Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication.
Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2.6- to 5.6-fold (NDFL) or 2.1- to 8.1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.
Zhao
Heng
H
Institute for Animal Science and Health (ID-Lelystad), Division of Infectious Diseases and Food Chain Quality, PO Box 65, 8200 AB Lelystad, The Netherlands.
Peeters
Ben P H
BPH
Institute for Animal Science and Health (ID-Lelystad), Division of Infectious Diseases and Food Chain Quality, PO Box 65, 8200 AB Lelystad, The Netherlands.
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular / Q000379:methods; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D009522:Newcastle disease virus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D013997:Time Factors; D014779:Virus Replication
D011994:Recombinant Proteins; D000469:Alkaline Phosphatase