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目的 测定从疫区捕获的宿主动物 -褐家鼠肺中分离的汉坦病毒 Pf2 6株的 S基因片段的全基因序列。方法 选择汉坦病毒 S基因序列保守区设计引物 ,用 RT- PCR方法将 S基因分两段扩增 ,再将扩增片段连接到 PU C19载体上 ,然后进行测序和分析。结果 Pf2 6株的 S基因片段的全序列长为 1776 nt。只有一个主要的编码 N蛋白的 ORF,起始位置为第 43 nt,终止于1332 nt,编码的蛋白长为 42 9aa。 Pf2 6株的 S基因片段核苷酸序列与 SEO型中的病毒株的同源性最高 ,而与汉坦病毒其他型的同源性较差。从种系发生树分析来看 ,Pf2 6株归结于汉坦病毒的SEO型。在 SEO型之内 ,从 S基因的种系发生树 (A)上看 ,Pf2 6株与 sr- 11株的亲缘关系比较近。但是 ,从编码的 N蛋白的种系发生树 (B)上看 ,Pf2 6株与 Z37株、Guo3株的亲缘关系比较近。Pf2 6株的 S基因和 N蛋白序列与汉坦病毒 SEO型分离株 sr- 11,Z37,Guo3株的同源性最高 ,分别为 :核苷酸同源性 ,97% ,96 % ,88% ;氨基酸同源性 ,97% ,98% ,98%。Pf2 6株与 sr- 11株 ,Guo3株 ,Z37株 N蛋白氨基酸变异主要发生在 N蛋白的前半部分。中国分离到的 Pf2 6 ,Guo3,Z37株与 sr- 11株氨基酸变异主要集中在 aa6 9- aa75位之间。结论 Pf2 6株为一株新的汉坦病毒 SEO型病毒株。
Objective To determine the whole gene sequence of the S gene fragment of Hantavirus Pf2 6 isolated from the host animal Rattus norvegicus captured in the epidemic area. Methods The conservative region of Hantavirus S gene was selected to design primers. The S gene was amplified by RT-PCR in two stages. The amplified fragment was ligated to PU C19 vector and sequenced and analyzed. Results The full length of S gene fragment of Pf2 6 strain was 1776 nt. There is only one major ORF encoding the N protein, starting at position 43 nt and ending at 1332 nt, encoding a protein of 42 9 aa. The nucleotide sequence of the S gene fragment of Pf2 6 strain has the highest homology with the virus strain in the SEO type and the poor homology with other Hantavirus strains. From phylogenetic tree analysis, 6 strains of Pf2 were attributed to Hantaan virus type SEO. Within the SEO type, from the phylogenetic tree (A) of the S gene, the relationship between the Pf2 6 strain and the sr-11 strain is relatively close. However, from the phylogenetic tree (B) of the encoded N protein, Pf2 6 strains are more closely related to Z37 and Guo3 strains. The S gene and N protein sequence of Pf2 6 strains shared the highest homology with the hantavirus SEO-type isolates sr-11, Z37 and Guo3 strains, with nucleotide homology of 97%, 96% and 88% ; Amino acid homology, 97%, 98%, 98%. The amino acid variation of N protein of Pf2 6, sr-11, Guo3 and Z37 mainly occurred in the first half of N protein. The amino acid variations of Pf2 6, Guo3, Z37 and sr-11 strains isolated in China mainly concentrated in the aa6 9-aa75 position. Conclusion Pf2 6 is a new hantavirus SEO type strain.