Production and characterization of monoclonal antibodies to Newcastle Disease Virus.

Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot.

α-Glucosidase Inhibitory Activity of 4-Aryl-N-(2,4-Thiazolidinedione-5-Acetyl)-1,3-Thiazol-2-Amines.

A series of N-(4-aryl-1,3-thiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides 3a-k and N- (1,3-benzothiazol-2-yl)-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamides 3l-n are synthesized and evaluated for their α-glucosidase inhibitory activity. N-[4-(m-Chlorophenyl)-1,3-thiazol-2yl]-2-(2,4-dioxo-1,3-thiazolidin-5- yl)acetamide (3g) and N-[4-(o-fluorophenyl)-1,3-thiazol-2-yl]-2-(2,4-dioxo-1,3-thiazolidin-5-yl)acetamide (3j) have shown very good inhibition. The remaining compounds have exhibited moderate to good activity ranging from 37- 63 % of α-glucosidase enzyme inhibition.

Development of dog mammary tumor xenograft in immunosuppressed Swiss albino mice.

Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and γ-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

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.

Oncolytic viruses & their specific targeting to tumour cells.

Cancer is one of the major causes of death worldwide. In spite of achieving significant successes in medical sciences in the past few decades, the number of deaths due to cancer remains unchecked. The conventional chemotherapy and radiotherapy have limited therapeutic index and a plethora of treatment related side effects. This situation has provided an impetus for search of novel therapeutic strategies that can selectively destroy the tumour cells, leaving the normal cells unharmed. Viral oncotherapy is such a promising treatment modality that offers unique opportunity for tumour targeting. Numerous viruses with inherent anti-cancer activity have been identified and are in different phases of clinical trials. In the era of modern biotechnology and with better understanding of cancer biology and virology, it has become feasible to engineer the oncolytic viruses (OVs) to increase their tumour selectivity and enhance their oncolytic activity. In this review, the mechanisms by which oncolytic viruses kill the tumour cells have been discussed as also the development made in virotherapy for cancer treatment with emphasis on their tumour specific targeting.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

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 × 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.

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

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 37ºC. The 6×His-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.

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line.

Parvoviruses are small, 260-Å-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.

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus.

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.

Newcastle disease virus as an oncolytic agent.

Cancer is a major cause of deaths in humans. Though there has been significant progress in cancer therapy, the limited efficacy and toxicities of current chemo- and radiotherapies have provided an impetus for the search of new therapeutics. A therapeutic approach, which uses viruses for the treatment of cancer termed, oncolytic virotherapy has recently emerged. Newcastle disease virus (NDV) is one such virus with an inherent oncolytic property. NDV causes a highly infectious disease in poultry worldwide. In humans it is reported to have oncolytic and immuno-stimulatory effects. It specifically replicates in tumour cells while sparing normal cells and cause oncolysis. For many years different strains of the NDV have been investigated for treatment of various human cancers. Recent advances in reverse genetics provided investigators the tools to produce recombinant NDV with improved oncolytic property.