H9N2亚型猪源性流感病毒在小鼠肺微血管内皮细胞内最佳增殖条件的研究
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摘要
试验旨在确定H9N2亚型猪源性流感病毒(SIV)在小鼠肺微血管内皮细胞(PMVEC)中增殖的最佳条件。将PMVEC解冻、复苏、培养,取长成单层的PMVEC,在不同浓度TPCK-胰蛋白酶维持液(0.1、0.2、0.3、0.4、0.6、0.8、1.0μg/mL)、不同H9N2亚型SIV(A/swine/HeBei/012/2008/(H9N2)接种剂量(1∶10、1∶100、1∶1 000、1∶10 000、1∶100 000、1∶1 000 000和1∶10 000 000)及不同病毒吸附时间(0.5、1.0、2.0和3.0h)条件下观察PMVEC形态变化,并测定细胞上清液中H9N2亚型SIV的HA滴度。在未加病毒的情况下,低于0.6μg/mL的TPCK-胰蛋白酶对PMVEC的生长未造成任何影响,但随着TPCK-胰蛋白酶浓度的增加,PMVEC开始出现肿胀、变圆,甚至脱落;采用含有0.6μg/mL TPCK-胰蛋白酶维持液将H9N2亚型SIV稀释为不同的浓度感染PMVEC,在0.3μg/mL TPCK-胰蛋白酶维持液、10-2病毒稀释倍数感染条件下48和72h HA滴度分别为4.6log2和6.4log2;病毒吸附时间为2h且中间震荡20s的条件下H9N2HA滴度最佳。结果表明,当TPCK-胰蛋白酶维持液浓度为0.3μg/mL、病毒接种浓度为10-2、吸附时间为2h且中间震荡20s时,H9N2亚型SIV在PMVEC中增殖最佳,达到6.8log2。
This study was aimed to determine the optimal proliferation conditions for culturing of H9N2 subtype swine influenza virus(SIV)in mouse pulmonary microvascular endothelial cell(PMVEC).The PMVECs were defrosted,recovered and cultured,then the morphological changes of PMVECs were observed and the HA titer of H9N2 subtype SIV of the cell culture supernate was determined by hemagglutination test after action with different concentrations of TPCK-trypsin maintenance media(0.1,0.2,0.3,0.4,0.6,0.8 and 1.0μg/mL),different doses of H9N2 subtype SIV(1∶10,1∶100,1∶1 000,1∶10 000,1∶100 000,1∶1 000 000 and 1∶10 000 000)and different viral adsorption time(0.5,1.0,2.0 and 3.0 h)on single layer of PMVECs.Whenthe dose of TPCK-trypsin was lower than 0.6μg/mL,there was no effect on morphology structure of PMVEC uninfected with H9N2 subtype SIV.However,the PMVEC began to swell,round and exfoliate with the increase of the TPCK-trypsin concentration;PMVECs were infected with different dose of H9N2 subtype SIV diluted with 0.6μg/mL TPCK-trypsin culture media,the viral titer were 4.6 log2 and 6.4 log2 on 48 and 72 hin 0.3μg/mL cell culture maintenance medium and the dose of 10-2 H9N2 subtype SIV;Meanwhile,the H9N2 subtype SIV titer was reached to peak value on condition of 2 hadsorption time and vibration 20 safter adsorption 1 h.When the TPCK-trypsin final concentration was 0.3μg/mL,H9N2 subtype SIV dose of 10-2,and 2 hviral adsorption and vibration 20 sin the middle of adsorption time,the H9N2 swine influenza virus had optimal proliferation in PMVEC cell,the viral titer was 6.8 log2.
