华南地区三株新城疫地方强毒株的序列测定及其系统发育分析

Sequencing of HN Gene of Three Newcastle Disease Virus Isolates from South China and Phylogenic Analysis

用设计的特异性引物,通过一步法RT-PCR扩增出5个毒株的897bp片段,对其中的3株病毒cDNA分析表明:各毒株的半胱胺酸位点和个数及糖基化位点基本没有改变。各毒株在抗原决定簇氨基酸位点HN基因346～353处的碱基突变甚多,且氨基酸也出现了部分的突变。针对HN基因897bp片段构建了相应的系统发育树,系统发育分析表明:中国(华南)地区NDV各毒株与欧美国家的Ⅱ至Ⅴ基因群明显分离,遗传规律较远;NDV WS1和NDV HY与台湾省NDV各毒株归属为一组,与中国毒株的遗传距离较近,均属新近出现的基因型Ⅶ型,NDⅦ型在远东和西欧(Ⅶb)和及中东和南非(Ⅶa)广泛存在,可能形成了ND的第4次大流行。这些一方面说明NDV的变异可能已突破种源屏障,在鸡与鹅之间进行相互传播;另一方面说明中国古老的NDV毒株仍然持续存在,并逐渐向强毒株突变,与其他新出现的基因型毒株"共循环"。

The hemagglutininneuraminidase(HN) is a major pathogenic and immunogenic protein of NDV.One step RT-PCR was employed to amplify a cDNA fraction (including for four of the five hemagglutinin determinants) of HN gene of NDVWS1,NDVFS2 and NDVHY strains using a pair of specific primers.The products were directly sequenced and cloned into the TT window of pGETEasy vector.Sequencing revealed that the HN gene fraction of all the strains contained 896 nucleotides,coding for 298 amino acids.Comparison of the nucleotide sequence and deduced amino acid sequence of HN of the isolates showed 89%～99%,84%～96% and 84%～96% homology respectively with other representative NDV strains;and 90%～98%,87%～91% and 88%～93% homology for the deduced amino acid sequence.A phylogenetic tree was constructed,and the result indicated that there existed great discrepancy between NDV isolates from South China and ⅡⅤ genotype in Western countries,but NDV strains from South China were of the same lineage as those from Taiwan,belonging to the novel genotype Ⅶ.The NDVFS2 isolated from chicken had the highest nucleotide sequence homology(99%) with the GPMV/QY971 isolated from geese,both of them belonging to the classical NDV genotype Ⅰ which indicated that the classical genotype strains still co circulated in South China.