登革2型病毒深圳分离株的鉴定及E基因序列分析

Identification and sequence analysis of E gene of Dengue virus type 2 strain isolated from patient serum in Shenzhen

目的对深圳市2005-2006年登革热病原体进行分离鉴定，从分子水平分析分离株的生物学特征，追踪其地域来源。方法采用酶联免疫吸附试验（ELISA）和胶体金免疫层析法检测60份疑似登革热患者血清中的特异性IgM、IgG抗体，其中9份标本抗体阳性。用C6／36细胞对9份早期病例血清进行病毒分离，以MGB荧光PCR方法鉴定。并用逆转录-半套式PCR（RT—semi—nested—PCR）方法和荧光PCR方法对其进行型别鉴定。采用RT—PCR方法扩增病毒E基因后进行序列测定，并与不同国家和地区的登革热毒株进行同源性比较和进化树分析。结果从9份抗体阳性的患者血清标本中成功分离到1株登革热病毒，经鉴定为登革2型病毒，将其命名为DEN2-SZ0521。深圳市登革2型病毒分离株SZ0521与登革2型国际标准株新几内亚（NGC）株在E基因上核苷酸和氨基酸的同源性分别为94．2％和98．2％，而与登革1、3、4型国际标准株HAWAII、H87、H241同源性分别为59．1％、57．2％、58．5％和68．1％、66．7％、63．2％。基因进化树显示SZ0521株与Malay0412a／Tw株亲缘关系最近，在进化树的同一分支上，核苷酸同源性最高，达99．8％，氨基酸同源性为100％，与Indonesia-76、Somalia-84和SriLanka-90同属基因Ⅳ亚型。结论首次从深圳市登革热患者血清中分离到1株登革2型病毒，证实该毒株可能来源于马来西亚。

Objective To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005--2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin. Methods IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed. Results Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94. 2% and 98.2% , but the homology with standard Dengue virus 1,3,4 in the same fragment were 59. 1% ,57.2% ,58.5% and 68.1% ,66. 7% , 63.2% ,respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99. 8% and 100% , respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90. Conclusion Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.