2017年云南省登革病毒Ⅰ型分离株NS1基因的鉴定及分析

Identification and analysis of NS1 gene of typeⅠdengue virus isolated in Yunnan Province,China in 2017

目的鉴定并分析2017年云南省登革病毒Ⅰ型(dengue virusⅠ,DENVⅠ)分离株的NS1基因。方法采用病毒RNA提取试剂盒提取登革热(dengue fever,DF)患者血清RNA,RT-PCR法鉴定病毒类型,并扩增DENVⅠ的NS1基因,进行测序。应用BIOEDIT软件比对NS1及DENVⅠ标准株(DQ672562.1)的核苷酸及氨基酸序列,并进行突变分析。应用MEGA 5.1软件中的NJ法(1000 Boot strap replicate)构建NS1基因的系统进化树。结果共分离获得16株DENVⅠ的NS1基因,经比对发现,201703、201704、201708、201709、201713分离株NS1基因第21位碱基由C变为T(无义突变);201705、201706、201712分离株NS1基因第289位碱基由T变为A(有义突变),氨基酸曲L变为M。16株分离株NS1基因其他突变位点均相同,共85处发生碱基突变,其中5个为有义突变。16株分离株与2011-China Donggua(JQ048541.1)、2008-Singapore(GU370049.2)、2014-Taiwan(KU365900.1)、2005-China Fujian(DQ193572.1)、2016-Malaysia(KX452065.1)、2007-Indonesia(KC762646.1)、2015-South Korea(KP406802.1)、2006-Japan(AB178040.1)、2007-Thailand(HM469966.1)、2013-singapore(KJ806945.2)有较近的亲缘关系。结论 2017年云南省16株分离毒株均为DENVⅠ,可能属于东南亚国家的输入性毒株。

Objective To identify and analyze the NS1 gene of dengue virus isolated in Yunnan Province,China in2017. Methods RNA was extracted directly from the serum of a patient with dengue fever（DF）by using the viral RNA extraction kit,of which the serotype was identified by RT-PCR,and NS1 gene was amplified and sequenced. The nucleotide and amino acid sequences of the NS1 were compared with those of standard DENVⅠ strain（DQ672562. 1）by using BIOEDIT software to analyze the mutation,based on which a phylogenetic tree was plotted by the NJ method（1 000 boot strap replicate） in MEGA 5. 1 software. Results NS1 gene was amplified from 16 DENV Ⅰ strains.Nucleotide mutation of C to T at site 21 of NS1 gene were found in isolates 201703,201704,201708,201709 and201713,all of which were nonsense mutation. However,nucleotide mutation of T to A at site 289 of NS1 gene were found in isolates 201705,201706 and 201712,which were sense mutation resulting in the amino acid mutation of leucine（L）to methionine（M）. The mutations at other sites of NS1 gene of the 16 strains were identical. A total of 85 nucleotide mutations were found,including five sense mutations. Phylogenetic analysis showed close relationship of the 16 strains to2011-China Dongguan（JQ048541. 1）,2008-Singapore（GU370049. 2）,2014-Taiwan（KU365900. 1）,2005-China Fujian（DQ-193572. 1）,2016-Malaysia（KX452065. 1）,2007-Indonesia（KC762646. 1）,2015-South Korea（KP406802. 1）,2006-Japan（AB178040. 1）,2007-Thailand（HM469966. 1）and 2013-singapore（KJ806945. 2）strains. Conclusion All the 16 isolates in Yunnan in 2017 were DENVⅠ,which might belong to the imported strains from the countries in Southeast Asia.