福建省白纹伊蚊中登革病毒的分离与鉴定
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摘要
近年来福建省登革热(Dengue fever,DF)输入性病例持续存在,且登革热的主要传播媒介白纹伊蚊在全省内广泛分布,为了解福建省福州市登革热的媒介白纹伊蚊携带登革病毒(Dengue virus,DENV)状况,2017年10月7日在福州市台江区元一花园小区内开展伊蚊监测,采用双层叠帐法捕获255只白纹伊蚊蚊体研磨液上清提取核酸后用实时荧光RT-PCR法检测DENV特异性核酸,将检测阳性的蚊体研磨液上清接种C6/36细胞进行病毒分离,成功分离到1株DENV病毒株mosquito13/Fujian/2017;经实时荧光RT-PCR法鉴定所分离病毒株的血清型为I型;利用型特异性引物通过RT-PCR扩增病毒E基因并测序进行分子遗传特性分析;E基因核苷酸和氨基酸同源性分析显示,该毒株与2017年10月17日同小区本地登革热病例血清中分离得到的登革毒株E基因序列完全一致,与越南2014年分离株KT825033/Vietnam/2014核苷酸(99.7%)和氨基酸(99.8%)同源性最高;系统进化树分析表明所分离登革病毒毒株的基因型为I型,与东南亚地区的越南,泰国,柬埔寨等国家进化关系相近,可能输入来源于东南亚国家。本研究证实了登革热外潜伏期的存在以及白纹伊蚊在登革热疫情传播过程中的媒介作用,提示在登革热的防控工作中媒介登革病毒监测、检测的重要性,也提示福建省需要加强输入来源监测,特别是东南亚入境人员的监测。
In recent years, imported cases of dengue fever(DF) persist in Fujian Province, China, and the main vector of DF, Aedes albopictus, is widely distributed in Fujian Province, China. To monitor the carrying status of the dengue virus(DEN V) in the DENV vector Aedes albopictus in Fuzhou City, Fujian Province, China,255 mosquitoes were collected in Yuanyi Garden Community, Taijiang District on October 7~(th) 2017. DENV was detected by real-time reverse transcription-polymerase chain reaction(RT-PCR) after extracting nucleic acid from mosquito-abrasive supernatants. A DENV strain, mosquito13/Fujian/2017, was isolated in C6/36 cells. The serotype of the isolated strain was identified as type I by real-time RT-PCR. The E-gene was amplified and sequenced by specific primers DENV1 E-F/DENV1 E-R. Homology analyses of the nucleotide and amino acids showed that this strain was completely consistent with the E-gene sequences of a later local case of Dengue fever(DF; October 17~(th) 2017), and had the highest homology with the nucleotides( 99.7%) and amino acids(99.8%) of KT825033/Vietnam/2014 isolated from Vietnam in 2014. Phylogenetic analyses showed that the genotype of mosquito13/Fujian/2017 was type I. The isolated strain had a similar evolutionary relationship with strains from Vietnam, Thailand, and Cambodia. The virus that caused a dengue fever outbreak in Fujian Province, China in 2017 might have transferred from the Southeast Asia region. In the present study, the resultsshowed the external incubation period of dengue fever, and further confirmed the role of Aedes albopictus in transmission of the dengue fever outbreak, which suggested that the prevention and control of dengue fever in Fujian Province, China must involve enhancement of the monitoring of Aedes albopictus and immigrants especially those from Southeast Asia.
In recent years, imported cases of dengue fever(DF) persist in Fujian Province, China, and the main vector of DF, Aedes albopictus, is widely distributed in Fujian Province, China. To monitor the carrying status of the dengue virus(DEN V) in the DENV vector Aedes albopictus in Fuzhou City, Fujian Province, China,255 mosquitoes were collected in Yuanyi Garden Community, Taijiang District on October 7~(th) 2017. DENV was detected by real-time reverse transcription-polymerase chain reaction(RT-PCR) after extracting nucleic acid from mosquito-abrasive supernatants. A DENV strain, mosquito13/Fujian/2017, was isolated in C6/36 cells. The serotype of the isolated strain was identified as type I by real-time RT-PCR. The E-gene was amplified and sequenced by specific primers DENV1 E-F/DENV1 E-R. Homology analyses of the nucleotide and amino acids showed that this strain was completely consistent with the E-gene sequences of a later local case of Dengue fever(DF; October 17~(th) 2017), and had the highest homology with the nucleotides( 99.7%) and amino acids(99.8%) of KT825033/Vietnam/2014 isolated from Vietnam in 2014. Phylogenetic analyses showed that the genotype of mosquito13/Fujian/2017 was type I. The isolated strain had a similar evolutionary relationship with strains from Vietnam, Thailand, and Cambodia. The virus that caused a dengue fever outbreak in Fujian Province, China in 2017 might have transferred from the Southeast Asia region. In the present study, the resultsshowed the external incubation period of dengue fever, and further confirmed the role of Aedes albopictus in transmission of the dengue fever outbreak, which suggested that the prevention and control of dengue fever in Fujian Province, China must involve enhancement of the monitoring of Aedes albopictus and immigrants especially those from Southeast Asia.
引文
[1] Guzman M G. Harris E. Dengue[J]. Lancet, 2015, 385(9966):453-465.
[2] Krameer M U,Sinka M E,Duda K A,Mylne A Q,Shearer F M, Barker C M, Moore C G, Carvalho R G,Coelho G E, Bortel W V,Hendrickx G,Schaffner F,Elyazar I R, Teng H J, Brady O J, Messina J P, Pigott D M,Scott T W,Smith D L,Wint G R W,Golding N,Hay S I.The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus[J/OL]. eLife,2015,4:e08347.
