肝素及硫酸软骨素抑制Ⅰ型与Ⅲ型草鱼呼肠孤病毒的体外增殖
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摘要
草鱼(Ctenopharyngodon idellus)是我国一类重要的经济鱼类,约占淡水养殖产量的20%,但每年由于病害原因造成的减产在30%以上。其中草鱼呼肠孤病毒(Grass carp reovirus,GCRV)危害最大,被中国农业农村部列为二类动物疫病,开发具有临床价值的环境友好型药物或疫苗对草鱼出血病的防控具有重要意义。近年来,有大量研究表明肝素分子能抑制多种病毒的感染。为了揭示肝素分子及其类似物是否也可以阻遏GCRV感染,本研究利用Ⅰ、Ⅲ型GCRV分别感染草鱼肾细胞CIK,通过real time RT-PCR及Western Blot技术分别揭示GCRV感染后细胞上清中的子代病毒扩增效率及细胞内病毒蛋白合成水平,评价了肝素及其类似物硫酸软骨素对GCRV感染的抑制效果。结果表明,肝素分子在100μg/mL以上浓度时即对GCRV有抑制效果,10mg/mL以上用量时,病毒蛋白合成效率及上清子代病毒扩增效率均显著降低;其类似物硫酸软骨素相比肝素抑制效果有所降低,但在20mg/mL以上用量时,病毒蛋白合成效率及上清子代病毒扩增效率也均有显著降低。研究结果表明肝素及其类似物对不同基因型的草鱼呼肠孤病毒的感染具有广谱抑制效应。本研究为开发肝素类似物作为拮抗草鱼出血病的环境友好型药物奠定了初步基础。
Grass carp(Ctenopharyngodon idellus)is an economic fish in China,which accounts for about 20%of freshwater fish production. However,there is more than 30% reduction due to disease. Grass carp reovirus(GCRV)causes grass carp hemorrhagic disease,which is defined the second class animal epidemic disease by Ministry of Agriculture and Rural Affairs,China. It is of great significance to develop environmental-friendly drugs in the prevention and control of grass carp hemorrhagic disease. Recently,heparin has been reported to inhibit viral infections in mammalian system. Here,in order to test the inhibitory effects of heparin and heparin analogue on GCRV infection,we employed a CIK/GCRV infection system to monitor the viral progeny production efficiency and viral protein-synthetic level by real time RT-PCR and Western Blot analysis,respectively. The results indicated that heparin had an inhibitory effect when the concentration was above100 μg/mL,and post treatment of 20 mg/mL heparin or heparin analogue,the viral replication level and viral protein synthesis were both inhibited significantly for type I grass carp reovirus. Interestingly,similar inhibitory results were achieved by chondroitin sulfate on type Ⅲ GCRV. Thus,this study paved the way for the development of heparin analogue-based environmental-friendly medicine against grass carp hemorrhagic disease.
Grass carp(Ctenopharyngodon idellus)is an economic fish in China,which accounts for about 20%of freshwater fish production. However,there is more than 30% reduction due to disease. Grass carp reovirus(GCRV)causes grass carp hemorrhagic disease,which is defined the second class animal epidemic disease by Ministry of Agriculture and Rural Affairs,China. It is of great significance to develop environmental-friendly drugs in the prevention and control of grass carp hemorrhagic disease. Recently,heparin has been reported to inhibit viral infections in mammalian system. Here,in order to test the inhibitory effects of heparin and heparin analogue on GCRV infection,we employed a CIK/GCRV infection system to monitor the viral progeny production efficiency and viral protein-synthetic level by real time RT-PCR and Western Blot analysis,respectively. The results indicated that heparin had an inhibitory effect when the concentration was above100 μg/mL,and post treatment of 20 mg/mL heparin or heparin analogue,the viral replication level and viral protein synthesis were both inhibited significantly for type I grass carp reovirus. Interestingly,similar inhibitory results were achieved by chondroitin sulfate on type Ⅲ GCRV. Thus,this study paved the way for the development of heparin analogue-based environmental-friendly medicine against grass carp hemorrhagic disease.
引文
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[18]Yu F,Wang L,Li W,Lu L.Identification of a novel membrane-associated protein from the S7 segment of grass carp reovirus[J].J Gen Virol,2019,100(3):369-379.
[19]Wang L,Yu F,Sun H,Lu L.Characterization of the interaction between outer-fiber protein VP55 of genotype III grass carp reovirus and Fibulin-4 of grass carp[J].Fish Shellfish Immunol,2019,86:355-360.
[20]Wang H,Liu W,Sun M,Chen D,Zeng L,Lu L,Xie J.Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus[J].Virol J,2018,15(1):92.
[21]Wang Q,Zeng W,Liu C,Zhang C,Wang Y,Shi C,Wu S.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRVin China[J].J Virol,2012,86(22):12466-12466.
[22]Holodniy M,Kim S,Katzenstein D,Konrad M,Groves E,Merigan T C.Inhibition of human immunodeficiency virus gene amplification by heparin[J].J Clin Microbiol,1991,29(4):676-679.
[23]Lin Y L,Lei H Y,Lin Y S,Yeh T M,Chen S H,Liu H S.Heparin inhibits dengue-2 virus infection of five human liver cell lines[J].Antivir Res,2002,56(1):93-96.
