福建省登革1型病毒基因组序列测定及进化分析

Complete genome sequencing and phylogenetic analysis of a Dengue virus type I strain isolated in Fujian province

目的对分离自福建省的1株登革1型病毒(FJ231/04)进行全基因组序列测定,并同其它病毒株全基因序列进行比较,了解其序列特征和可能的传播来源。方法采取分段扩增、克隆、测序的方案,设计10对引物分别扩增不同的片段,基因组5’末端序列采用RACE技术进行扩增,扩增产物随后克隆到质粒载体并测序。通过末端重叠序列进行拼接后组成全长病毒基因组序列。结果拼接后的病毒全基因组全长10735nt,编码一个长的开放读码框。参考已公布的登革1型病毒的全基因序列,对该病毒株进行进化分析。序列的进化分析表明,该福建省分离株属登革1型病毒中的第1群(基因1型)病毒。在遗传距离上,该毒株和2002年的泰国分离株最为接近(差异为0.53%)。根据病毒发现时间及其核酸差异,推测该病毒可能是通过在东南亚一带感染者或病人带入福建。结论通过对登革病毒全基因组序列的测定可以获得毒株的特征。此外病毒序列的进化分析,还可为了解病毒可能的来源以及传播途径,正确了解登革病毒在我省的流行病学概况提供重要的线索。

To characterize the Dengue virus type Ⅰ strain （Fj231/04） isolated in Fujian province and explore the possible viral origin and transmission route,ten primer pairs were designed and the fragments varied in molecular size and different location in viral genome were amplified by using these primers. By the technique of rapid amplification eDNA ends （RACE）, the viral 5＇ terminal was amplified. All the obtained products were purified for subsequent cloning and sequencing, and the sequencing results were spliced and assembled into a viral complete genomic sequence by the terminals overlapped each other. The complete genome was composed of 10735 nt and comprised a large open reading frame （ORF） encoded the viral polyprotein. The viral genomic sequence data was submitted to the GenBank （Accession No. DQ193572）. For phylogenetic analysis, the viral genomic sequence was aligned with other published Dengue type Ⅰ genomic sequences available from GenBank, and calculated the genetic distance and reconstructed the phylogenetic tree. The phylogenetic analysis demonstrated that the FJ231/04 isolate was most genetically closed to the NIID02-20 strain, found in Thailand in 2002 but isolated in Japan （divergence is 0.53 %）. The genetic identity and the interval of isolation between two virus strains, suggested that there were viruses, genetically resemble to FJ231/04 strain, circulating in the Southeast Asia region until 2004. The viruses caused Dengue fever outbreak in Fujian in 2004 might be transferred from Southeast Asian region, through the Dengue virus human carrier, to Fujian province. This deduction requires more evidence, despite supported by the epidemiological investigation. Complete genome sequencing could characterize the Dengue virus isolate and the results could be used for the phylogenetic analysis with other viruses. This technique facilitates the investigation of the possible viral origin or the transmission route, and provides clues to measures for Dengue virus-associated disease control and prevention.