禽呼肠孤病毒感染DF1细胞后干扰素刺激基因在转录水平表达量的动态变化

doi:10.16431/j.cnki.1671-7236.2021.04.030

摘要/Abstract

摘要： 为了解干扰素（interferon，IFN）和干扰素刺激基因在禽呼肠孤病毒（ARV）感染DF1细胞后的表达情况，试验将ARV病毒感染DF1细胞，观察细胞病变，收集感染后0、6、12、24、36、48、72、96 h的细胞样品，抽提反转录成cDNA，通过实时荧光定量PCR技术检测干扰素IFN-α和IFN-β及9种禽源常见干扰素刺激基因在感染后不同时间点在转录水平表达量的动态变化规律。结果显示，在ARV感染DF1细胞后，DF1细胞出现典型的细胞病变，感染后12 h病毒开始快速增殖，在36~96 h维持在较高的水平；IFN-α和IFN-β在转录水平的表达量在感染后均表现为显著下调（P<0.05；P<0.01）；IFI6、OAS、IFIT5、ISG12在转录水平表达量变化规律相似，均呈现显著上调表达（P<0.05；P<0.01），在感染后96 h达到峰值；其中IFIT5的上调幅度最大，感染后96 h的表达量是0 h的19.62倍（P<0.01）；而Mx、IFITM3、PKR、Viperin、ZAP的表达量变化规律相似，均表现为显著下调表达（P<0.05；P<0.01），其中Mx、IFITM3、Viperin的下调幅度较大，PKR和ZAP下调幅度很小。说明在ARV感染DF1细胞后，干扰素及多种干扰素刺激基因在转录水平呈现规律性变化，与病毒在DF1细胞中复制存在一定的联系。结果表明，ARV感染后可以诱导多种干扰素刺激基因的表达，这些干扰素刺激基因在抵御ARV病毒的入侵，抑制ARV的复制、释放及病毒的清除中发挥着重要作用。本研究为今后深入研究ARV的致病机理和宿主的抗病毒免疫应答提供了参考。

关键词: 禽呼肠孤病毒(ARV); DF1细胞; 干扰素; 干扰素刺激基因; 转录水平

Abstract: In order to reveal the expression of interferon (IFN) and interferon stimulated gene in DF1 cells infected with Avian reovirus (ARV),infect DF1 cells with ARV virus,observe cytopathic changes,collect cell samples at 0,6,12,24,36,48,72 and 96 h after infection,and extract reverse transcription into cDNA.Real-time PCR assays was used to detect the dynamic changes of interferon IFN-α and IFN-β and 9 common avian interferon-stimulating genes at the transcription level at different time points after infection.The results showed that after ARV infected DF1 cells,DF1 cells appeared typical cytopathic effect.The virus began to proliferate rapidly at 12 h after infection and maintained at a high level from 36-96 h.The expression of IFN-α and IFN-β at the transcriptional level was significantly down-regulated after infection (P<0.05;P<0.01);IFI6,OAS,IFIT5,and ISG12 had similar changes in the expression level of transcription,and all showed significant up-regulation (P<0.05;P<0.01),reaching peak at 96 h after infection.Among them,IFIT5 had the largest up-regulation,and its expression at 96 h after infection was 19.62 times than that at 0 h (P<0.01).While the expression changes of Mx,IFITM3,PKR,Viperin,and ZAP were similar,and all showed significant down-regulation (P<0.05;P<0.01),among them,the downward adjustment of Mx,IFITM3 and Viperin was larger than that of PKR and ZAP.The results showed that interferon and various interferon-stimulating genes showed regular changes at the transcription level after ARV infection in DF1 cells,which was related to virus replication in DF1 cells.The results showed that ARV infection could induce the expression of variety of interferon-stimulating genes,which played an important role in resisting ARV virus invasion,inhibiting ARV virus replication,release,and virus clearance.This study provided a further research direction of ARV pathogenesis and virus-inhibiting mechanism responses to ARV infection.

Key words: Avian reovirus (ARV); DF1 cell; interferon; interferon stimulated gene; transcrip-tional level

中图分类号:

S831.7

王盛, 万丽军, 谢芝勋, 谢丽基, 罗思思, 范晴, 张艳芳, 曾婷婷, 黄娇玲, 张民秀, 谢志勤, 邓显文. 禽呼肠孤病毒感染DF1细胞后干扰素刺激基因在转录水平表达量的动态变化[J]. 中国畜牧兽医, 2021, 48(4): 1423-1430.

WANG Sheng, WAN Lijun, XIE Zhixun, XIE Liji, LUO Sisi, FAN Qing, ZHANG Yanfang, ZENG Tingting, HUANG Jiaoling, ZHANG Mingxiu, XIE Zhiqin, DENG Xianwen. Altered Gene Expression Profiles of the Interferon Stimulated Genes in DF1 Cells Infected with Avian Reovirus[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1423-1430.

