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:
shahiduaf@gmail.com
Abstract
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.
http://dx.doi.org/10.29261/pakvetj/2022.059