RESUMEN
Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors.
Asunto(s)
Virus Defectuosos/genética , Prueba de Complementación Genética , Virus de la Parainfluenza 3 Bovina/genética , Animales , Bovinos , Técnicas de Cultivo de Célula , Línea Celular , Prueba de Complementación Genética/instrumentación , Prueba de Complementación Genética/métodos , Vectores Genéticos , Virus de la Parainfluenza 3 Bovina/crecimiento & desarrollo , Proteínas Virales de Fusión/genética , Proteínas de la Matriz Viral/genética , Replicación ViralRESUMEN
The bovine parainfluenza virus type 3 BN-CE vaccine strain was obtained by serial passage of the BN-1 strain in chicken embryonic fibroblasts (CEF). We previously identified a substitution (L288I) in the fusion (F) protein between the two strains. To examine the effect of the substitution on CEF adaptation and attenuation, we generated a recombinant BN-1 strain with the L288I substitution in the F protein (FL288I-EGFP). FL288I-EGFP replicated more efficiently than a recombinant BN-1 strain (wt-EGFP) in semi-suitable cell lines, suggesting that the L288I substitution was established in the BN-1 strain during the process of adaptation in CEF.