我国分离的西尼罗病毒与乙脑病毒生物学表型的比较研究
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摘要
我国于2011年在新疆维吾尔自治区自然界采集的蚊虫标本分离到西尼罗病毒(West Nile virus,WNV),这是我国首次在自然界蚊虫标本分离到该病毒,但是一直缺少对该病毒生物学特性研究的报道。本研究利用细胞培养和动物实验的方法研究了我国首次在蚊虫分离的WNV(XJ11129株)对不同细胞系和动物的致病性,以及通过中和试验测定了病毒滴度及其与乙脑病毒(Japanese encephalitis virus,JEV)P3株之间的交叉抗原特性。通过病毒感染细胞的超薄切片观察到XJ11129株WNV呈有包膜的球形颗粒,直径约为40～60nm;XJ11129株WNV对哺乳动物细胞(BHK-21和Vero)和蚊虫细胞(C6/36)细胞均可以引起细胞病变;接种BHK-21细胞显示WNV感染产生的蚀斑直径较JEV大;XJ11129株病毒接种C6/36细胞滴度可达108PFU/100μL;颅内接种2日龄乳鼠第2～3d即开始出现明显发病特征,第4d出现动物的肢体瘫痪并开始死亡,至第5d全部死亡。WNV和JEV的交叉中和试验结果显示,XJ11129株病毒对自身病毒株的中和抗体滴度可达1∶1 280,但对JEV的交叉中和抗体滴度仅有1∶10。综合以上结果提示,从我国新疆维吾尔自治区蚊虫分离的WNV XJ11129株对哺乳动物具有较强致病性,而目前尚无针对WNV感染有效的治疗方法和疫苗,因此,加强我国新疆维吾尔自治区WNV在人间传播的检测和监测具有重要意义。
The West Nile virus(WNV)was isolated ?rst in mainland China from mosquito specimens in south-ern Xinjiang Uygur Autonomous Region of China in 2011. However,studies on the biologic characteristics of WNV have been lacking. Using in vitro and in vivo tests,the biologic phenotype and cross-immunoreactions were compared between P3 Japanese encephalitis virus(JEV) and WNV(XJ11129) strains isolated from China. The XJ11129 virus strain was observed as spherical particles with capsules,and the diameter of the virus was about 40～60 nm. The XJ11129 virus could cause cytopathic changes in mammalian cells(BHK and Vero)and mosquito cells(C6/36). The titers of the XJ11129 virus were ≤108 PFU/100 μL,and the titer of the virus began to decrease 168 h after infecting C6/36 cells. Upon intracranial inoculation of the XJ11129 virus in 2 -day-old suckling mice,disease characteristics were seen clearly at days 2～3,paralysis began at day 4,and all mice died at day 5. The XJ11129 virus could produce a high - titer neutralization antibody response to its own strains(1∶1,280 dilution),but produced only a 1∶10 cross-neutralization PRNT90 titer against the JEV. These data show that the XJ11129 strain isolated from mosquitoes in Xinjiang Uygur Autonomous Region of China was strongly pathogenic to mammalian cells and mouse.
The West Nile virus(WNV)was isolated ?rst in mainland China from mosquito specimens in south-ern Xinjiang Uygur Autonomous Region of China in 2011. However,studies on the biologic characteristics of WNV have been lacking. Using in vitro and in vivo tests,the biologic phenotype and cross-immunoreactions were compared between P3 Japanese encephalitis virus(JEV) and WNV(XJ11129) strains isolated from China. The XJ11129 virus strain was observed as spherical particles with capsules,and the diameter of the virus was about 40～60 nm. The XJ11129 virus could cause cytopathic changes in mammalian cells(BHK and Vero)and mosquito cells(C6/36). The titers of the XJ11129 virus were ≤108 PFU/100 μL,and the titer of the virus began to decrease 168 h after infecting C6/36 cells. Upon intracranial inoculation of the XJ11129 virus in 2 -day-old suckling mice,disease characteristics were seen clearly at days 2～3,paralysis began at day 4,and all mice died at day 5. The XJ11129 virus could produce a high - titer neutralization antibody response to its own strains(1∶1,280 dilution),but produced only a 1∶10 cross-neutralization PRNT90 titer against the JEV. These data show that the XJ11129 strain isolated from mosquitoes in Xinjiang Uygur Autonomous Region of China was strongly pathogenic to mammalian cells and mouse.
引文
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[23]Andreadis T G.The contribution of Culex pipiens com-plex mosquitoes to transmission and persistence of West Nile virus in North America[J].J Am Mosq Control As-soc,2012,28(4 Suppl):137-151.
[2]Kramer L D,Styer L M,Ebel G D.A global perspec-tive on the epidemiology of West Nile virus[J].Annu Rev Entomol,2008,53:61-81.
[3]Kilpatrick A M.Globalization,land use,and the inva-sion of West Nile virus[J].Science,2011,334(6054):323-327.
[4]Colpitts T M,Conway M J,Montgomery R R,Fikrig E.West Nile Virus:biology,transmission,and human infection[J].Clin Microbiol Rev,2012,25(4):635-648.
