蝙蝠体表蛛蝇中肯科伊病毒的分离鉴定  

Isolation and Molecular Characterization of Kaeng Khoi Virus from Eucampsipoda Sundaica Bat Flies

作　　者：冯云[1] 付士红[2] 张海林[1] 杨卫红[1] 章域震[1] 梁国栋[2] FENG Yun;FU Shihong;ZHANG Hailin;YANG Weihong;ZHANG Yuzhen;LIANG Guodong(Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention ,Yunnan Institute of Endemic Diseases Control and Prevention , Dali , Yunnan 6 71000 , China;State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention ,Beijing 102206 ,China)

机构地区：[1]云南省地方病防治所、云南省自然疫源性疾病防控技术重点实验室,云南大理671000 [2]中国疾病预防控制中心病毒病预防控制所、国家传染病预防和控制重点实验室,北京102206

出　　处：《病毒学报》2018年第4期461-470,共10页Chinese Journal of Virology

基　　金：国家自然科学基金/地区基金(项目号:31560049),题目:蝙蝠体表吸血昆虫携带病毒及其与蝙蝠和其他人畜动物感染关系研究;云南省应用基础研究计划项目/面上项目(项目号:2016FB029),题目:蝙蝠体表吸血昆虫携带病毒及其与蝙蝠和其他人畜动物感染关系研究~~

摘　　要：为调查云南省蝙蝠体表寄生虫蛛蝇中病原体的携带情况。采集蝙蝠体表蛛蝇标本,用细胞培养法分离病毒,对阳性分离物进行分子生物学鉴定,用RT-PCR（Reverse transcription PCR）、TCID（50）（Tissue culture infective dose）、免疫荧光等实验对病毒的特性进行研究。2012年从励腊县的棕果蝠（Rousettus leschenaulti）体表采集蛛蝇183只,利用节肢动物线粒体细胞色素氧化酶I（COI）的通用引物用PCR方法鉴定为蛛蝇科的Eucampsipoda sundaica。从这些蛛蝇中分离到1株阳性分离物MLBC1215,能引起金黄地鼠肾细胞（Golden hamster kidney cell,BHK-21）和非洲绿猴肾细胞（African green monkey kidney cell,VeroE6）出现圆缩、脱落的细胞病变（Cytopathic effect,CPE）。用RT-PCR方法扩增序列,测序结果显示MLBC1215病毒株为三节段RNA病毒,各基因组编码区序列长度分别为S基因为975核苷酸,M基因为4 568核苷酸,L基因为6 866核苷酸,3个基因节段核苷酸序列所形成的进化树拓扑构图相似,MLBC1215病毒株的S、M和L基因均与1970年泰国分离的肯科伊病毒（Kaeng Khoi orthobunyavirus,KKV）（PSC-19）处于同一进化分支。MLBC1215株的S、M、L片段与泰国PSC-19株的核苷酸的同源性分别为97.9%、94.4%和95.3%;氨基酸的同源性分别为98.7%、96.5%和98.4%。MLBC1215株在BHK-21细胞生长到60h达到最高滴度,TCID_（50）为10^（4.8）/0.1ml,随后病毒滴度逐渐下降,至144h TCID_（50）为10^（3.6）/0.1ml。MLBC1215株对小白鼠的主要器官均能引起病理改变,从而导致小白鼠发病死亡。采集的33份人血清中有9份IgG抗体与MLBC1215株有反应,检测结果为阳性,抗体滴度为4份1：20,4份1：40,1份1：80。本次研究证实云南省腊勐县蝙蝠体表吸血昆虫蛛蝇携带布尼亚病毒目KKV。To investigate the distribution patterns of Nycteribiid and pathogens of flies in bats in Yunnan Province,China.The virus was isolated from positive samples cultured in Baby hamster kidney cell（BHK-21）and African green monkey kidney cell（VeroE6）and then identified by molecular biology technique,reverse transcription-polymerase china reaction（RT-PCR）,tissue culture and immunofluorescence.In this way,we could undertake molecular characterization of a newly isolated virus strain.This virus strain,MLBC1215,was isolated from 183 flies fromRousettusleschenaultia,which was classified as Eucampsipoda sundaica by PCR with arthropods mitochondrial cytochrome oxidase universal primer.All samples were collected from Mengla Town in 2012.The specimen of MLBC1215 showed cytopathic effect（CPE）in both BHK-21 and VeroE6 cells at the second post-infection,which involved rounding up and exfoliation.As the genome amplification by RT-PCR showed,the whole genome of MLBC1215 contained three RNA segments：the small gene（S）,975 nucleotides long;the medium gene（M）,4,568 nucleotides long;and the large gene（L）,6,866 nucleotides long.Phylogenetic analyses based on S,M and L segments suggested that MLBC1215 belonged within the same clade as the KKV strain PSC-19,which was isolated in Thailand in 1970.The nucleotide homology of MLBC1215 and PSC-19 in S,M and L segments was 97.9%,94.4% and 95.3%,respectively;the amino-acid homology was 98.7%,96.5%and 98.4%,respectively.The MLBC1215 strain reached the highest titer in BHK-21 cells in 60 hand the TCID50 was 104.8/0.1 ml;then the virus decreased gradually in titer to 144 hand the TCID50 was 103.6/0.1 ml.MLBC1215 could cause pathological changes to the main organs of experimental mice,followed by their death.Out of 33 human serum samples collected in Mengla,9 of them were tested positive by indirect immunofluorescence assay（IFA）against immunoglobulin-G（IgG）,with different antibody titers of 1∶20（4 samples）,1∶40（4 samples）,and 1∶80（1 samples）.Thi

关 键 词：肯科伊病毒(KKV) 蛛蝇 勐腊 

分 类 号：R373.3[医药卫生—病原生物学]