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

Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

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

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References (38)

Publisher
Springer Journals
Copyright
Copyright © Springer-Verlag 2011
Subject
Biomedicine; Virology ; Infectious Diseases; Medical Microbiology
ISSN
0304-8608
eISSN
1432-8798
DOI
10.1007/s00705-011-0932-0
pmid
21327786
Publisher site
See Article on Publisher Site

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.

Journal

Archives of VirologySpringer Journals

Published: Jun 1, 2011

Keywords: Newcastle Disease Virus; Allantoic Fluid; Chicken Embryo Fibroblast; Newcastle Disease Virus Strain; Bovine Respiratory Syncytial Virus

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