests that none of the individual E4 Orfs are essential for viral replication. Moreover, successful deletion of a 1.5-kb fragment in the BAV3 E4 region increased the available insertion capacity of replication-competent BAV3 vector (E3-E4 deleted) to approximately 4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to approximately 5.0 kb. This is extremely useful for the construction of BAV3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.


Assuntos
Proteínas E4 de Adenovirus/genética , Genes Virais/fisiologia , Mastadenovirus/genética , Fases de Leitura Aberta/fisiologia , Proteínas Virais/genética , Infecções por Adenoviridae/sangue , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/virologia , Proteínas E4 de Adenovirus/imunologia , Proteínas E4 de Adenovirus/fisiologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Modelos Animais de Doenças , Mastadenovirus/imunologia , Mastadenovirus/fisiologia , Mutagênese , Ratos , Infecções Respiratórias/sangue , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Sigmodontinae , Proteínas Virais/imunologia , Proteínas Virais/fisiologia , Replicação Viral
3.
Virology ; 278(1): 234-43, 2000 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-11112498

RESUMO

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.


Assuntos
Adenoviridae/genética , Vírus da Diarreia Viral Bovina/imunologia , Sigmodontinae/imunologia , Proteínas E3 de Adenovirus/genética , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/biossíntese , Antígenos Virais/genética , Antígenos Virais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Células Cultivadas , DNA Recombinante/imunologia , Vírus da Diarreia Viral Bovina/química , Vírus da Diarreia Viral Bovina/genética , Feminino , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/imunologia , Imunização , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Masculino , Mucosa Nasal/imunologia , Ratos , Proteínas Recombinantes/biossíntese , Sigmodontinae/sangue , Sigmodontinae/virologia , Transcrição Gênica , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
4.
Virology ; 261(1): 143-52, 1999 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10441562

RESUMO

Early region 4 (E4) of bovine adenovirus type 3 (BAV-3) was analyzed by Northern blotting, RT-PCR analysis, cDNA sequencing, and S1 nuclease protection assays. The transcriptional map of the E4 region of BAV-3 has marked dissimilarities from those of mouse adenovirus-1, ovine adenovirus-287, and human adenovirus-2, for which the transcriptional maps have been constructed. The E4 region of BAV-3, located between 98.6 and 89.8 MU transcribes seven distinct classes of bovine adenovirus type 3 mRNA. The seven mRNA species formed by the removal of one to three introns share both the 3' end and a short 5' leader (25 nucleotides). The E4 mRNAs can encode at least five unique polypeptides, namely, 143R1, 69R, 143R2, 268R, and 219R. Isolation of a replication-competent recombinant "BAV404" containing 1.9-kb insertion [glycoprotein (gD) of bovine herpesvirus 1, under the control of a SV40 early promoter and poly(A)] in the region between E4 and the right ITR suggested that this region is nonessential for BAV-3 replication. Expression of gD by BAV404 recombinant virus was confirmed by immunoprecipitation with gD-specific monoclonal antibodies. Analysis of the kinetics of protein expression indicated that gD is expressed at both early and late times postinfection. These results suggest that: (a) E4 produces seven 5'-3' coterminal mRNAs and (b) the right terminal region of BAV-3 can be used for the expression of vaccine antigens.


Assuntos
Proteínas E4 de Adenovirus/genética , Mastadenovirus/genética , Transcrição Gênica , Proteínas Virais/genética , Proteínas E4 de Adenovirus/metabolismo , Animais , Northern Blotting , Bovinos , DNA Complementar , Mastadenovirus/metabolismo , Camundongos , Plasmídeos , Testes de Precipitina , Proteínas Recombinantes/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Transfecção , Proteínas Virais/biossíntese
5.
Avian Dis ; 43(1): 16-21, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10216755

RESUMO

Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age. The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine. The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age. However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC. Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively. The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.


Assuntos
Vírus da Varíola das Aves Domésticas/imunologia , Varíola Aviária/prevenção & controle , Vacinas Virais/imunologia , Cultura de Vírus/métodos , Animais , Anticorpos Antivirais/biossíntese , Células Cultivadas , Embrião de Galinha , Galinhas , Linfócitos T/imunologia
6.
Virus Genes ; 17(1): 99-100, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9778793

RESUMO

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/genética
7.
Virus Genes ; 16(3): 313-6, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9654686

RESUMO

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 DNA
8.
J Virol ; 72(2): 1394-402, 1998 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9445040

RESUMO

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.


Assuntos
Mapeamento Cromossômico , Genoma Viral , Mastadenovirus/genética , Sequência de Aminoácidos , Animais , Bovinos , Genes Virais , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Transcrição Gênica
9.
Virus Res ; 40(1): 33-45, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8725119

RESUMO

The murine intranasal (i.n.) infection model was used to study the molecular distribution of equine herpesvirus-1 (EHV-1) during acute infection, latency and following a reactivation stimulus. After inoculation, infectious virus was detected in lungs, nasal turbinates, brains and olfactory bulbs during the acute phase. A nested PCR (nPCR) readily detected virus in these tissues and, in addition, virus was detected in spleens and (in the second round of nPCR) in peripheral blood mononuclear cells (PBMC). A digoxigenin-labelled in situ hybridization probe detected EHV-1 DNA in bronchiolar and vascular endothelium in the lungs and in and around germinal centres in the spleens. One month later, although infectious virus was absent from all tissues, the trigeminal ganglia, olfactory bulb and PBMC remained positive for virus DNA although this was detected only on the second round of nPCR. Furthermore, in situ hybridization, using either DNA or RNA probes, suggested that little or no transcription of virus occurred in neural tissues during the 'latent phase'. Following a reactivation stimulus, infectious virus was not isolated from any tissues, however, EHV-1 DNA was detected on the first round of nPCR in olfactory bulb, trigeminal ganglia and PBMC. This suggested a quantitative increase in EHV-1 DNA occurred following reactivation stimulus. The significance of these results is discussed in relation to the molecular state of EHV-1 in different tissues at various stages of infection and the validity of the murine model for studying latency and reactivation of EHV-1 in the horses.


Assuntos
DNA Viral/análise , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/isolamento & purificação , RNA Viral/análise , Doença Aguda , Animais , Linhagem Celular , Modelos Animais de Doenças , Infecções por Herpesviridae/patologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Latência Viral
10.
J Gen Virol ; 76 ( Pt 12): 3113-8, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8847517

RESUMO

Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares a similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.


Assuntos
Herpesvirus Equídeo 1/fisiologia , RNA Mensageiro/análise , RNA Viral/análise , Gânglio Trigeminal/virologia , Latência Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Núcleo Celular/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Humano 1/genética , Cavalos , Hibridização In Situ , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Sondas RNA , RNA Antissenso/análise , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Eliminação de Partículas Virais
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