Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both.
Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric beta-galactosidase gene. Subsequently, using the BoHV-5 gE virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE) and TK + gE (BoHV-5 gE/TK) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE, BoHV-5 TK and BoHV-5 gE/TK) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.
Brum
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Setor de Virologia, Departamento de Microbiologia e Parasitologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil.
Weiblen
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Flores
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Chowdhury
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D000818:Animals; D002417:Cattle; D003673:Defective Viruses / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics; D025881:Herpesvirus 5, Bovine / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D015151:Immunoblotting; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology; D014774:Virulence / Q000235:genetics
D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C414993:glycoprotein E, bovine herpesvirus type 5; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase