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Oncolytic Properties Of Newcastle Disease Virus And Its Recombinant Hemagglutininneuraminidase Protein

KrishiKosh

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Title Oncolytic Properties Of Newcastle Disease Virus And Its Recombinant Hemagglutininneuraminidase Protein
 
Creator Bhanudas, Jagtap Ramesh
 
Contributor Raja, A.
Kumanan, K.
Sridhar, R.
Kirubaharan, J. John
 
Subject Newcastle disease virus
Oncolytic properties
Cancer cell lines
MTT assay
Flow cytometry
Real time RT-PCR
Nanoparticles
Canine mammary tumor cells
 
Description Oncolytic properties of the Indian isolates, velogenic NDV 2K17, mesogenic
NDV 2K36 and 2K3 were studied. To study the oncolytic properties of NDV isolates,
three cancer cell lines, MCF-7 (Human mammary adenocarcinoma), HeLa (Human
cervical carcinoma) and A72 (Canine carcinoma) were used. Each NDV isolate was
infected into these cell lines individually and the virus induced cytopathic changes in
these cell lines were recorded by staining the cells with H&E stain and Giemsa stain.
Transmission electron microscopy was used to confirm the presence of NDV in the
cancer cell lines.
NDV isolates were titrated in MCF-7 cancer cell line. The log TCID50 titre of
velogenic isolate NDV 2K17 was 10^3.7 TCID50 per 50 μl of virus inoculums. Whereas,
TCID50 titers of mesogenic isolates NDV 2K36 and NDV 2K3 were 10^4.5 and 10^4.3
TCID50 per 50 μl of virus inoculums in MCF-7 cell line.
Each cancer cell line was infected with 1, 0.5 and 0.25 MOIs and the cytotoxicity
was measured by MTT assay. At 1 MOI, NDV 2K36 produced the least cell viability of
21.71%, 27.83% and 48.07% at 72h p.i in MCF-7, HeLa and A72 cell line respectively
when compared to other two NDV isolates.
In flow cytometry, NDV 2K36 isolate produced more apoptotic percentage,
70.87%, 89.56% at 72h p.i in MCF-7 and HeLa cells over the other two isolates whereas
in A72 cells, NDV 2K3 induced slightly higher percentage of apoptosis, 74.87% at 72h
p.i when compared to NDV 2K36, 71.83% at 72h p.i. Among the three cell lines studied,
NDV isolates induced more apoptosis in HeLa cell line than the other two cell lines. In
real time RT-PCR, the expression levels of bax and caspase-3 genes were in time
dependent manner. The NDV-NP gene expression was also showed that NDV isolates
also increased their mRNA expression levels in time dependent manner. This indicates
that all three NDV isolates induced apoptosis is bax dependent and both intrinsic and
extrinsic pathways of apoptosis could be activated during oncolysis.
In gold nanoparticle coupled NDV, NDV 2K36 produced significantly higher
percentage of apoptosis (85.31%) when compared to the other two NDV isolates in HeLa
cells. There was no significant difference between the gold nanoparticle coupled NDV
2K36 and uncoupled NDV 2K36 induced apoptosis. In calcium phosphate nanoparticle
coupled NDV isolates on HeLa cell line, NDV 2K36 produced significantly higher
percentage of apoptosis (84.51%) when compared to the other two NDV isolates. There
was no significant difference between the calcium phosphate nanoparticle coupled NDV
2K36 and uncoupled NDV 2K36 induced apoptosis.
NDV HN protein gene containing plasmid (pHN) coupled with gold
nanoparticles induced significantly higher percentage of apoptosis (22.36%) than the
pHN transfected by using lipofectamine and control plasmid in HeLa cells.
Ten canine mammary tumors were collected from the clinical cases and the
tumor cell suspension was infected with NDV 2K36 isolate. The apoptotic percentage
ranged from 2.35 to 12.6 with a mean of 4.5 for 24h p.i, 5.37 to 14.92 with a mean of
9.07 for 48h p.i and 11.63 to 26.20 with a mean of 17.59 for 72h p.i. and in case of
infected cells, the apoptotic percentage ranged from 18.66 to 41.80 with a mean of 25.44
for 24h p.i, 34.05 to 55.04 with a mean of 39.55 for 48h p.i and 52.70 to 76.14 with a
mean of 51.65 for 72h p.i.
 
Date 2016-05-23T16:50:55Z
2016-05-23T16:50:55Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66237
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University