从致倦库蚊和骚扰阿蚊标本中分离寨卡病毒的比较研究
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摘要
由不同蚊虫分离到的寨卡病毒的生物学表型研究未见报道。本研究首次对从致倦库蚊和骚扰阿蚊中分离的两株寨卡病毒进行生物学表型及病毒分子特征开展研究。分别观察两株病毒在多种组织培养细胞中的致病变效应、空斑形成及病毒滴度等,并对两株病毒基因序列进行分子遗传分析。结果显示,致倦库蚊分离株(GZDJ1685)在BHK-21细胞、C6/36细胞和Vero细胞中的病变时间分别为48h、48h和144h;而骚扰阿蚊分离株(GZDJ1666-2)在BHK-21细胞中的病变时间为144h,该毒株不引起Vero细胞和C6/36细胞病变。两毒株均可在BHK-21细胞中形成空斑。GZDJ1685毒株在BHK-21细胞、C6/36细胞和Vero细胞中的扩增滴度分别为10~(4.2)PFU/mL、10~(5.17) PFU/mL和10~(4.6) PFU/mL,而GZDJ1666-2毒株在以上3种细胞中的扩增滴度分别为10~(3.5) PFU/mL、10~(5.49) PFU/mL和10~(6.08)PFU/mL。病毒分子遗传系统进化分析发现,GZDJ1685和GZDJ1666-2毒株处于共同的进化分支,均属于亚洲Ⅱ型寨卡病毒。寨卡病毒编码区氨基酸位点分析提示,GZDJ1685和GZDJ1666-2毒株在结构基因的氨基酸位点(S139N、D683E、V763M、T777M)和非结构基因NS1基因的(A188V)变异与从埃及伊蚊和寨卡病毒感染患者分离的病毒完全相同。研究结果提示,虽然从致倦库蚊和骚扰阿蚊分离的寨卡病毒株对组织培养细胞致病变作用和病毒滴度存在巨大差异,但从致倦库蚊和骚扰阿蚊分离的寨卡病毒株均具备与2016年以来在南美洲流行的寨卡病毒相同的分子基础。
The biological phenotype of Zika virus strains isolated from different mosquitoes has not been reported. This study is the first to study the biological phenotype and molecular characteristics of the two Zika virus strains isolated from Culex quinquefasciatus and Armigeres subalbatus. The cytopathic effects(CPEs), plaque formation and virus titration of the two strains of virus in various tissue culture cells were investigated, and the genetic sequences of the two strains of virus were analyzed by molecular genetic analysis,The results showed that the CPEs of Zika viruses isolated from C. quinquefasciatus(GZDJ1685) were observed at 48 h, 48 h, and 144 hrs in BHK-21, C6/36, and Vero cells, respectively. The CPEs of Zika viruses isolated from A. subalbatus(GZDJ1666-2) were observed at 144 hrs in BHK-21 cells. However, GZDJ1666-2 did not induce CPEs in C6/36 or Vero cells, even though viral genes were amplified. The amplification titers of GZDJ1685 strain in BHK-21 cells, C6/36 cells and Vero cells were 10~(4.2) PFU/mL,10~(5.17) PFU/mL and 10~(4.6)PFU/mL, respectively, while the amplification titers of GZDJ1666-2 strain in the above three cells were 10~(3.5)PFU/mL, 10~(5.49) PFU/mL and 10~(6.08) PFU/mL, respectively. Virus phylogenetic analyses found that GZDJ1685 and GZDJ1666-2 strains shared the same evolutionary branch, all belong to Asia Ⅱ Zika virus. Amino-acid analyses of the coding region of Zika viruses indicated that GZDJ1685 virus and GZDJ1666-2 virus had the same variation in amino acid site of structural genes(S139N, D683E, V763M, T777M) and non-structural genes NS1 gene(A188V) as those isolated in A. aegypti and Zika virus infected patients. The results indicated that although there were significant differences in the CPEs and viral titers of Zika viruses isolated from C. quinquefasciatus and A. subalbatus on tissue culture cells, the Zika virus strains isolated from C. quinquefasciatus and A. subalbatus had the same molecular basis as the Zika virus that has been prevalent in South America since2016.