[24]Seki Y,Mizukura M,Ichimiya T,Suda Y,Nishihara S,Masuda M,Takase-Yoden S.O-sulfate groups of heparin are critical for inhibition of ecotropic murine leukemia virus infection by heparin[J].Virology,2012,424(1):56-66.
[2]章漳,王硕,邱宏,王峥涛,丁侃.硫酸乙酰肝素蛋白聚糖与病毒感染[J].中国医疗前沿,2009,4(3):1-3.
[3]赵建勇,独军政,高闪电,丛国正,林彤,邵军军,伏小平,常惠芸.硫酸乙酰肝素受体介导的病毒感染作用[J].中国人兽共患病学报,2008,24(9):874-877.
[4]Khanna M,Ranasinghe C,Jackson R,Parish C R.Heparan sulfate as a receptor for poxvirus infections and as a target for antiviral agents[J].J Gen Virol,2017.DOI:10.1099/jgv.0.000921.[Epub ahead of print]
[5]方勤,朱作言.水生呼肠孤病毒研究进展[J].中国病毒学,2003,18(1):82-86.
[6]王方华,李安兴.草鱼病毒性出血病研究进展[J].南方水产,2006,2(3):66-71.
[7]郭帅,李家乐,吕利群.草鱼呼肠孤病毒的致病机制及抗病毒新对策[J].渔业现代化,2010,37(1):37-42.
[8]张敏利,盛佳璐,喻飞,王浩,吕利群.草鱼Fibulin-4蛋白与草鱼呼肠孤病毒外衣壳蛋白相互作用的研究[J].病毒学报,2018,34(4):557-564.DOI:10.13242/j.cnki.bingduxuebao.003423
[9]Fang Qin,Ke Lihua.Growth characteristics and high titer culture of grass carp hemorrhage virus(GCHV)-873 in vitro[J].Virol Sin,1989,3:315-319.
[10]张超,王庆,石存斌.草鱼呼肠孤病毒HZ08株的分离与鉴定[J].中国水产科学,2010,17(6).
[11]Fan Y,Rao S,Zeng L,Ma J,Zhou Y,Xu J,Zhang H.Identification and genomic characterization of a novel fish reovirus,Hubei grass carp disease reovirus,isolated in 2009 in China[J].J Gen Virol,2013,94(Pt 10):2266-2277.
[12]Zhang Q,Gui J F.Virus genomes and virus-host interactions in aquaculture animals[J].Sci China Life Sci,2015,58(2):156-169.
[13]HR.L,YG.L,WW.Z,YY.W,Q.W,SQ.W.Pathogenicity and tissue distribution of grass carp reovirus after intraperitoneal administration[J].Virol J,2014,11:178.
[14]Wang T,Li J,Lu L.Quantitative in vivo and in vitro characterization of co-infection by two genetically distant grass carp reoviruses[J].J Gen Virol,2013,94(Pt 6):1301-1309.
[15]Ye X,Tian Y Y,Deng G C,Chi Y Y,Jiang X Y.Complete genomic sequence of a reovirus isolated from grass carp in China[J].Virus Res,2012,163(1):275-283.
[16]Pei C,Ke F,Chen Z Y,Zhang Q Y.Complete genome sequence and comparative analysis of grass carp reovirus strain 109(GCReV-109)with other grass carp reovirus strains reveals no significant correlation with regional distribution[J].Arch Virol,2014,159(9):2435-2440.
[17]方勤,Sanket Shah.草鱼呼肠毒的三维重构与衣壳蛋白特性[J].中国科学C辑生命科学,2005,35(3):231-237.
[18]Yu F,Wang L,Li W,Lu L.Identification of a novel membrane-associated protein from the S7 segment of grass carp reovirus[J].J Gen Virol,2019,100(3):369-379.
[19]Wang L,Yu F,Sun H,Lu L.Characterization of the interaction between outer-fiber protein VP55 of genotype III grass carp reovirus and Fibulin-4 of grass carp[J].Fish Shellfish Immunol,2019,86:355-360.
[20]Wang H,Liu W,Sun M,Chen D,Zeng L,Lu L,Xie J.Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus[J].Virol J,2018,15(1):92.
[21]Wang Q,Zeng W,Liu C,Zhang C,Wang Y,Shi C,Wu S.Complete genome sequence of a reovirus isolated from grass carp,indicating different genotypes of GCRVin China[J].J Virol,2012,86(22):12466-12466.
[22]Holodniy M,Kim S,Katzenstein D,Konrad M,Groves E,Merigan T C.Inhibition of human immunodeficiency virus gene amplification by heparin[J].J Clin Microbiol,1991,29(4):676-679.
[23]Lin Y L,Lei H Y,Lin Y S,Yeh T M,Chen S H,Liu H S.Heparin inhibits dengue-2 virus infection of five human liver cell lines[J].Antivir Res,2002,56(1):93-96.
[24]Seki Y,Mizukura M,Ichimiya T,Suda Y,Nishihara S,Masuda M,Takase-Yoden S.O-sulfate groups of heparin are critical for inhibition of ecotropic murine leukemia virus infection by heparin[J].Virology,2012,424(1):56-66.