云南省2006-2015年狂犬病病毒核蛋白基因进化特征分析

摘要

目的 阐明云南省2006-2015年t18株狂犬病病毒(Rabies virus,RABV)核蛋白(Nucleoprotein,N)基因进化特征及流行病学特点.方法 在云南省狂犬病流行区采集狂犬、可疑犬和病牛脑组织以及狂犬病病例脑组织、唾液和脑脊液标本,用直接免疫荧光法进行病毒抗原检测,阳性标本提取病毒核酸,通过RT-PCR扩增RABV的N基因序列并进行序列测定,用MEGA5.0软件邻接法(Neighbor-Joining)进行系统进化分析.结果 经RT-PCR和序列测定获得2012-2015年云南省91株RABV的N基因序列,并与2006-2011年获得的云南27株RABV及邻省和东南亚地区的29株RABV的N基因序列进行系统进化分析.结果表明,云南RABV进化群可分为YN-A(105株)、YN-B(6株)和YN-C(7株),并分别属于中国进化群中的China-Ⅰ、China-Ⅵ和China-Ⅱ.YN-A流行株在2006-2015年间每年均有分布,其中,2006-2011年14株,2012-2015年91株,并分布于13个州(市);YN-B和YN-C分别仅分布于2006-2010年和2008-2011年,地区分布也较为局限.YN-A和YN-C与相邻四川、贵州、广西和湖南省的China-Ⅰ或China-Ⅱ进化群流行株亲缘关系较近,YN-B与缅甸、泰国、越南和柬埔寨流行株亲缘关系较近.结论 云南省RABV存在不同传播来源的3个进化群.YN-A为优势进化群,在2006-2015年间广泛分布于云南省狂犬病流行区,并具有较强的传播速率.2012年以来云南省未再发现YN-B和YN-C进化群的流行.%Objective To understand the molecular evolution characteristics of the nucleoprotein (N) genes and epidemiological feature of 118 rabies virus (RABV) strains isolated in Yunnan province,China from 2006 to 2015.Methods The brain tissue samples from mad dogs,suspicious sick dogs,sick cow,and human brain tissue,saliva and CSF samples from rabies patients were collected in Yunnan province to detect the viral antigen by direct immunofluorescence assay (DFA).The viral RNA from positive samples was extracted.Coding region of N gene was amplified by RT-PCR and sequenced.The phylogenetic tree was constructed by Neighbor-Joining method of MEGA5.0 software.Results The sequences of N genes of 91 RABV strains in Yunnan from 2012 to 2015 were obtained.With the sequences of N genes of 27 RABV strains in Yunnan from 2006 to 2011 and 29 RABV strains from Southeast Asian Countries,the phylogenetic analysis was performed.RABV strains in Yunnan were divided into clades YN-A (105 strains),YN-B (6 strains),YN-C (7 strains),which belonged to clades China-Ⅰ,China-Ⅵ,China-Ⅱ respectively.Clade YN-A was epidemic every year from 2006 to 2015,of them,14 strains from 2006 to 2011 and 91 strains from 2012 to 2015 were distributed in 13 prefectures (cities) of Yunnan.Clades YN-B and YN-C were epidemic only from 2006 to 2010 and from 2008 to 2011 respectively.The regional distribution of clades YNoB and YN-C was limited.The strains of YN-A and YN-C were closely related to the strains of clades China-Ⅰ and China-Ⅱ from neighboring Sichuan,Guizhou,Guangxi and Hunan provinces.The strains of YN-B were closely related to the strains from Myanmar,Laos,Vietnam and Cambodia.Conclusions Three RABV clades with multiple transmission sources were identified in Yunnan.Clade YN-A was widely distributed in rabies endemic area in Yunnan from 2006 to 2015,and it has strong ability to spread as principal clade in Yunnan.Since 2012,clades YN-B and YN-C were not found again in Yunnan.