Generation Of Virulent Newcastle Disease Virus Strain ZJI Isolated From An Outbreak In The Goose From Genomic CDNA Clone

Newcastle disease virus (NDV) is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. It is the causative agent of Newcastle disease (ND), which is widely distributed and has inflicted substantial economic costs to poultry industry worldwide. Aquatic birds are usually considered to be resistant even to most virulent NDV strains for chickens, however, outbreaks of clinical ND in goose flocks in Southern and Eastern China have been reported frequently since late 1990. This disease entity has posed a great threat to goose production and other poultry industry and attracted much attention. Therefore, it is highly imperative to elucidate systematically the genesis, characteristics, genetical evolution and pathogenicity of the pathogens. More importantly, it is in urgent need to develop a novel and effective vaccine to prevent the infection and circulation of the virulent NDV in both goose and chicken flocks. Reverse genetics manipulation has been established in recent years based on cloned cDNA of RNA viruses. It has gradually become a very significant method for the research of molecular virology, especially for the research of virus functional genome, construction of chimeric viruses and expression of heterologous genes.Several virulent NDV strains from outbreaks in goose flocks have been isolated and characterized pathotypically and genotypically in recent years in our laboratory. The results demonstrated that all the isolates are virulent strains and belong to the genotype Vlld. The complete genomic sequence of one of these goose strains, ZJI, has been determined to be 15192 nts rather than 15186 nts of the published length of La Sota, CloneSO, Beaudette C and Hitchner Bl. An additional six nucleotides (nt) motif is present in the noncoding region of NP gene of ZJI genome and this characteristic isshared by ND viruses of genotype V-IX. It is of crucial importance to explore the role played by the six nts motif insertion in NDV evolution. Reverse genetics technique is a powerful tool to study genomic structures and functions. For this purpose, three expression plasmids, or helper plasmids, containing NP, P or L gene from NDV strain ZJI respectively were constructed. In addition, the function of the helper plasmids was identified and evaluated by the constructed NDV mini-genome. Site-directed mutagenesis was conducted to modify the full-length cDNA clone. The modified full-length cDNA of ZJI and the helper plasmids originated from La Sota were cotransfected into BSR-T7/5 cells. Infectious NDV LaZJIR was successfully generated and identified. The rescued virus was indistinguishable from the wild-type ZJI with respect to its virulence and growth characteristics in cell culture and in embryonated chicken eggs. The results proved that the recovered virus is a faithful copy of the parent NDV strain ZJI. 1. Construction of expression vectors with NP, P and L genes of ZJI andidentification of their functionThree pairs of primers were designed on the basis of the published cDNA sequence of NDV strain ZJI. NP, P and L gene fragments of 1.54kb, 1.3kb and 6.7kb in length respectively, containing corresponding entire open reading frame (ORF) were amplified by high-fidelity reverse-transcription polymerase chain reaction (RT-PCR). The PCR products were analyzed by gel electrophoresis and then cloned into pGEM-T easy vector. The fragments of interest were removed from the recombinants and subcloned into eukaryotic expression vector pCI-neo to form the recombinants pCI-NP, pCI-P and pCI-L, respectively. The constructed recombinants were identified by restriction enzymes digestion and the results revealed that the fragments were successful inserted. The green fluorescent protein (GFP) gene in the vector pEGFP was removed and inserted into the downstream of pCI-P, thereby the GFP gene was fused with P gene to form recombinant pCI-P-GFP. The recombinant was then transfected into chicken embryo fibroblast (CEF) and COS-1 cells respectively. Specific fluorescence in the transfected cell cultures wa...