The biological phenotype of Zika virus strains isolated from different mosquitoes has not been reported. This study is the first to study the biological phenotype and molecular characteristics of the two Zika virus strains isolated from Culex quinquefasciatus and Armigeres subalbatus. The cytopathic effects(CPEs), plaque formation and virus titration of the two strains of virus in various tissue culture cells were investigated, and the genetic sequences of the two strains of virus were analyzed by molecular genetic analysis,The results showed that the CPEs of Zika viruses isolated from C. quinquefasciatus(GZDJ1685) were observed at 48 h, 48 h, and 144 hrs in BHK-21, C6/36, and Vero cells, respectively. The CPEs of Zika viruses isolated from A. subalbatus(GZDJ1666-2) were observed at 144 hrs in BHK-21 cells. However, GZDJ1666-2 did not induce CPEs in C6/36 or Vero cells, even though viral genes were amplified. The amplification titers of GZDJ1685 strain in BHK-21 cells, C6/36 cells and Vero cells were 10~(4.2) PFU/mL,10~(5.17) PFU/mL and 10~(4.6)PFU/mL, respectively, while the amplification titers of GZDJ1666-2 strain in the above three cells were 10~(3.5)PFU/mL, 10~(5.49) PFU/mL and 10~(6.08) PFU/mL, respectively. Virus phylogenetic analyses found that GZDJ1685 and GZDJ1666-2 strains shared the same evolutionary branch, all belong to Asia Ⅱ Zika virus. Amino-acid analyses of the coding region of Zika viruses indicated that GZDJ1685 virus and GZDJ1666-2 virus had the same variation in amino acid site of structural genes(S139N, D683E, V763M, T777M) and non-structural genes NS1 gene(A188V) as those isolated in A. aegypti and Zika virus infected patients. The results indicated that although there were significant differences in the CPEs and viral titers of Zika viruses isolated from C. quinquefasciatus and A. subalbatus on tissue culture cells, the Zika virus strains isolated from C. quinquefasciatus and A. subalbatus had the same molecular basis as the Zika virus that has been prevalent in South America since2016.
引文
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[18]Liu Y,Liu J,Du S,Shan C,Nie K,Zhang R,Li X F,Zhang R,Wang T,Qin C F,Wang P,Shi PY,Cheng G.Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes[J].Nature,2017,545(7655):482.
[19]Diallo D,Sall A A,Diagne C T,Faye O,Faye O,Ba Y,Hanley K A,Buenemann M,Weaver S C,Diallo M.Zika virus emergence in mosquitoes in southeastern Senegal,2011[J/OL].PLoS One,2014,9(10):e109442.
[20]Guedes D R,Paiva M H,Donato M M,Barbosa P P,Krokovsky L,Rocha SWDS,Saraiva K,Crespo M M,Rezende T M,Wallau G L,Barbosa R M,Oliveira CM,Melo-Santos M A,Pena L,Cordeiro M T,Franca RFO,Oliveira A L,Peixoto C A,Leal W S,Ayres CF.Zika virus replication in the mosquito Culex quinque-fasciatus in Brazil[J/OL].Emerg Microbes Infect,2017,6(8):e69.
[21]Guo X X,Li CX,Deng Y Q,Xing D,Liu Q M,Wu Q,Sun A J,Dong Y D,Cao W C,Qin C F,Zhao TY.Culex pipiens quinquefasciatus:a potential vector to transmit Zika virus[J/OL].Emerg Microbes Infect,2016,5(9):e102.
[2]Duffy M R,Chen T H,Hancock W T,Powers A M,Kool J L,Lanciotti R S,Pretrick M,Marfel M,Holz-bauer S,Dubray C,Guillaumot L,Griggs A,Bel M,Lambert A J,Laven J,Kosoy O,Panella A,Bigger-staff B J,Fischer M,Hayes E B.Zika virus outbreak on Yap Island,Federated States of Micronesia[J].N Engl J Med 2009,360(24):2536-43.
[3]Franca R F,Neves M H,Ayres C F,Meloneto O P,Filho S P.First International Workshop on zika virus held by Oswaldo Cruz Foundation FIOCRUZ in North-east Brazil March 2016-A Meeting Report[J/OL].PLoS Negl Trop Dis,2016,10(6):e0004760.
[4]Mlakar J,Korva M,Tul N,Popovi?M,Polj?ak-Pri-jatelj M,Mraz J,Kolenc M,Resman Rus K,Vesnaver Vipotnik T,Fabjan Vodu?ek V,Vizjak A,Vizjak J,Petrovec M,Av?i??upanc T.Zika virus associated with microcephaly[J].N Engl J Med,2016,374(10):951-958.
[5]Caolormeau V M,Blake A,Mons S,Lastère S,Roche C,Vanhomwegen J,et al.Guillain-Barre Syndrome outbreak associated with Zika virus infection in French Polynesia:a case-control study[J].Lancet,2016,387(10027):1531-1539.
[6]Enserink,M.INFECTIOUS DISEASES.An obscure mosquito-borne disease goes global[J].Science,2015,350:1012-1013.
[7]WHO.WHO Director-General summarizes the outcome of the Emergency Committee regarding clusters of micro-cephaly and Guillain-Barrésyndrome[EB/OL].Avail-able at http://www.who.int/mediacentre/news/state-ments/2016/emergency-committee-zika-microce-phaly/en/(accessed 1 February 2016).
