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Recovery Of Newcastle Disease Virus (NDV-D58 strain) From cDNA

KrishiKosh

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Title Recovery Of Newcastle Disease Virus (NDV-D58 strain) From cDNA
 
Creator Gururaj, K.
 
Contributor Kirubaharan, J. John
Chandran, N. Daniel Joy
Kumanan, K.
Ganesan, P.I.
 
Subject Newcastle disease
Reverse genetics
Genome
Gene expression
DNA sequencing
Phylogenetic tree
Plasmids
Transfection
BSR T7/5
 
Description The focus of the present study is to develop a plasmid vector backbone to clone
the cDNA of the whole genome of NDV D58 strain and to develop a system based
on reverse genetics to recover recombinant NDV D58 strain. So that the major
need for developing DIVA vaccine is made ready. The objectives of the work
were to generate a cDNA library for D58 strain of lentogenic Newcastle disease
virus, to construct an anti-genome NDV expression plasmid and expression
plasmid to carry out transfection in mammalian cells and to generate recombinant
NDV and biological characterization of recombinant NDV by Mean Death Time
(MDT).
1. The D58 strain of NDV is identified for the present study as it is a
naturally occurringenteric strain of lentogenic pathotype. It has been
thermostabilized and used as a vaccine strain. The vaccine has been named
as “ND Unique- Ranikhet disease vaccine, Live, Lentogenic,
TANUVAS D58 strain Freeze dried, I.P”
(http://globionindia.com/globivacndunique.html). The strain has been
characterized both by biological and genome analysis protocols.
2. The entire genome including the leader and trailer sequences have been
sequenced. The virus was found to contain 15,186 nucleotides including
55 leader and 114 trailer sequences. These sequences have been found to
be highly conserved. Based on the phylogenetic tree constructed using
neighbour joining algorithm at a boot strap value of 4000, the D58 strain
was found to be positioned in the clade containing lentogenic strains like
LaSota, B1 and Clone 30.
3. The bacterial plasmid pBR322 has been used for cloning the cDNA
fragments of whole genome of NDV D58 strain. The plasmid has been
subjected to three modifications to include T7 terminator, Hepatitis delta
virus ribozyme and a polylinker. The polylinker was added to facilitate
multiple cloning. The resultant modified plasmid is named as pBR322/JKRL.
4. The genome has been divided into seven fragments namely D1 – D7 of
varying length and synthesized using primers with restriction sites to clone
them into pBR322/JK-RL. These fragments have been cloned in sequential
manner and the plasmid with entire genome is named as pNDVD58fl. The
presence of the entire genome is confirmed by insert release PCR.
5. To recover the virus from this plasmid, a modified BHK21 cell constantly
expressing T7 polymerase - BSR T7/5 (a generous gift from Dr.
Conzelmann, Germany) was used. To facilitate the release,the mammalian
cell expression plasmid pCIneo was used.Three plasmids were generated
using this plasmid carrying the genes of RNP namely NP, P and L. The
generated plasmids are named as pCIneo-NP, pCIneo-P and pCIneo-L.
6. The BSR T7/5 cells were transfected simultaneously with pNDVD58fl and
pCIneo-NP, pCIneo-P and pCIneo-L at 2:1:1:1 ratio. The supernatant was
checked for viral proteins from 24 hours post transfection. The M protein
could be identified by qPCR from 24 hours onwards. However, the
haemagglutination activity was observed at 72 hours post transfection.
7. The supernatant was inoculated into embryonated chicken eggs at 48 and
72 hours post infection and the harvested virus used for estimation of mean
death time. The recombinant virus was found to have MDT more than 60
hours as parent virus.
 
Date 2016-05-24T12:38:01Z
2016-05-24T12:38:01Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66267
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University