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Arch Virol. 2011 Jun;156(6):979-86. doi: 10.1007/s00705-011-0932-0. Epub 2011 Feb 17.

Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

Author information

1
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Grazing Animal Diseases of Ministry of Agriculture, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu 730046, People's Republic of China.

Abstract

A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7.

PMID:
21327786
DOI:
10.1007/s00705-011-0932-0
[Indexed for MEDLINE]

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