[Show abstract][Hide abstract] ABSTRACT: The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-a, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-a of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 1.21; 5.22 0.60%, and TNF-a gene expression was found to be 15.44 0.42; 6.51 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-a act synergistically in the induction of apoptosis in HeLa cells.
[Show abstract][Hide abstract] ABSTRACT: The Non-Structural protein 1 of Canine Parvovirus – 2 (CPV2.NS1) plays a major role in viral cytotoxicity and pathogenicity. CPV2.NS1 has been proven to cause apoptosis in HeLa cells in vitro in our laboratory. Here we report that CPV2.NS1 has no toxic side effects on healthy cells but regresses skin tumors in Wistar rats. Histopathological examination of tumor tissue from CPV2.NS1 treated group revealed infiltration of mononuclear and polymorphonuclear cells with increased extra cellular matrix, indicating signs of regression. Tumor regression was also evidenced by significant decrease in mitotic index, AgNOR count and PCNA index, and increase in TUNEL positive apoptotic cells, in CPV2.NS1 treated group. Further, CPV2.NS1 induced anti-tumor immune response through significant increase in CD8+ and NK cell population in CPV2.NS1 treated group. These findings suggest that CPV2.NS1 can be a possible therapeutic candidate as an alternative to chemotherapy for treatment of cancer.
Research in Veterinary Science 08/2014; · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The use of chemotherapy and/or radiotherapy for treatment of cancer is limited due to genotoxic side effects on healthy
cells, involvement of anti-apoptotic signal transduction pathways that prevent cell death, and requirement of functional p53
for induction of apoptosis in cancerous cells. Efforts are beings made worldwide to develop new anticancer therapies as an
alternative to chemotherapy. And viral gene therapy is one of the most potent therapeutics that is being ventured worldwide.
Canine parvovirus-2 (CPV-2) is one of those viruses that have an inherent oncolytic property. The non-structural protein-1
(NS1 protein) of CPV-2 plays a major role in parvoviral cytotoxicity and pathogenicity in permissive cells. The oncolytic
potential of CPV2-NS1 has been established in vitro. Prior to taking up the in vivo studies, the present study was undertaken
to clone Canine Parvovirus NS1 gene and reporter gene GFP in eukaryotic expression vector pVIVO2-mcs, and to
characterize the double construct in mammalian cells. The genes were successfully cloned in pVIVO2-mcs and
characterized for their expression as demonstrated by fluorescence microscopy and immunofluorescence staining. This
characterized double gene construct will further be used to evaluate the oncolytic potential of CPV-2 NS1 in experimentally
induced in vivo tumour model.
Indian Journal of Biotechnology 01/2014; 13:41-46. · 0.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the "new CPV-2a" in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.
Applied biochemistry and biotechnology 10/2013; · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, Indian Newcastle disease virus (NDV)
isolates UP/3 and UP/4 collected from suspected field samples
were characterized to be velogenic by pathogenic and
phylogenetic analysis. Phylogenetic analysis based on the
nucleotide sequences of the F gene indicated that UP/3 and UP/4
isolates belong to genotype VII and genotype IV, respectively.
These isolates possessed the amino acid sequence 112R/K-R-QK/
R-R-F117 in the F0 cleavage site, which is typical of velogenic
NDV pathotype. All the NDV strains analyzed have shown to
have non-synonymous to synonymous base substitution rate in the
F gene ranging between 0.074-0.197, demonstrating the presence
of purifying selection. HN gene of the isolates was found to have
an open reading frame encoding 571 amino acids. The deduced
amino acid sequences of the HN glycoprotein revealed that the
cysteine residues essential for intra-molecular disulphide bonds to
stabilize the HN molecules, antigenic sites and key residues for
receptor binding were all conserved in Indian isolates
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.
[Show abstract][Hide abstract] ABSTRACT: Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.
Indian journal of experimental biology 02/2013; 51(2):116-23. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.
Indian journal of experimental biology 09/2012; 50(9):618-24. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer is one of the killer diseases in humans and needs alternate curative measures despite recent improvement in modern treatment modalities. Oncolytic virotherapy seems to be a promising nonconventional way to treat cancers. Newcastle disease virus (NDV), a poultry virus, is nonpathogenic to human and domestic animals and has a long history of being used in oncotherapy research in several preclinical studies. The ability of NDV to successfully infect and destroy cancer cells is dependent on the strain and the pathotype of the virus. Adaptation of viruses to heterologous hosts without losing its replicative and oncolytic potential is prerequisite for use as cancer virotherapeutics. In the present study, velogenic NDV was adapted for replication in HeLa cells, and its cytotoxic potential was evaluated by observing morphological, biochemical, and nuclear landmarks of apoptosis. Our results indicated that the NDV-induced apoptosis in HeLa cells was dependent on upregulation of TNF-related apoptosis-inducing ligand (TRAIL) and caspases activation. Different determinants of apoptosis evaluated in the present study indicated that this strain could be a promising candidate for cancer therapy in future.
Applied biochemistry and biotechnology 05/2012; 167(7):2005-22. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.
Indian journal of experimental biology 05/2012; 50(5):325-31. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parvoviruses are small, 260-A-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.
Indian journal of experimental biology 09/2011; 49(9):654-9. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine.
