Effect Of V Protein Of NDV Yulin Strian On Type I IFN Signaling Pathway Related Factors And Construction Of Full-length CdNA Of Strain Yulin

Newcastle disease(ND) is one of the highly contagious avain diseases. It causes severe economic losses in poultry industry worldwide. The etiological agent is Newcastle disease virus(NDV), which is a member of genus Avulavirus in family Paramyxoviridae. It has a single-stranded, negative-sense RNA genome that encodes six proteins: Nucleoprotein(NP), Phosphoprotein(P), Matrix protein(M), Fusion protein(F), Haemagglutinin-neuraminidase(HN), large RNA-dependent RNA polymerase(L). In addition, NDV generates two additional proteins V and W by P gene RNA editing. P/V/W proteins have common residues in the N-terminus, but they are different in their C-terminus. The V protein is responsible for blocking the antiviral action of type I IFN(IFNα and β). It can block the IFN signal pathway by targeting STAT1 via proteasome-mediated degradation and inhibition phosphorylation of STAT1. The mechanism of NDV V protein antagonizing IFN signal is unclear. In this study, we use reporter gene system and real time PCR to detect the mRNA expression levels of IFN signaling pathway related genes in the NDV strain Yulin infection and V protein transfected cells. Meanwhile, full-length cDNA of stain Yulin was successfully constructed. It would lay the foundation of NDV reverse genetic system for researching the function of NDV V protein. The major results are following:1. The ability of Vprotein of NDV antagonism type I interferonTo detect the role of V protein of NDV in type I IFN signaling pathway, this study use dual-luciferase assay to detect the activity of ISRE(IFN-stimulated response element), which is specifically related to type I IFN signaling pathway. The results showed that NDV V protein inhibited the activity of ISRE. Velogenic strain Yulin inhibited the activity of ISRE is stronger than lentogenic strain La Sota.2. Effect of NDV V protein on typeⅠIFN signaling pathway related factorsAfter Newcastle disease virus strains Yulin and La Sota infected Hep-2 cells, we used real-time PCR to detect the mRNA expression levels of the related factors in typeⅠ signaling pathway. The results showed that the mRNA expression levels of ISG56, ISG15 and STAT1 were increased significantly. After transfection the V protein of NDV strains Yulin and La Sota, the mRNA expression levels of ISG15 and STAT1 significantly were reduced.3. Construction of full-length cDNA of NDV strain YulinIn order to obtain the full-length cDNA of strain Yulin, a segmented genome cloning strategy was used. Eight fragments(F1 to F8) were amplified by high-fidelity RT-PCR. The T7 RNA polymerase promoter was introduced in upstream of the F1 fragment. T7 RNA polymerase termination and hepatitis Delta virus ribozyme sequence were introduced to the downstream of F8 fragment by fusion PCR. All eight fragments were successively cloned into the modified pBR322 vector. Compared with the original genome sequences, there were four mutations on the plasmid, three of which were synonymous mutations. Another mutation caused amino acid change, but it also existed in the other NDV strains, suggesting that this mutation could not affect subsequent virus rescue. The full-length cDNA construct lays the foundation for NDV reverse genetics system. It would be further used to research the mechanism of IFN antagonism of NDV V protein.