[8]Epelboin Y,Talaga S,Epelboin L,Dusfour I.Zika vi-rus:An updated review of competent or naturally infect-ed mosquitoes[J/OL].PLoS Negl Trop Dis,2017,11(11):e0005933.
[9]Louren?o-De-Oliveira R,Failloux A B.Lessons learned on Zika virus vectors[J/OL].PLoS Negl Trop Dis,2017,11(6):e0005511.
[10]Song S,Li Y,Fu S,Liu H,Li X,Gao X,Xu Z,Liu G,Wang D,Tian Z,Zhou J,He Y,Lei W,Wang H,Wang B,Lu X,Liang G.Could Zika virus emerge in Mainland China?Virus isolation from nature in Culex quinquefasciatus,2016[J/OL].Emerg Microbes Infect,2017,6(11):e93.
[11]Fu S,Song S,Liu H,Li Y,Li X,Gao X,Xu Z,Liu G,Wang D,Tian Z,Zhou J,He Y,Lei W,Wang H,Wang B,Lu X,Liang G.ZIKA virus isolated from mos-quitoes:a field and laboratory investigation in China,2016[J].Sci China Life Sci,2017,60(12):1-8.
[12]Lei W,Guo X,Fu S,Feng Y,Tao X,Gao X,Song J,Yang Z,Zhou H,Liang G.The genetic characteris-tics and evolution of Tembusu virus[J].Vet Microbiol,2017,201:32-41
[13]Mutebi J P,Hughes H R,Burkhalter K L,Kothera L,Vasquez C,Kenney J L.Zika virus MB16-23 in mosqui-toes,Miami-Dade County,Florida,USA,2016[J].Emerg Infect Dis,2018,24(4):808-810.
[14]Cao L,Fu S,Gao X,Li M,Cui S,Li X,Cao Y,Lei W,Lu Z,He Y,Wang H,Yan J,Gao GF,Liang G.Low protective efficacy of the current japanese encephali-tis vaccine against the emerging genotype 5 japanese en-cephalitis virus[J/OL].PLoS Negl Trop Dis,2016,10(5):e0004686.
[15]Bonaldo M C,Ribeiro I P,Lima N S,Dos Santos AA,Menezes L S,da Cruz S O,de Mello I S,Furtado N D,de Moura E E,Damasceno L,da Silva K A,de Castro M G,Gerber A L,de Almeida L G,Louren?o-de-Oliveira R,Vasconcelos A T,Brasil P.Isolation of infective Zika virus from urine and saliva of patients in Brazil[J/OL].PLoS Negl Trop Dis,2016,10(6):e0004816.
[16]Haddow A D,Schuh A J,Yasuda C Y,Kasper M R,Heang V,Huy R,Guzman H,Tesh R B,Weaver S C.Genetic characterization of Zika virus strains:geographic expansion of the Asian lineage[J/OL].PLoS Negl Trop Dis,2012,6(2):e1477.
[17]Pettersson J H,Eldholm V,Seligman S J,Lundkvist?,Falconar A K,Gaunt M W,Musso D,Nougairède A,Charrel R,Gould E A,de Lamballerie X.How did Zika virus emerge in the Pacific Islands and Latin Ameri-ca[J/OL]mBio,2016,7(5):e01239-16.
[18]Liu Y,Liu J,Du S,Shan C,Nie K,Zhang R,Li X F,Zhang R,Wang T,Qin C F,Wang P,Shi PY,Cheng G.Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes[J].Nature,2017,545(7655):482.
[19]Diallo D,Sall A A,Diagne C T,Faye O,Faye O,Ba Y,Hanley K A,Buenemann M,Weaver S C,Diallo M.Zika virus emergence in mosquitoes in southeastern Senegal,2011[J/OL].PLoS One,2014,9(10):e109442.
[20]Guedes D R,Paiva M H,Donato M M,Barbosa P P,Krokovsky L,Rocha SWDS,Saraiva K,Crespo M M,Rezende T M,Wallau G L,Barbosa R M,Oliveira CM,Melo-Santos M A,Pena L,Cordeiro M T,Franca RFO,Oliveira A L,Peixoto C A,Leal W S,Ayres CF.Zika virus replication in the mosquito Culex quinque-fasciatus in Brazil[J/OL].Emerg Microbes Infect,2017,6(8):e69.
[21]Guo X X,Li CX,Deng Y Q,Xing D,Liu Q M,Wu Q,Sun A J,Dong Y D,Cao W C,Qin C F,Zhao TY.Culex pipiens quinquefasciatus:a potential vector to transmit Zika virus[J/OL].Emerg Microbes Infect,2016,5(9):e102.