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An Efficient Approach for the Recovery of LaSota Strain of Newcastle Disease Virus from Cloned cDNA by the Simultaneous use of Seamless PCR Cloning Technique and RNA-POL II Promoter
Faisal Masoud1, Muhammad Shahid Mahmood*1, Sajjad-ur-Rahman1 and Rao Zahid Abbas2

1Institute of Microbiology, University of Agriculture, Faisalabad
2Department of Parasitology, University of Agriculture, Faisalabad
*Corresponding author:


The reverse genetic system for the LaSota strain of Newcastle disease virus (NDV) based on RNA POL II promoter is currently unavailable. This study was designed to produce the LaSota virus from the cDNA in the laboratory. The cDNA was cloned in mammalian expression vector pcDNA 3.1 and it was flanked by hammerhead and hepatitis delta virus ribozyme. The virus was rescued from the Vero cell line by simultaneous in-vitro transcription of full-length LaSota strain clone along with support plasmids i.e. NP, P, and L genes. The RNA polymerase was produced by cell lines themselves; there was no need to provide it by outside means. The progeny of the recombinant NDV shows the presence of an artificially introduced genetic marker (A set of three nucleotides) in the intergenic space of the F and HN genes. The haemagglutinin titer (HA) of rescued and parenteral viruses indicates values of 28 and 27; while the mean death time assay (MDT) score of the viruses was 106 and 104 hrs, respectively. The embryo infective dose (EID50/mL) was lower in recombinant virus (109.10) as compared to the wild virus (109.24). The growth curve of both viruses was indistinguishable from each other, which shows that recombinant LaSota is a faithful copy of the parenteral virus. The results predict that; this system can facilitate the easy, efficient and robust rescue of viruses, for their use as a vaccine vector.

To Cite This Article: Masoud F, Mahmood MS, Sajjad-ur-Rahman, Abbas RZ 2022. An efficient approach for the recovery of LaSota strain of Newcastle disease virus from cloned cDNA by the simultaneous use of seamless PCR cloning technique and RNA-POL II promoter. Pak Vet J, 42(3): 346-351.


ISSN 0253-8318 (Print)
ISSN 2074-7764 (Online)