目的 对2013年浙江省登革热病例进行实验室确诊与病原的分子溯源。方法 对患者血清样本同时进行登革热病毒IgM抗体和核酸检测及病毒分离,阳性分离株采用RT-PCR方法扩增E基因,进行核苷酸序列测定和同源性与进化分析。结果 18例患者发病前均有在境外登革热流行区暴露史,从患者18份血清样本中检测到登革热病毒IgM抗体阳性15份(83.3%),核酸阳性8份(44.4%),分离到登革热1型病毒6株,4型1株。6株登革热1型株之间E基因核苷酸和氨基酸同源性分别为90.9%～99.7%和96.8%～100%,与浙江省2004年登革热1型株之间核苷酸和氨基酸同源性分别为91.4%～98.5%和96.6%～99.0%;E基因进化树显示,登革热1型株位于GⅠ、GⅣ和GⅤ基因亚型上,登革热4型株位于GⅠ亚型上,2013年7株登革热病毒与浙江省2004-2009年登革热病毒之间亲缘关系较远。结论 证实2013年浙江省登革热病例均为输入性,输入国为菲律宾、安哥拉、斯里兰卡和巴西等;登革热1型株基因亚型分别为亚洲型、南太平洋型和美洲/非洲型,登革热4型株为东南亚型;登革热4型病毒为浙江省首次分离。

Objective To confirm the laboratory diagnosis of dengue fever and trace the molecular origin of the pathogen in Zhejiang province, China, 2013. Methods The serum samples from the dengue fever patients were used to detect the dengue IgM antibodies and viral nucleic acids, and to isolate the virus. The envelope (E) gene of the isolated strains was amplified by RT-PCR. The amplicons were sequenced and subjected to homologous and phylogenetic analyses. Results All 18 dengue fever patients had been to endemic areas of dengue fever overseas. Fifteen (83.3%) out of 18 serum samples from the patients were positive for anti-dengue IgM, and 8 (44.4%) were positive for dengue viral nuclei acids. Six strains of Dengue virus type 1 (DV-1) and 1 strain of DV-4 were isolated. The nucleotide sequence homology of the E gene and the amino acid sequence homology were 90.9%-99.7% and 96.8%-100%, respectively, among the 6 DV-1 strains, and 91.4%-98.5% and 96.6%-99.0%, respectively, between the 6 DV-1 strains and the DV-1 stains isolated in 2004 in Zhejiang province. The phylogenetic analysis of E gene showed that the gene subtypes of DV-1 strains were genotypeⅠ (GⅠ), GⅣ and GⅤ, and the subtype of DV-4 strain was GⅠ. All the 7 strains of dengue virus isolated in 2013 had a far phylogenetic relationship with the strains isolated from 2004 to 2009 in Zhejiang province. Conclusion All the dengue fever cases in Zhejiang province in 2013 are imported from countries including Philippines, Angola, Sri Lanka, and Brazil. The DV-1 strains are derived from the subtypes in Asia, South Pacific and America/Africa. The DV-4 strain is derived from the subtype in Southeast Asia, and it is the first detection in Zhejiang province.