RESUMO
Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Engenharia Genética/métodos , Genoma Viral , Herpesvirus Canídeo 1/genética , Animais , Proteínas de Bactérias , Linhagem Celular , Cães , Escherichia coli/genética , Vetores Genéticos/genética , Herpesvirus Canídeo 1/crescimento & desenvolvimento , Recombinação Genética , Timidina Quinase/genética , TransfecçãoRESUMO
Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.
Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Bovino 1/crescimento & desenvolvimento , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Proteínas do Envelope Viral/genética , Animais , Células 3T3 BALB , Western Blotting , Primers do DNA , Herpesvirus Bovino 1/genética , Proteínas Imediatamente Precoces/genética , Camundongos , Microscopia de Fluorescência , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Transativadores , Transfecção , Ubiquitina-Proteína Ligases , Proteínas ViraisRESUMO
Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.
