牛副流感病毒3型感染MDBK细胞后影响天然免疫基因表达的筛选与验证
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摘要
为筛选与验证牛副流感病毒3型(BPIV3)感染MDBK细胞后天然免疫相关基因的差异表达变化,通过细胞病变观察、Western blot验证病毒HN蛋白表达,以及病毒增殖复制动力学曲线,确定病毒复制情况。利用转录组测序技术,对1MOI基因C型SD2014BPIV3毒株感染MDBK细胞的12h对照组与感染组的差异表达基因进行筛选,最后收集BPIV3感染MDBK不同时间点细胞样本,应用RT-qPCR在转录水平验证了天然免疫相关显著差异基因的表达变化情况。结果表明,BPIV3毒株在MDBK细胞上有较强的复制能力,从病毒感染12h的转录组数据中获得了1 401个显著差异基因,经过筛选获得了9个与天然免疫相关的基因,最后在转录水平验证了MDA5、IRF7、STAT1、ISG65、TRIM25等9个与天然免疫相关基因的表达变化,并验证了其上、下游通路或同一家族基因的表达变化。结果表明,筛选与验证的BPIV3感染MDBK细胞差异表达天然免疫相关基因,为宿主细胞影响病毒复制的分子机制研究奠定了基础。
The present study determined differentially expressed innate immunity genes after bovine parainfluenza virus 3 infection(BPIV3)in MDBK cells.The cytopathic effect,expression of viral HN protein and the dynamics of viral replication were verified by cytopathic observation,Western blot and TCID50 determination.The differentially expressed genes of MDBK cells infected with 1 MOI genotype C SD2014 BPIV3 were screened by transcriptome sequencing.Then,MDBK cells samples infected with 1 MOI BPIV3 at different time points were collected.RT-qPCR assay was used to validate the expression changes of significantly different innate immunity genes.The results showed that BPIV3 strain has strong replication ability in MDBK cells.The 1 401 differentially expressed genes were obtained from transcriptome data of 12 hours after virus infection and nine innate immunity genes were screened.Nine innate immunity genes,such as MDA5,IRF7,STAT1,ISG65 and TRIM25,were identified at the transcriptional level,and the expression changes of upstream and downstream pathways or genes of the same family were also verified.This study provided differentially expressed innate immune-related genes towards understanding the infection mechanisms of BPIV3,which offers the basic data for further study on the molecular mechanism of host cells influencing virus replication.
The present study determined differentially expressed innate immunity genes after bovine parainfluenza virus 3 infection(BPIV3)in MDBK cells.The cytopathic effect,expression of viral HN protein and the dynamics of viral replication were verified by cytopathic observation,Western blot and TCID50 determination.The differentially expressed genes of MDBK cells infected with 1 MOI genotype C SD2014 BPIV3 were screened by transcriptome sequencing.Then,MDBK cells samples infected with 1 MOI BPIV3 at different time points were collected.RT-qPCR assay was used to validate the expression changes of significantly different innate immunity genes.The results showed that BPIV3 strain has strong replication ability in MDBK cells.The 1 401 differentially expressed genes were obtained from transcriptome data of 12 hours after virus infection and nine innate immunity genes were screened.Nine innate immunity genes,such as MDA5,IRF7,STAT1,ISG65 and TRIM25,were identified at the transcriptional level,and the expression changes of upstream and downstream pathways or genes of the same family were also verified.This study provided differentially expressed innate immune-related genes towards understanding the infection mechanisms of BPIV3,which offers the basic data for further study on the molecular mechanism of host cells influencing virus replication.
引文
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[22] Zhang L,Zhao X,Zhang M,et al.Ubiquitin-specific protease2bnegatively regulates IFN-beta production and antiviral activity by targeting TANK-binding kinase 1[J].J Immunol,2014,193(5):2230-2237.
[2] Jonas W,Siamak Z,Branka H,et al.Mononegaviruses of veterinary importance:Molecular epidemiology and control(Volume2)[M].CABI Publish:Wallingford,2013:98-105.
