我国分离的甲病毒YN87448毒株全部结构区基因核苷酸的序列测定及分析

THE COMPLETE NUCLEOTIDE SEQUENCE OF THE STRUCTURAL REGION GENE OF THE ALPHAVIRUS YN87448 STRAIN ISOLATED IN CHINA AND ITS RELATIONSHIP TO OTHER SINDBIS VIRUSES

Ｙ Ｎ８７４４８ 毒株１９８６ 年分离自云南傣族５２ 岁女发热病人的血液标本，曾用血清学方法鉴定为披膜病毒科甲病毒属。用我们创立的单引物差异 Ｒ Ｔ－ Ｐ Ｃ Ｒ 方法及简并引物 Ｒ Ｔ－ Ｐ Ｃ Ｒ 法均已证明：该病毒为甲病毒属的辛德毕斯样病毒（ Ｓｉｎｄｂｉｓ － ｌｉｋｅ ｖｉｒｕｓ） 。本研究从甲病毒属的病毒基因序列的共同保守区，设计４ 对引物。用 Ｒ Ｔ－ Ｐ Ｃ Ｒ 方法扩增，再将扩增片段分别连结克隆到ｐ Ｇ Ｅ Ｍ－ Ｔ 载体。通过对４ 个克隆的测序完成了对 Ｙ Ｎ８７４４８ 毒株的全部结构区基因序列的测定，结果表明：（１） Ｙ Ｎ８７４４８ 病毒的全部结构区基因序列长度为４ ０５９ 个核苷酸（ 不包括多聚腺苷酸尾） ；（２） Ｙ Ｎ８７４４８ 病毒的全部结构区基因与辛德毕斯病毒家族中毒力最强的、唯一能致成年小鼠１００ ％ 死亡的、南非分离的辛德毕斯样病毒 Ｓ Ａ Ａ Ｒ８６ 株相应基因的核苷酸序列同源性为９９ ％ ，但 Ｙ Ｎ８７４４８ 毒株不致成年小鼠死亡；（３） 二者基因结构上有一点大的差异是， Ｙ Ｎ８７４４８ 病毒位于基因组８ ６４１ｂｐ 处的结构区基因 Ｅ２ 和 Ｅ３ 之间多出３ 个核苷酸（ Ａ Ａ Ａ） ；（４） 与其它甲属病毒的进化树比较来看?

YN87448 virus was isolated from a feverish female patient (52 years old) in Yunnan Province in 1986, and was identified as a member of Alphavirus by the serological test. Prior to sequence analysis of YN87448 virus, it was identified by the single primer differential RT-PCR display and degenerate primers RT-PCR as a sindbis-like virus. Then, the complete nucleotide sequence of the structural gene of YN87448 virus was determined with four clones, which were obtained through virus reverse transcription, 4 pair primer polymerase chain reaction (RT-PCR) and linking of the four overlapping fragments into pGEM-T vector respectively. The results obtained were as follows. 1. The complete nucleotide sequence of structural gene of YN87448 virus is 4059 nucleotides long excluding the 3′-terminal poly(A); 2. The homogeneity of the nucleotide sequence between YN87448 virus and sindbis-like virus S.A.AR86 is 99%; 3. There is a 3-nucleotide (AAA) insertion between E3 and E2 region of the YN87448 virus; 4. Phylogenetic tree shows that YN87448 may be a new sindbis-like virus.