[5]May F J,Davis C T,Tesh R B,Barrett A D.Phylo-geography of West Nile virus:from the cradle of evolu-tion in Africa to Eurasia,Australia,and the Americas[J].J Virol,2011,85(6):2964-2974.
[6]Weaver S C,Reisen W K.Present and future arboviral threats[J].Antiviral Res,2010,85(2):328-345.
[7]David S,Abraham A M.Epidemiological and clinical as-pects on West Nile virus,a globally emerging pathogen[J].Infect Dis(Lond),2016,48(8):571-586.
[8]Sejvar J J,Lindsey N P,Campbell G L.Primary causes of death in reported cases of fatal West Nile Fever,Unit-ed States,2002-2006[J].Vector Borne Zoonotic Dis,2011,11:161-164.
[9]Guarner J,Shieh W J,Hunter S,Paddock C D,Morken T,Campbell G L,Marfin A A,Zaki S R.Clin-icopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis[J].Hum Pathol,2004,35(8):983-990.
[10]Lu Z,Fu SH,Cao L,Tang C Z,Zhang S,Li Z X,Tusong M,Yao X H,Zhang H L,Wang P Y,Mumai-er M,Yuan X Y,Li M H,Zhu C Z,Fu L P,Liang GD.Human infection with West Nile Virus,Xinjiang,China,2011[J].Emerg Infect Dis,2014,20:1421-1423.
[11]Li X L,Fu S H,Liu W B,Wang H Y,Lu Z,Tong SX,Li Z X,Nasci R S,Kosoy O,Cui Y,Liang G D.West Nile virus infection in Xinjiang,China[J].Vector Borne Zoonotic Dis,2013,13:131-133.
[12]Cao L,Fu SH,Lv Z,Tang C Z,Cui S H,Li X L,Gao X Y,Li M H,Cao Y X,Lei W W,He Y,Wang H Y,Liang G D.West Nile virus infection in suspected febrile typhoid cases in Xinjiang,China[J/OL].Emerg Microbes Infect,2017,6(6):e41.
[13]Pan XL,Liu H,Wang H Y,Fu SH,Liu H Z,Zhang H L,Li M H,Gao X Y,Wang J L,Sun X H,Lu X J,Zhai Y G,Meng W S,He Y,Wang H Q,Han N,Wei B,Wu Y G,Feng Y,Yang D J,Wang L H,Tang Q,Xia G,Kurane I,Rayner S,Liang G D.Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia[J].J Virol,2011,85(19):9847-9853.
[14]Cao L,Fu S H,Gao X Y,Li M H,Cui S H,Li X L,Cao Y X,Lei W W,Lu Z,He Y,Wang H Y,Yan JH,Gao G F,Liang G D.Low protective efficacy of the current Japanese encephalitis vaccine against the emerg-ing genotype 5 Japanese encephalitis virus[J/OL].PLoS Negl Trop Dis,2016,10(5):e0004686.
[15]Kaiser J A,Wang T,Barrett A D.Virulence determi-nants of West Nile virus:how can these be used for vac-cine design[J].Future Virol,2017,12(5):283-295.
[16]Zhang S,Li L,Woodson S E,Huang C Y,Kinney RM,Barrett A D,Beasley D W.A mutation in the enve-lope protein fusion loop attenuates mouse neuroinvasive-ness of the NY99 strain of West Nile virus[J].Virolo-gy,2006,353(1):35-40.
[17]Fall G,Di Paola N,Faye M,Dia M,Freire C C M,Loucoubar C,Zanotto P M A,Faye O,Sall A A.Bio-logical and phylogenetic characteristics of West African lineages of West Nile virus[J/OL].PLoS Negl Trop Dis,2017,11(11):e0006078.
[18]Hanna S L,Pierson T C,Sanchez M D,Ahmed A A,Murtadha M M,Doms R W.N-linked glycosylation of West Nile virus envelope proteins influences particle as-sembly and infectivity[J].J Virol,2005,79(21),13262-13274.
[19]Campbell G L,Marfin A A,Lanciotti R S,Gubler D J.West Nile virus[J].Lancet Infect Dis,2002,2(9):519-529.
[20]Mansfield K L,Horton D L,Johnson N,Li L,Barrett A D,Smith D J,Galbraith S E,Solomon T,Fooks AR.Flavivirus-induced antibody cross-reactivity[J].JGen Virol,2011,92(Pt 12):2821-2829.
[21]Lustig Y,Sofer D,Bucris E D and Mendelson E.Sur-veillance and diagnosis of West Nile virus in the face of flavivirus cross-reactivity[J].Front Microbiol,2018,9:2421.
[22]Higgs S,Snow K,Gould E A.The potential for West Nile virus to establish outside of its natural range:a con-sideration of potential mosquito vectors in the United Kingdom[J].Trans R Soc Trop Med Hyg,2004,98(2):82-87.
[23]Andreadis T G.The contribution of Culex pipiens com-plex mosquitoes to transmission and persistence of West Nile virus in North America[J].J Am Mosq Control As-soc,2012,28(4 Suppl):137-151.