Veterinary Immunology and Immunopathology 07/2011; · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HN gene of NDV is known to possess the inherent ability to induce apoptosis in transformed cells. It is reported that HN is a potent inducer of interferons and capable of upregulating the TNF-α related apoptosis inducing ligand (TRAIL).Tumor necrosis factor- has been shown to exhibit direct anti tumor activity, killing some tumor cells and reducing the rate of proliferation of others while sparing normal cells. Apoptotic response of TNF-α is mediated by the activation of caspase-8. The present study was under-taken to determine the oncolytic effect of HN protein and TNF-α when used in combination on N-methyl-N-nitroso urea induced mammary tumor in rats. For the assessment of oncolytic effect of HN and TNFα, mammary tumor were produced in 30 female Sprague Dawley rats (age 6-7 weeks) by injecting 50 mg/kg body weight of N-methyl-N-nitroso urea intra-peritonealy. The rats were grouped in 5 groups (n=6) viz I, II, III, IV and V. The recombinant pcDNA-nd.hn.kozak was injected intratumorally @100 µg/rat in group I, and group II rats were treated with pcDNA.tnf α ozak.rat. The group III rats were treated with both the recombinant clones. The group IV rats were taken as vector control while group V was taken as mock control. Three doses of recombinants were given at a gap of one week. All rats were sacrificed humanely on 7 th day from last treatment and peripheral blood and tumor tissues were collected from these rats for the assessment of immune response and oncolytic effect by assessing proliferation of CD4 +
[Show abstract][Hide abstract] ABSTRACT: Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.
Indian journal of experimental biology 02/2011; 49(2):140-5. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granulocyte-macrophage colony stimulating factor (GMCSF), a multifunctional cytokine can enhance immune responses when administered along with DNA vaccine. Aim of the present study was to clone and express the chicken GMCSF cytokine for use as 'genetic adjuvant'. Chicken GMCSF gene 435bp was amplified using specific primers in which restriction sites of BamHI and HindIII were at forward and reverse primers respectively. The PCR product was cloned into eukaryotic expression vector pcDNA 3.1(+) and clones were confirmed by restriction digestion and nucleotide sequencing. Functional activity of recombinant GMCSF was checked by expression of GMCSF specific mRNA in transfected Vero cells by RT-PCR of total RNA isolated from transfected Vero cells. The recombinant plasmid can be used as genetic adjuvant in chicken.
Indian journal of experimental biology 12/2010; 48(12):1175-80. · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine characterized by high fever and multiple haemmorhages. The E2 glycoprotein of CSFV is immunogenic and induces neutralizing antibodies against CSFV. In the present study, complete coding region of the E2 gene from Indian virulent field isolate (Mathura) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently cloned into a mammalian expression vector; pcDNA3.1(+) at BamHI and XbaI site. The recombinant plasmid; pcDNA.E2.CSFV. was confirmed by restriction enzyme digestion. The pcDNA.E2.CSFV. transfected Vero cell expressed E2 protein which was confirmed by western blotting, immunoperoxidase and indirect immunofluorescent tests. Additionally, flow cytometry analysis also confirmed that 15% of transfected Vero cells expressed the E2 glycoprotein compared to mock or vector alone transfected cells. Further study is under way to evaluate recombinant pcDNA.E2.CSFV. Mathura clone as DNA vaccine against CSFV.
Indian Journal of Virology 06/2010; 21(1):69-75. · 0.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine
characterized by high fever and multiple haemmorhages. The E2 glycoprotein of CSFV is immunogenic and induces neutralizing
antibodies against CSFV. In the present study, complete coding region of the E2 gene from Indian virulent field isolate (Mathura)
was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently cloned into a mammalian expression
vector; pcDNA3.1(+) at BamHI and XbaI site. The recombinant plasmid; pcDNA.E2.CSFV. was confirmed by restriction enzyme digestion. The pcDNA.E2.CSFV. transfected
Vero cell expressed E2 protein which was confirmed by western blotting, immunoperoxidase and indirect immunofluorescent tests.
Additionally, flow cytometry analysis also confirmed that 15% of transfected Vero cells expressed the E2 glycoprotein compared
to mock or vector alone transfected cells. Further study is under way to evaluate recombinant pcDNA.E2.CSFV. Mathura clone
as DNA vaccine against CSFV.
KeywordsClassical swine fever (CSF)-Classical swine fever virus (CSFV)-Reverse transcription-polymerase chain reaction (RT-PCR)
Indian Journal of Virology 01/2010; 21(1):69-75. · 0.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Newcastle disease virus (NDV) causes economically significant Newcastle disease (ND) in almost all birds worldwide. Previous studies have shown that NDV induces caspase dependent apoptotic pathways in infected cells. In the present study, time course induction of apoptotic pathways in Vero cells is described. In NDV-infected cells, caspase-8 activity, percentage of cells showing TRAIL expression was higher at 24h p.i. (post-infection) compared to 48 h p.i. In contrast, caspase-9 activity, efflux of cytochrome c, loss of mitochondrial membrane potential was higher at 48 h compared to 24h p.i. The caspase-3 activity was high both times. Based on these results, it was concluded that at 24h p.i., NDV induces apoptosis through extrinsic apoptotic pathway while at 48 h p.i. predominantly through intrinsic apoptotic pathway.
Virus Research 07/2009; 144(1-2):350-4. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways.