藏鸡NDV的分离鉴定及F基因的遗传进化分析

摘要

【Objective】The study was to investigate the Tibetan chicken of Newcastle disease virus virulence and phylogenetic characteristics of the F gene.【Method】From the acquisition of the disease compound isolation and identification of NDV,MDT and ICPI were used to determine the virulence of the isolates.RT-PCR was used to clone Tibet chicken NDV isolates F gene,and then sequencing and evolutionary analysis were conducted.【Result】We isolated and identified 8 NDV as F5,FX10,F20,F74,F75,F17,F72,FH2,etc,of which F17,F72,FH2 were the virulent strains,the remaining five attenuated strains.F gene sequences showed that the eight virus F gene ORF are 1662bp,encoding 553 amino acids,the virulent strain contains 12 cysteine residues,attenuated strain of 13 cysteine residues than the strong strain in 27 (virulent strain C^27 into R^27) more than one cysteine residue;There are six potential glycosylation sites.The separation of eight NDV in GenBank 20 NDV reference strains showed that the F protein amino acid sequence variants are more focused in 8-30,97-124,45,385-386,402-403,479-494,509-520 aa department,the virulent strain of AA substitution frequency is more concentrated,while the attenuated strain is the conserved region of more AA substitution dispersed focus.Separation between the NDV strains NDV F gene coding region of nucleotide sequence homology is 83.5% to 99.9%,of which 95.2% to 99.4% homology are for the virulent strains,99.4 % to 99.9% for attenuated strains.Separation between NDV strains F gene coding region of amino acid homology is 87.5% to 99.5%,of which 95.3% to 98.2% are the virulent strains;homology between the attenuated strain is 98.6% to 99.5%.Phylogenetic analysis showed that three strong strains are genotype Ⅶ,and five weak strains are genotype Ⅱ.【Conclusion】Eight NDV were separated,of which three virulent strains belong to genotype Ⅶ,and five attenuated strains belong to genotype Ⅱ.%【目的】探讨藏鸡NDV的毒力和F基因的遗传进化特征。【方法】从采集的藏鸡病料中分离鉴定NDV，通过测定鸡胚平均致死时间（MDT）和1日龄雏鸡脑内接种致死指数（ICPI）来确定分离株的毒力。采用RTPCR方法克隆藏鸡NDV分离株F基因，测序后进行序列分析，并进行进化分析。【结果】共分离鉴定出F5、FX10、F20、F74、F75、F17、F72、FH2等8株NDV，其中F17、F72、FH2为强毒株，其余5株为弱毒株。8株病毒F基因ORF均为1 662 bp，编码553个氨基酸，强毒株ORF编码产物含有12个半胱氨酸残基，弱毒株有13个半胱氨酸残基，比强毒株在27位（强毒株C^27变为R^27）多了1个半胱氨酸残基；有6个潜在的糖基化位点。分离的8株NDV与GenBank中发表的20株NDV参考毒株比较结果表明，F蛋白氨基酸序列变异位点较多，主要集中在8-30，97-124，45，385-386，402-403，479-494，509-520位氨基酸处，强毒株AA替代较集中，而弱毒株保守区较多且AA替代较分散。分离NDV株之间F基因编码区核苷酸序列同源性为83.5%～99.9%，其中强毒株之间同源性为95.2%～99.4%，弱毒株之间同源性为99.4%～99.9%。分离NDV株之间F基因编码的氨基酸同源性为87.5%～99.5%，其中强毒株之间同源性为95.3%～98.2%，弱毒株之间同源性为98.6%～99.5%。进化分析显示，3株强毒株均为基因Ⅶ型，5株弱毒株均为基因Ⅱ型。【结论】分离了8株藏鸡NDV，其中3株为强毒株，属于基因Ⅶ型；5株为弱毒株，属于基因Ⅱ型。