This study was aimed to determine the optimal proliferation conditions for culturing of H9N2 subtype swine influenza virus(SIV)in mouse pulmonary microvascular endothelial cell(PMVEC).The PMVECs were defrosted,recovered and cultured,then the morphological changes of PMVECs were observed and the HA titer of H9N2 subtype SIV of the cell culture supernate was determined by hemagglutination test after action with different concentrations of TPCK-trypsin maintenance media(0.1,0.2,0.3,0.4,0.6,0.8 and 1.0μg/mL),different doses of H9N2 subtype SIV(1∶10,1∶100,1∶1 000,1∶10 000,1∶100 000,1∶1 000 000 and 1∶10 000 000)and different viral adsorption time(0.5,1.0,2.0 and 3.0 h)on single layer of PMVECs.Whenthe dose of TPCK-trypsin was lower than 0.6μg/mL,there was no effect on morphology structure of PMVEC uninfected with H9N2 subtype SIV.However,the PMVEC began to swell,round and exfoliate with the increase of the TPCK-trypsin concentration;PMVECs were infected with different dose of H9N2 subtype SIV diluted with 0.6μg/mL TPCK-trypsin culture media,the viral titer were 4.6 log2 and 6.4 log2 on 48 and 72 hin 0.3μg/mL cell culture maintenance medium and the dose of 10-2 H9N2 subtype SIV;Meanwhile,the H9N2 subtype SIV titer was reached to peak value on condition of 2 hadsorption time and vibration 20 safter adsorption 1 h.When the TPCK-trypsin final concentration was 0.3μg/mL,H9N2 subtype SIV dose of 10-2,and 2 hviral adsorption and vibration 20 sin the middle of adsorption time,the H9N2 swine influenza virus had optimal proliferation in PMVEC cell,the viral titer was 6.8 log2.
引文
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[13]ZENG H,PAPPAS C,BELSER J A,et al.Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses:Possible involvement in the pathogenesis of human H5N1virus infection[J].Journal of Virology,2012,86(2):667-678.
[14]张增峰,樊晓晖,陈晓燕,等.禽流感病毒H9N2在人肺组织的复制[J].病毒学报,2013,29(2):206-209.ZHANG Z F,FAN X H,CHEN X Y,et al.Avian influenza virus subtype H9N2replication in human lung tissue[J].Chinese Journal of Virology,2013,29(2):206-209.(in Chinese)
[15]XU T,QIAO J,ZHAO L,et al.Acute respiratory distress syndrome induced by avian influenza A(H5N1)virus in mice[J].American Journal of Respiratory&Critical Care Medicine,2006,174(6):1011-1017.
[16]刘永法,黄玉梅,张宗尧,等.一株H5N6亚型禽流感病毒对鸭和小鼠的致病性研究[J].中国畜牧兽医,2015,42(8):2176-2182.LIU Y C,HUANG Y M,ZHANG Z Y.et al.Pathogenicity of a strain of H5N6subtype avian influenza virus in ducks and mice[J].China Animal Husbandry&Veterinary Medicine,2015,42(8):2176-2182.(in Chinese)
[17]徐明举,利凯,徐彤,等.H9N2亚型猪流感病毒诱导小鼠肺损伤模型的建立[J].畜牧兽医学报,2011,42(6):838-844.XU M J,LI K,XU T,et al.Study on the model of acute lung injury induced by swine influenza H9N2in mice[J].Acta Veterinaria et Zootechnica Sinica,2011,42(6):838-844.(in Chinese)
[18]冯婷,殷仲伟,李晋蓉,等.流感病毒H1N1在MDCK细胞的培养及纯化条件的优化[J].中国病原生物学杂志,2012,7(7):493-496.FENG T,YIN Z W,LI J R,et al.Optimization of culture conditions for culturing of influenza virus H1N1in MDCK cells and its purification[J].Journal of Pathogen Biology,2012,7(7):493-496.(in Chinese)
[19]陈润莉,周海涛,侯红斌,等.流感病毒在MDCK细胞上的培养条件优化[J].实用预防医学,2011,18(7):1203-1205.CHEN R L,ZHOU H T,HOU H B,et al.Optimization of MDCK cell culturing condition for the cultivation of influenza virus[J].Practical Preventive Medicine,2011,18(7):1203-1205.(in Chinese)
[20]史爱华,姜北宇,沈佳,等.胰蛋白酶浓度对H9亚型禽流感病毒在MDCK细胞上增殖的影响[J].安徽农业科学,2011,39(35):21813-21815.SHI A H,JIANG B Y,SHEN J,et al.Effects of trypsin on proliferation of avian influenza virus H9N2subtype in MDCK cell[J].Journal of Anhui Agricultural Sciences,2011,39(35):21813-21815.(in Chinese)
[21]史爱华,姜北宇,沈佳,等.H9N2亚型禽流感病毒在MDCK细胞中增殖最佳条件研究[J].动物医学进展,2011,32(11):42-45.SHI A H,JIANG B Y,SHEN J,et al.The optimal proliferation condition of H9N2subtype avian influenza virus in MDCK cells[J].Progress in Veterinary Medicine,2011,32(11):42-45.(in Chinese)
[22]刘丽玲,王秀荣,陈化兰,等.不同亚型禽流感病毒在MDCK细胞上的生长特性[J].中国兽医杂志,2006,42(10):18-20.LIU L L,WANG X R,CHEN H L,et al.The growth characteristics of different subtypes of avian influenza virus in MDCK cells[J].Chinese Journal of Veterinary Medicine,2006,42(10):18-20.(in Chinese)
[23]李春艳,肖晶,李曦,等.微载体规模化培养MDCK细胞增殖H9N2亚型禽流感病毒的研究[J].中国人兽共患病学报,2009,25(12):1149-1153.LI C Y,XIAO J,LI X,et al.Research on multiplication of the H9N2subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system[J].Chinese Journal of Zoonoses,2009,25(12):1149-1153.(in Chinese)
[2]PEIRIS J S,GUAN Y,MARKWELL D,et al.Co-circulation of avian H9N2and contemporary“human”H3N2influenza A viruses in pigs in South-Eastern China:Potential for genetic reassortment?[J].Journal of Virology,2001,75(20):9679-9686.
