Impact Of Fusion Protein On The Virulence Of Newcastle Disease Virus

Newcastle disease is one of the most serious poultry diseases. It caused byNewcastle disease virus (NDV). NDV belongs to rubulavirus of paramyxovirinae inparamyxoviridae. NDV is consisted of six genes encoding six proteins. The toxicity ofNDV is related with F gene. Study found that the number of basic amino acid of thecleavage site of the F protein was closely related of NDV toxicity. So it is important tostudy the nucleotide sequence encoding the cleavage site amino acids.In this study,329F gene nucleotide sequences of fragment length1662bp, whichbelong to isolates of China were downloaded from National Center for BiotechnologyInformation (NCBI). And the sequences were analyzed phylogenetically and theamino acid sequences of F protein were analyzed in this study. The cleavage sites ofthe F protein were related with the toxicity through the study of Amino acid of the Fprotein. In order to verify the above results,18China NDV isolates were analyzedphylogenetically and the sequences of their F protein genes were compared with thoseof other published NDV strains from different regions of the globe. And the aminoacid sequences of F protein were analyzed in this study. The result showed that Chinaisolates belonged to two branches: Class Ⅰa nd Class Ⅱ, ClassⅡ branch had the mostisolates. the amount of genotype Ⅰ, Ⅱ, Ⅵ, Ⅶ andⅨ strains accounted for about99%. The largest number of strains was Genotype Ⅶ, especially sub-genotype Ⅶd.This article set a theoretical foundation for the study of NDV virulence molecularmechanisms and control of Newcastle disease outbreak.F and HN proteins are two kinds envelope protein of the Newcastle disease virus.They related with the fusion of the virus with the host cell membrane. Wherein the Fprotein is the main reason for the virulence of different NDV, and the cleavage site ofthe F protein has played an important role. In this study F、HN genes were cloned, andnucleotides of the F gene cleavage sites were mutated as the same nucleotides as thelentogenic and velogenic cleavage points. And all the genes were connected to eukaryotic expression vector of pcDNA3.0. Then the plasmids grouping transfectedinto BHK-21cells was observed cell fusion. In the same time PEGFP-N3-T7andpGFP-T7plasmids were transfected into BHK-21cells of every groups. Theexpression within the transcriptional activity of T7RNA polymerase made the F,HN, GFP gene co-expression. Through the experimental results can be found that Fprotein cleavage site is indeed closely related to the cell membrane fusion.RNA virus reverse genetics system can be oriented to change the gene sequence ofthe virus and detected the artificial virus phenotype. In this study F5-pMD18-Tplasmid containing Mukteswar vaccine strains F protein cleavage site gene wasmutated to lentogenic F protein cleavage site gene. F5-pMD18-T and F6-pMD18-Tplasmids were digested with SpeⅠ andEcoRⅠ. Then the results were connected toFR5-6-pMD18-T plasmid. FR5-6-pMD18-T plasmid was digested with XbaⅠ、StuⅠ、Vsp Ⅰ, F1-4-pMC18plasmid was digested with XbaⅠ、StuⅠ. Then the resultwas connected to FR5-6-pMD18-T plasmid with T4ligase. The results were detectedwith three digested verify group, they were Xba Ⅰ andStuⅠ,SnaB Ⅰa ndStu Ⅰ,SnaB Ⅰ andEco72Ⅰ. Then F1-6-pMC18was digested with SnaB Ⅰ andXbaⅠ.F6-11-HT-pMC18was digested with XbaⅠ, SnaB Ⅰa ndVsp Ⅰ. The results wasTheresults were detected with six digested verify group, they were SnaB Ⅰa ndXba Ⅰ,NcoⅠ andXbaⅠ, SacⅡ a ndNco Ⅰ,Mlu Ⅰ,BstE Ⅱ, NsiⅠ.The results showed thatthe full-length cDNA of Mukteswar vaccine strains was spliced. This study made afoundation of the construction of Newcastle disease virus reverse genetics system.This study also provides theoretical support to build artificial Metro vaccine.