Virulence Research, Complete Genome Sequencing, Construction Of Full Length CDNA Of Newcastle Disease Virus

Newcastle disease （ND） is a badly infectious disease caused by Newcastle disease virus（NDV）andpossesses a great threat to the poultry farming. NDV, a member of Avulavirus in Paramyxoviridae family, isa non-segmented, single-stranded, negative-sense RNA virus with about15kb genome. It’s structure is3’Leader-NP-P-M-F-HN-L-5’Trailer, which encodes six structural proteins and two nonstructural proteins.Although NDV only has one serotype, it could be divieded into two classes-class I and class II-based onF gene or complete genome. The class I and class II consists of genotype I^IX and I^XI, respectively. NDVhas a wide arrange of hosts and it can infect about240kinds of wild birds. Wild birds are as a naturalreservoir of NDV, which plays a pivotal role in heredity, evolution and virulence change of NDV. In2008,two NDV strains were isolated from wild birds by my lab at North of Qinling Mountains，Shaanxi，China.One of strains was from Eurasian Blackbird and another was from Spotted-necked Dove. Previous researchindicated that both strains belonged to genotype Ⅸ, while most of strains from poultry were subgenotypeⅦd. According HN gene analyse，the homogeneous of both strains was beyond99%. However, ICPI ofvirus from Blackbird amounted to1.638, while that of virus from Spotted-necked dove just accounted for0.425. Therefore, researching virulence of both viruses, sequencing complete genome of these strains, andconstructing reverse genetic system of wild birds origin NDV, will offer important information forresearch of heredity, evolution and virulence of NDV. Two NDV strains（Blackbird/China/08andSpottedDove/China/08） were enployed as the the material of this project, which was isolated from wildbirds by my laboratory in QinLing Mountains in2008. This work began with research of virulencecombining complete genome sequence of both strains, and then analysed the heredity and evolution ofNDV and major factors that contribute to the viral virulence. Eventually, the full length cDNA of one of thestrains was constructed, which laid a foundation for the further construction of NDV’s reverse geneticsystem, research of viral mechanism and development of vaccine. The major results are following:1. The gap of ICPI （intracerebral pathogenicity index） value of both strains were studied anddifferences has been detected: the value of ICPIof Blackbird/China/08was1.55that belonged to thestandard of velogenic stains, while SpottedDove/China/08was just o.425that situated in the range oflentogenic strains. After injecting virus, the death rate of chicks was higher in the group ofBlackbird/China/08than that of SpottedDove/China/08. All chicks in the group of Blackbird/China/08were died at the sixth day after injecting virus, while there were two chicks died until the third day after injectingvirus in the group of SpottedDove/China/08and total three chicks died finally. The proliferous ability ofBlackbird/China/08was stronger than that of SpottedDove/China/08on DF-1cells. The titer ofBlackbird/China/08in the culture solution of cells was1.1×10⁵PFU/mL at the12th hour after infection,while it just was7.0×103PFU/mL for SpottedDove/China/08. The fusion ability and ability to form celllesions of Blackbird/China/08was stronger than that of SpottedDove/China/08. At the24th hour ofpost-infection, the number of cell nucleus of syncytium formed by Blackbird/China/08was more thanthat of SpottedDove/China/08, and cell pathological change （CPE） of Blackbird/China/08was moreobvious than that of SpottedDove/China/08.2. Complete genome of two wild birds origin NDV strains were successfully constructed by RT-PCR（Reverse Transcriptional PCR）. The complete genome length of the two strains were both15192bp. Afteranalysis the complete genome sequence, we found the cleavage site of F protein of both strains was¹¹²R-R-Q-R-R-F¹¹⁷, and the number of amino acid residues of their HN protein was571, and the functionalregion of L protein was⁷⁴⁶CMVQGDNQVI⁷⁵⁵, and there were7cysteine residues in the C terminal of Vprotein. Though alignment of both strains complete genome, homology of both strains arrived at99.9%,and there were17and9differences in terms of their genome and structural proteins, respectively. Amongthe differences of their structural proteins, two differences situated in the77th and10⁵th amino acidresidues of P protein, and one difference was in the16th of M protein, and three differences were in the110th,468th, and522th of HN protein, and three differences were in1076th,1728th, and1876th of Lprotein. Both strains belonged to the genotype IX based on phylogenetic tree of partial F gene and completegenome. Furthermore, they were closed to virulent NDV of the genotype Ⅸ and Ⅲ and the homologybetween those strains arrived at91.7%⁹9.8%.3. The annealing temperature, expanded time and reaction system of fusion PCR was modified andedstablished in this project, as a consquence, the full length cDNA of one strain was successfullyconstructed, which laid a foundation for the further construction of reverse genetic system of NDV. Toconstruct the complete genome of NDV, several days or even months is required depended on restrictionenzyme. However, using this modified fusion PCR, it not only saved the expensive high fidelity DNApolymerase with highly efficient amplification, but also reduce the construction time of the completegenome of NDV to two days. This method saved lots time and speed up the establishment of revers geneticsystem and also affords a quick way to research and development of vaccine, especially that of emergentinfectious disease.