[3]刘健,杨显超,徐锋,等.2株弱血凝活性H9N2亚型禽流感病毒特性的研究[J].中国畜牧兽医,2016,43(6):1624-1629.LIU J,YANG X C,XU F,et al.Study on the characteristics of two low hemagglutinating activity H9N2AIV strains[J].China Animal Husbandry&Veterinary Medicine,2016,43(6):1624-1629.(in Chinese)
[4]殷斌,孙文博,杜以军,等.山东H9N2亚型猪流感病毒的分离鉴定及遗传进化分析[J].南方农业学报,2015,46(9):1691-1697.YIN B,SUN W B,DU Y J,et al.Isolation,identification and genetic evolution analysis of H9N2subtype swine influenza virus in Shandong province[J].Journal of Southern Agriculture,2015,46(9):1691-1697.(in Chinese)
[5]CONG Y L,WANG C F,YAN C M,et al.Swine infection with H9N2influenza viruses in China in2004[J].Virus Genes,2008,36(3):461-469.
[6]何军,刘丽萍,侯赛,等.安徽省2株人感染H9N2流感病毒基因特征[J].中华流行病学杂志,2016,37(5):708-713.HE J,LIU L P,HOU S,et al.Genomic characteristics of 2strains of influenza A(H9N2)virus isolated from human infection cases in Anhui province[J].Chinese Journal of Epidemiology,2016,37(5):708-713.(in Chinese)
[7]王玥莲,鲁丽莉,冯琛,等.2016年四川省首例人感染禽流感H9N2危重症临床救治转运路径[J].世界最新医学信息文摘(电子版),2016,16(88):152-153.WANG Y L,LU L L,FENG C,et al.Discussion on clinical pathway of the first human avian influenza H9N2critical disease in Sichuan province,2016[J].World Latest Medicine Information(Electronic Version),2016,16(88):152-153.(in Chinese)
[8]游胜,冯秀,刘晓青,等.江西省首例人感染H9N2禽流感病例流行病学调查[J].现代预防医学,2017,44(5):782-784.YOU S,FENG X,LIU X Q,et al.Epidemiological investigation on the first case of human infection with H9N2avian influenza in Jiangxi[J].Modern Preventive Medicine,2017,44(5):782-784.(in Chinese)
[9]NINOMIYA A,TAKADA A,OKAZAKI K,et al.Seroepidemiological evidence of avian H4,H5,and H9influenza A virus transmission to pigs in Southeastern China[J].Veterinary Microbiology,2002,88(2):107-114.
[10]MATROSOVICH M N,KRAUSS S,WEBSTER R G.H9N2influenza A viruses from poultry in Asia have human virus-like receptor specificity[J].Virology,2001,281(2):156-162.
[11]LI K S,XU K M,PEIRIS J S,et al.Characterization of H9subtype influenza viruses from the ducks of Southern China:A candidate for the next influenza pandemic in humans?[J].Journal of Virology,2003,77(12):6988-6994.
