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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.

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.

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.

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.

Newcastle disease virus causes (NDV) apoptotic death of infected cells. In the present study, the stimulus that provoked the induction of apoptosis in infected cells was examined. Vero cells infected with NDV developed apoptosis as characterized by DNA fragmentation and decreased DNA content. In presence of ammonium chloride, infected cells did not show reduced DNA content indicating the requirement of virus entry for the induction of apoptosis. UV-inactivated NDV did not induce apoptosis in cells suggesting the need of virus replication. Although cycloheximide blocked NDV-induced apoptosis, actinomycin-D did not, suggesting that de-novo viral protein synthesis was critical for the induction of apoptosis. In addition, activation of caspases was also detected by flowcytometry, indirect fluorescent and colorimetric assays. Based on the results, it was concluded that NDV-induced apoptosis in Vero cells required virus replication, de-novo protein synthesis and caspase activation.

1. The immune responses induced by recombinant plasmids containing Newcastle disease virus (NDV) F (pVAX.nd.f) or HN (pcDNA.nd.hn) genes separately or in combination in bi-cistronic (pIRES.nd.hn.f) constructs were evaluated in maternal antibody-positive commercial chicks. 2. Immunofluorescence and immunoperoxidase tests demonstrated the expression of both F and HN proteins in Vero cells. Real-time PCR analysis revealed the expression of HN and/or F genes in muscle, peripheral blood mononuclear cells (PBMC), spleen and liver after immunisation. 3. Chicks inoculated intramuscularly thrice (two booster doses) with pVAX.nd.f and pcDNA.nd.hn did not develop detectable haemagglutination inhibiting (HI) antibodies. In contrast, an increase in a NDV-specific cell-mediated immune response was demonstrated. 4. After challenge with virulent NDV, chicks immunised with the recombinant plasmids as well as those in control groups succumbed to Newcastle disease. 5. Based on these results, it is concluded that DNA vaccines containing HN and/or F genes fail to protect commercial chicks, possibly due to interference from maternal antibodies.

Newcastle disease virus (NDV), an avian paramyxovirus, induces apoptosis in chicken embryo fibroblast (CEF) cells. In the present investigation, the ability of haemagglutinin-neuraminidase (HN) protein of NDV to cause apoptosis in CEF cells was examined. The results revealed that cells expressing the HN protein demonstrated decreased DNA content, phosphatidylserine exposure and increased cytoplasmic vacuolation. Up-regulation of caspase-1, -9, -8, -3, loss of mitochondrial transmembrane potential and an increase in oxidative stress were also observed in cells expressing the HN protein. Based on the above results it can be concluded that HN protein of NDV causes apoptosis in CEF cells.