[3]王海勇,童钦,王炜,等.我国牛副流感病毒3型血清学调查[J].中国预防兽医学报,2014,36(2):154-156.
[4] Fulton R W,Neill J D,Saliki J T,et al.Genomic and antigenic characterization of bovine parainfluenza-3viruses in the United States including modified live virus vaccine(MLV)strains and field strains from cattle[J].Virus Res,2017,235:77-81.
[5]杨建乐,赵贵民,侯佩莉,等.牛副流感病毒3型抗体间接ELISA检测方法的建立与初步应用[J].动物医学进展,2016,37(11):19-24.
[6]朱远茂,马磊,杨婷,等.牛副流感的流行、危害及其防控[J].中国预防兽医学报,2016,38(8):667-671.
[7] Vareille M,Kieninger E,Edwards M R,et al.The airway epithelium:soldier in the fight against respiratory viruses[J].Clin Microbiol Rev,2011,24(1):210-229.
[8] Barik S.What Really Rigs Up RIG-I?[J].J Innate Immun,2016,8(5):429-436.
[9] Schneider W M,Chevillotte M D,Rice C M.Interferon-stimulated genes:a complex web of host defenses[J].Annu Rev Immunol,2014,32:513-545.
[10] Villalba M,Fredericksen F,Otth C,et al.Transcriptomic analysis of responses to cytopathic bovine viral diarrhea virus-1(BVDV-1)infection in MDBK cells[J].Mol Immunol,2016,71:192-202.
[11]赵贵民,侯佩莉,贾春涛,等.牛副流感病毒3型(BPIV3)P蛋白磷酸化位点与功能性结构域分析[J].中国兽医学报,2017,37(2):231-237.
[12]朱彤,赵贵民,沈付娆,等.牛肠道病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及初步应用[J].病毒学报,2015,31(5):488-493.
[13] Neill J D,Ridpath J F,Valayudhan B T.Identification and genome characterization of genotype B and genotype C bovine parainfluenza type 3viruses isolated in the United States[J].BMC Vet Res,2015,11:112.
[14]赵红丽,刘智慧,李婷婷,等.牛副流感病毒3型在不同细胞中的复制动力学分析[J].中国生物制品学杂志,2016,29(12):1255-1257.
[15] Maidana S S,Lomonaco P M,Combessies G,et al.Isolation and characterization of bovine parainfluenza virus type 3from water buffaloes(Bubalus bubalis)in Argentina[J].BMC Vet Res,2012,8:83.
[16] Komatsu T,Takeuchi K,Gotoh B.Bovine parainfluenza virus type 3accessory proteins that suppress beta interferon production[J].Microbes Infect,2007,9(8):954-962.
[17] Eberle K C,McGill J L,Reinhardt T A,et al.Parainfluenza virus 3blocks antiviral mediators downstream of the interferon lambda receptor by modulating stat1phosphorylation[J].J Virol,2015,90(6):2948-2958.
[18] Rabbani M A,Ribaudo M,Guo J T,et al.Identification of interferon-stimulated gene proteins that Inhibit human parainfluenza virus type 3[J].J Virol,2016,90(24):11145-11156.
[19] Schoggins J W,MacDuff D A,Imanaka N,et al.Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity[J].Nature,2014,505(7485):691-695.
[20] Laurent-Rolle M,Morrison J,Rajsbaum R,et al.The interferon signaling antagonist function of yellow fever virus NS5protein is activated by type I interferon[J].Cell Host Microbe,2014,16(3):314-327.
[21] Sanchez-Aparicio M T,Feinman L J,Garcia-Sastre A,et al.Paramyxovirus V proteins interact with the RIG-I/TRIM25regulatory complex and inhibit RIG-I signaling[J].J Virol,2018,92(6):e01960-17.
[22] Zhang L,Zhao X,Zhang M,et al.Ubiquitin-specific protease2bnegatively regulates IFN-beta production and antiviral activity by targeting TANK-binding kinase 1[J].J Immunol,2014,193(5):2230-2237.