[3]谢汉国,张山鹰,林岩,方义亮,林耀莹,欧阳榕.福建省不同地区白纹伊蚊种群密度与生态特征调查[J].医学动物防制,2013, 29(2):123-124.
[4]严延生,洪荣涛,沈晓娜,翁育伟,蔡少健,徐保海,李世清,何家鑫,许龙善,林云钦,郑能雄,林茂,林淑华.福州市2004年登革热流行病学和病原学特征分析[J].中华流行病学杂志,2006,5(27):371-374.
[5] Wang J, Chen H, Huan M, Zhang Y, Xie J, Yan Y,Zheng K, Weng Y. Epidemiological and etiological investigation of dengue fever in the Fujian province of China during 2004-2014[J]. Sci China Life Sci, 2017, 60(1), 72-80.
[6]李世清,何似,陈润,徐保海,许龙善.福建省首次从病人血清及蚊体分离出登革病毒的研究[J].中国人兽共患病杂志,2000,16(1):55-56.
[7]郑能雄,王宗汉,张晓阳,郑高,陈黄河,李文最,陈红光,陈敏红,林云钦,赵松涛.福州市白纹伊蚊的孳生状况季节消长及其影响因素[J].海峡预防医学杂志,2001,7(6):6-9.
[8] Rico-Hesse R. Molecular evolution and distribution of Dengue viruses type 1 and 2 in nature[J]. Virology,1990,174(2):479-493.
[9] Fahri S, Yohan B, Trimarsanto H, Sayono S, Hadisaputro S,Dharmana E,Syafruddin D,Sasmono R T.Molecular surveillance of dengue in Semarang, Indonesia revealed the circulation of an old genotype of dengue virus serotype-1[J/OL].PLoS Negl Trop Dis,2013,7(8):e2354.
[10]张瑾,徐翮飞,薛晓宁,朱可,陈晓光,张娟,林元,张齐.两例输人性登革病毒合并基孔肯雅病毒感染实验室检测.[J].病毒学报,2017, 33(2):200-204. DOI:10.13242/j.cnki.bingduxuebao.003122.
[11] Sun J,Wu D,Zhou H,Zhang H,Guan D,He X,Cai S, Ke C, Lin J. The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong,China,in2014[J]. J Infect,2016,72(1):80-90.
[12] Poornima P,Patrick C,Yolanda T,Nunez A,Ryan E M,Malboeuf C,Levin J Z,Lennon N J,Balmaseda A, Harris E, Henn M R.Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity[J]. J Virol,2012,86(16):8546-8558.
[2] Krameer M U,Sinka M E,Duda K A,Mylne A Q,Shearer F M, Barker C M, Moore C G, Carvalho R G,Coelho G E, Bortel W V,Hendrickx G,Schaffner F,Elyazar I R, Teng H J, Brady O J, Messina J P, Pigott D M,Scott T W,Smith D L,Wint G R W,Golding N,Hay S I.The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus[J/OL]. eLife,2015,4:e08347.
[3]谢汉国,张山鹰,林岩,方义亮,林耀莹,欧阳榕.福建省不同地区白纹伊蚊种群密度与生态特征调查[J].医学动物防制,2013, 29(2):123-124.
[4]严延生,洪荣涛,沈晓娜,翁育伟,蔡少健,徐保海,李世清,何家鑫,许龙善,林云钦,郑能雄,林茂,林淑华.福州市2004年登革热流行病学和病原学特征分析[J].中华流行病学杂志,2006,5(27):371-374.
[5] Wang J, Chen H, Huan M, Zhang Y, Xie J, Yan Y,Zheng K, Weng Y. Epidemiological and etiological investigation of dengue fever in the Fujian province of China during 2004-2014[J]. Sci China Life Sci, 2017, 60(1), 72-80.
[6]李世清,何似,陈润,徐保海,许龙善.福建省首次从病人血清及蚊体分离出登革病毒的研究[J].中国人兽共患病杂志,2000,16(1):55-56.
[7]郑能雄,王宗汉,张晓阳,郑高,陈黄河,李文最,陈红光,陈敏红,林云钦,赵松涛.福州市白纹伊蚊的孳生状况季节消长及其影响因素[J].海峡预防医学杂志,2001,7(6):6-9.
[8] Rico-Hesse R. Molecular evolution and distribution of Dengue viruses type 1 and 2 in nature[J]. Virology,1990,174(2):479-493.
[9] Fahri S, Yohan B, Trimarsanto H, Sayono S, Hadisaputro S,Dharmana E,Syafruddin D,Sasmono R T.Molecular surveillance of dengue in Semarang, Indonesia revealed the circulation of an old genotype of dengue virus serotype-1[J/OL].PLoS Negl Trop Dis,2013,7(8):e2354.
[10]张瑾,徐翮飞,薛晓宁,朱可,陈晓光,张娟,林元,张齐.两例输人性登革病毒合并基孔肯雅病毒感染实验室检测.[J].病毒学报,2017, 33(2):200-204. DOI:10.13242/j.cnki.bingduxuebao.003122.
[11] Sun J,Wu D,Zhou H,Zhang H,Guan D,He X,Cai S, Ke C, Lin J. The epidemiological characteristics and genetic diversity of dengue virus during the third largest historical outbreak of dengue in Guangdong,China,in2014[J]. J Infect,2016,72(1):80-90.
[12] Poornima P,Patrick C,Yolanda T,Nunez A,Ryan E M,Malboeuf C,Levin J Z,Lennon N J,Balmaseda A, Harris E, Henn M R.Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity[J]. J Virol,2012,86(16):8546-8558.