信鸽新城疫病毒P/Anhui/1/09株囊膜糖蛋白基因的 遗传进化及氨基酸差异性位点分析

为分析流行于信鸽群中新城疫病毒致病株P/Anhui/1/09的分子遗传特性及其囊膜糖蛋白的差异性,采用RT-PCR法扩增其F和HN基因开放阅读框(ORF),并与已公布的主要代表性毒株序列进行遗传进化分析及615个全长F基因和480个全长HN基因推导氨基酸序列比对分析,同时对P/Anhui/1/09株进行型内遗传关系分析。结果显示:P/Anhui/1/09株F和HN基因开放阅读框长度分别为1 662 bp和1 716 bp,GenBank登录号分别为JQ290284和JQ305097,其F基因与NDV05-029、PG/CH/JS/1/06、CH/98-1鸽源毒株亲缘关系最近,同源率在98.2%～99.5%,同属基因VIb亚型,但与传统疫苗株LaSota、V4、B1和Mukteswar的遗传距离较远,同源率仅在84%～86.3%。HN基因ORF进化树分支拓扑结构与F基因相似。型内遗传关系分析显示,VIb亚型可进一步为VIb1～VIb5 5个进化分支,其中我国分离株都位于VIb1和VIb4进化分支中。氨基酸序列比对及糖基化位点分析显示,P/Anhui/1/09的F蛋白裂解位点氨基酸序列为112K-R-Q-K-R-F117,与NDV强毒株分子特征相符,并发现在七肽重复区旁497位新增一个潜在N-糖基化位点,除2个基因III型日本分离株(Miyadera和MIY/51)外,所有含497位新增糖基化位点的毒株都位于VIb1分支。包含P/Anhui/1/09株在内的大部分鸽源毒株在F蛋白aa132S(融合肽区)、aa259H和HN蛋白aa3H(胞质区)、aa122R、aa347G、aa349E存在一定特征性。

Evolution analysis and variability of amino acid sites of envelope glycoprotein of strain NDV P/Anhui/1/09 isolated from racing-pigeon

To analyze the molecular genetic characteristics and variability of envelope glycoprotein of pathogenic strain of Newcastle disease virus P/Anhui/1/09 epidemic in pigeon flock, the sequences of open reading frame (ORF) of F and HN gene were amplified by RT-PCR. Evolution analysis was performed with the representative strains and P/Anhui/1/09. The published 615 F full-length gene and 480 HN full-length gene were deduced to amino acid sequence, and then were analyzed. Genetic relationship of strains from genotype VI containing P/Anhui/1/09 was analyzed. The results showed that F and HN genes ORF length of P/Anhui/1/09 were 1 662 bp and 1 716 bp, and their GenBank accession number were JQ290284 and JQ305097, respectively. P/Anhui/1/09 was closest to NDV05-029, PG/CH/JS/1/06, CH/98-1 strains of pigeon-origin based on F gene belonged to VIb subtype, and their homologous rates were from 98.2% to 99.5%. However, it was fruther to the traditional vaccine strains of LaSota, V4, B1 and Mukteswar with 84% to 86.3% in homology. Phylogenetic tree topology of the HN gene ORF was similar to its F gene ORF. VIb subtype could be divided into five evolutionary branches as VIb1-VIb5 based on genetic relationships, and strains belonged to VIb subtype isolated from China were all located in VIb1 and VIb4 branches. The F protein cleavage site amino acid sequence of P/Anhui/1/09 was ¹¹²K-RQKR-F¹¹⁷ based on amino acid sequence analysis, which was consistent with virulent strains of NDV. A new potential N-glycosylation site 497 adjacent heptad repeat region was discovered in strains belonged to VIb1 branch containing P/Anhui/1/09 except two genotype III strains (Miyadera and MIY/51) isolated from Japan. There existed a certain characteristic on F protein aa132S (region of fusion peptide), aa259H, and HN protein aa3H (cytomere), aa122R, aa347G, aa349E at most strains of pigeon-origin containing P/Anhui/1/09.