登革2型病毒04株基因组全序列的测定

Complete sequence analysis of the entire genome of the Dengue type 2 virus 04 strain isolated in China

目的 对我国登革 2型病毒 0 4株 (D2 0 4)基因组进行全序测定及分析 ,为了解其基因组结构与生物功能的关系及研究开发登革病毒新型疫苗奠定基础。方法 根据登革 2型国际标准株NGC的序列 ,设计 13对重叠引物 ,通过RT PCR扩增出D2 0 4株不同的cDNA片段 ,分别克隆到pGEM T载体 ,转化受体菌DH5α ,挑取阳性克隆进行PCR、酶切鉴定及序列测定。结果 D2 0 4株的基因组全长 10 72 3个碱基 ,从 97到 10 2 6 9位为一个长的开放读码框 ,编码 3391个氨基酸。将它与其它登革 2型病毒株NGC、JAM、PR15 9(S1)、16 6 81及它的候选疫苗株PDK 5 3进行比较 ,其核酸序列同源性分别为 95 .0 %、97.6 %、89.8%、93.8%和 93.7% ,氨基酸序列的类似性分别为 97.8%、98.6 %、96 .7%、97.6 %和 97.5 %。结论 我国D2 0 4株的基因组全序列基本类似于已发表的其它登革 2型病毒株。在比较的 5株登革 2型病毒株中 ,D2 0 4株更类似于JAM株 ,其次是NGC株 ,与S1株的类似性略低。比较的结果表明 ,D2 0 4株与JAM株紧密相关 ,它们可能属于同一拓扑型。D2 0 4株全序的测定 ,对分析我国毒株来源及研制适于我国人群的登革疫苗具有一定意义。

Objective To sequence the entire genome of Dengue 2 virus 04 (D2 04) strain, provide direct information about the genomic structure and its possible relationships to the biological functions, and aid in the development of new Dengue vaccines. Methods Thirteen pairs of primers were designed according to the sequence of Dengue 2 virus prototype strain NGC. Using RT PCR, cDNA fragments of D2 04 strain were acquired from infected C6/36 cells. The cDNA fragments were cloned into the vector pGEM T and then transformed to competent DH5α cells. Positive clones were screened and amplified by PCR, and then the products were determined by enzyme digestion. The sequences of inserted fragment were determined by PRISM TM ABI 377 automated sequencer. Results Sequence analysis showed that the entire genome of D2 04 strain consisted of 10?723 nucleotides(nt) and contained a single open reading frame(ORF) of 10?173 nt which encoded a polyprotein of 3?391 amino acids(aa). The nucleotide sequence and the deduced amino acid sequence of D2 04 strain were compared with those of other Dengue 2 virus strains such as NGC, JAM, PR159(S1),16681 and its attenuated vaccine derivative PDK 53. The results revealed that the homology of nucleotide sequences among the five strains was 95.0%,97.6%,89.8%,93.8% and 93.7%, respectively, and the similarity of their amino acid sequences was 97.8%, 98.6%, 96.7% ,97.6% and 97.5%,repectively. The genomic organization of D2 04 strain was similar to that of other reported Dengue 2 virus strains. The amino acid sequence of D2 04 strain polypeptide revealed 28 cysteine residues conserved within the Dengue 2 virus, as well as 7 potential glycosylation sites at Asn 69 of PrM protein; Asn 67 and Asn 153 of E protein; Asn 130, Asn 207, Asn 359 and Asn 399 of NSI protein. Conclusions Among the five Dengue 2 virus strains D2 04 strain is more similar to JAM (97.6% similarity) than NGC, and it is less similar to S1. Comparative data reveal that D2 04 strain appears to be closely related to JAM strain and they may belong to the same topotype. The sequence analysis of D2 04 strain would aid in understanding the origin of Dengue virus and developing Dengue vaccine in China.