[12]吕嘉文,胡国栋.肺微血管内皮细胞屏障功能调控与修复机制研究进展[J].广东医学,2017,38(8):1287-1290.LV J W,HU G D.Progress on barrier function regulation and repair mechanism of pulmonary microvascular endothelial cells[J].Guangdong Medical Journal,2017,38(8):1287-1290.(in Chinese)
[13]ZENG H,PAPPAS C,BELSER J A,et al.Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic avian influenza viruses:Possible involvement in the pathogenesis of human H5N1virus infection[J].Journal of Virology,2012,86(2):667-678.
[14]张增峰,樊晓晖,陈晓燕,等.禽流感病毒H9N2在人肺组织的复制[J].病毒学报,2013,29(2):206-209.ZHANG Z F,FAN X H,CHEN X Y,et al.Avian influenza virus subtype H9N2replication in human lung tissue[J].Chinese Journal of Virology,2013,29(2):206-209.(in Chinese)
[15]XU T,QIAO J,ZHAO L,et al.Acute respiratory distress syndrome induced by avian influenza A(H5N1)virus in mice[J].American Journal of Respiratory&Critical Care Medicine,2006,174(6):1011-1017.
[16]刘永法,黄玉梅,张宗尧,等.一株H5N6亚型禽流感病毒对鸭和小鼠的致病性研究[J].中国畜牧兽医,2015,42(8):2176-2182.LIU Y C,HUANG Y M,ZHANG Z Y.et al.Pathogenicity of a strain of H5N6subtype avian influenza virus in ducks and mice[J].China Animal Husbandry&Veterinary Medicine,2015,42(8):2176-2182.(in Chinese)
[17]徐明举,利凯,徐彤,等.H9N2亚型猪流感病毒诱导小鼠肺损伤模型的建立[J].畜牧兽医学报,2011,42(6):838-844.XU M J,LI K,XU T,et al.Study on the model of acute lung injury induced by swine influenza H9N2in mice[J].Acta Veterinaria et Zootechnica Sinica,2011,42(6):838-844.(in Chinese)
[18]冯婷,殷仲伟,李晋蓉,等.流感病毒H1N1在MDCK细胞的培养及纯化条件的优化[J].中国病原生物学杂志,2012,7(7):493-496.FENG T,YIN Z W,LI J R,et al.Optimization of culture conditions for culturing of influenza virus H1N1in MDCK cells and its purification[J].Journal of Pathogen Biology,2012,7(7):493-496.(in Chinese)
[19]陈润莉,周海涛,侯红斌,等.流感病毒在MDCK细胞上的培养条件优化[J].实用预防医学,2011,18(7):1203-1205.CHEN R L,ZHOU H T,HOU H B,et al.Optimization of MDCK cell culturing condition for the cultivation of influenza virus[J].Practical Preventive Medicine,2011,18(7):1203-1205.(in Chinese)
[20]史爱华,姜北宇,沈佳,等.胰蛋白酶浓度对H9亚型禽流感病毒在MDCK细胞上增殖的影响[J].安徽农业科学,2011,39(35):21813-21815.SHI A H,JIANG B Y,SHEN J,et al.Effects of trypsin on proliferation of avian influenza virus H9N2subtype in MDCK cell[J].Journal of Anhui Agricultural Sciences,2011,39(35):21813-21815.(in Chinese)
[21]史爱华,姜北宇,沈佳,等.H9N2亚型禽流感病毒在MDCK细胞中增殖最佳条件研究[J].动物医学进展,2011,32(11):42-45.SHI A H,JIANG B Y,SHEN J,et al.The optimal proliferation condition of H9N2subtype avian influenza virus in MDCK cells[J].Progress in Veterinary Medicine,2011,32(11):42-45.(in Chinese)
[22]刘丽玲,王秀荣,陈化兰,等.不同亚型禽流感病毒在MDCK细胞上的生长特性[J].中国兽医杂志,2006,42(10):18-20.LIU L L,WANG X R,CHEN H L,et al.The growth characteristics of different subtypes of avian influenza virus in MDCK cells[J].Chinese Journal of Veterinary Medicine,2006,42(10):18-20.(in Chinese)
[23]李春艳,肖晶,李曦,等.微载体规模化培养MDCK细胞增殖H9N2亚型禽流感病毒的研究[J].中国人兽共患病学报,2009,25(12):1149-1153.LI C Y,XIAO J,LI X,et al.Research on multiplication of the H9N2subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system[J].Chinese Journal of Zoonoses,2009,25(12):1149-1153.(in Chinese)