Phylogenetic and pathotypic characterization of newcastle disease virus in Tibetan chickens, China

Chickens are considered to be potential reservoirs of Newcastle disease virus (NDV). In this study, six Newcastle disease virus strains were isolated and characterized in Tibetan chickens. The HN gene was sequenced, and phylogenetic relationship to reference strains was studied. The phylogenetic analysis demonstrated that these six isolated strains were closely related to NDV isolates of the reference strains GQ245823, KT002186, KU527561, KJ563939, AY225110, EU305607, KM056357, Y18898, GQ245832, AF077761 and lasota strain. Among them, EU305607, KJ563939 and KM056357 were isolated from India, while lasota strain came from attenuated vaccine widely used in China. Then, mean death time (MDT) and intracerebral pathogenicity index (ICPI) were used to estimate the pathogenicity of the isolates. Pathogenicity experiment showed HNH1 and HN17 to be virulent. Our results indicated that genetically diverse viruses circulate in Tibetan chickens, and based upon the phlogeographic analysis, we estimated the origin of ancestral viruses of the isolates and its sister strains located in India and China (lasota strain). It indicates the importance of continuous surveillance to enhance current understanding of the genetic evolution of the NDV strains.(AU)

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.

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

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.