参考文献

[1] VAN DER HEIDE L.The history of avian reovirus[J].Avian Diseases,2000,44(3):638-641.
[2] 方勤,朱作言.呼肠孤病毒结构与功能研究进展[J].病毒学报,2003,19(4):381-384. FANG Q,ZHU Z Y.Progress in Avian reovirus structure and function reovirus[J].Chinese Journal of Virology,2003,19(4):381-384.(in Chinese)
[3] DANDAR E,BALINT A,KECSKEMETI S,et al.Detection and characterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease[J].Archives of Virology,2013,158(12):2583-2588.
[4] GREEN A M,BEATTY P R,HADJILAOU A,et al.Innate immunity to Dengue virus infection and subversion of antiviral responses[J].Journal of Molecular Biology,2014,426(6):1148-1160.
[5] IWASAKI A,PILLAI P S.Innate immunity to influenza virus infection[J].Nature Reviews Immuno-logy,2014,14(5):315-328.
[6] SECOMBES C J,ZOU J.Evolution of interferons and interferon receptors[J].Frontiers in Immunology,2017,8:209.
[7] MORAGA I,HARARI D,SCHREIBER G,et al.Receptor density is key to the alpha2/beta interferon differential activities[J].Molecular and Cellular Biology,2009,29(17):4778-4787.
[8] SCHNEIDER W M,CHEVILLOTTE M D,RICE C M.Interferon-stimulated genes:A complex web of host defenses[J].Annual Review of Immunology,2014,32:513-545.
[9] NIGG P E,PAVLOVIC J.Oligomerization and GTP-binding requirements of MxA for viral target recogni-tion and antiviral activity against influenza a virus[J].Journal of Biological Chemistry,2015,290(50):29893-29906.
[10] BENFIELD C T,LYALL J W,TILEY L S.The cytoplasmic location of chicken mx is not the determining factor for its lack of antiviral activity[J].PLoS One,2010,5(8):e12151.
[11] BENFIELD C T,LYALL J W,KOCHS G,et al.Asparagine 631 variants of the chicken Mx protein do not inhibit influenza virus replication in primary chicken embryo fibroblasts or in vitro surrogate assays[J].Journal of Virology,2008,82(15):7533-7539.
[12] BRASS A L,HUANG I C,BENITA Y,et al.The IFITM proteins mediate cellular resistance to influenza a H1N1 virus,west nile virus,and dengue virus[J].Cell,2009,139(7):1243-1254.
[13] SMITH S E,GIBSON M S,WASH R S,et al.Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro[J].Journal of Virology,2013,87(23):12957-12966.
[14] TAG-EI-DIN-HASSAN H T,SASAKI N,MORITOH K,et al.The chicken 2'-5' oligoadenylate synthetase A inhibits the replication of West nile virus[J].Japanese Journal of Veterinary Research,2012,60(2-3):95-103.
[15] AMICI C,LA FRAZIA S,BRUNELLI C,et al.Inhibition of viral protein translation by indomethacin in Vesicular stomatitis virus infection:Role of eIF2alpha kinase PKR[J].Cellular Microbiology,2015,17(9):1391-1404.
[16] LOSTALE-SEIJO I,MARTINEZ-COSTAS J,BENAVENTE J.Interferon induction by Avian reovirus[J].Virology,2016,487:104-111.
[17] 黄莉,谢芝勋,谢丽基,等.禽呼肠孤病毒σA基因对鸡胚成纤维细胞天然免疫相关基因转录水平的调控影响[J].中国兽医科学,2019,49(5):611-618. HUANG L,XIE Z X,XIE L J,et al.Regulation of Avian reovirus σA gene on expression of innate immune genes in chicken embryo fibroblasts[J].Chinese Veteri-nary Science,2019,49(5):611-618.(in Chinese)
[18] ZHANG B H,LIU X Y,CHEN W,et al.IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways[J].Acta Biochimica et Biophysica Sinica,2013,45(10):867-874.
[19] WANG X,HINSON E R,CRESSWELL P.The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts[J].Cell Host Microbe,2007,2(2):96-105.
[20] LI X,JIA Y,LIU H,et al.High level expression of ISG12(1) promotes cell apoptosis via mitochondrial-dependent pathway and so as to hinder Newcastle disease virus replication[J].Veterinary Microbiology,2019,228:147-156.
[21] KAPCZYNSKI D R,JIANG H J,KOGUT M H.Characterization of cytokine expression induced by Avian influenza virus infection with real-time RT-PCR[J].Methods in Molecular Biology,2014,1161:217-233.
[22] CHEN S,LUO G,YANG Z,et al.Avian tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways[J].Veterinary Research,2016,47(1):74.
[23] XIE L,XIE Z,WANG S,et al.Altered gene expression profiles of the MDA5 signaling pathway in peripheral blood lymphocytes of chickens infected with avian reovirus[J].Archives of Virology,2019,164(10):2451-2458.
[24] SMITH S E,CIBSON M S,WASH R S,et al.Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro[J].Journal of Virology,2013,87(23):12957-12966.
[25] ABDALLAH F,HASSANIN O.Positive regulation of humoral and innate immune responses induced by inactivated Avian influenza virus vaccine in broiler chickens[J].Veterinary Research Communications,2015,39(4):211-216.
[26] DAVIET S,VAN BORM S,HABYARIMANA A,et al.Induction of Mx and PKR failed to protect chickens from H5N1 infection[J].Viral Immunology,2009,22(6):467-472.
[27] 谢智文,曹艳杰,何怡宁,等.干扰素刺激基因在鸡传染性支气管炎病毒感染雏鸡气管黏膜中表达的动态变化[J].动物医学进展,2016,37(3):19-24. XIE Z W,CAO Y J,HE Y N,et al.Dynamic changes in expression of interferon stimulated genes in trachea mucosa of chickens infected with IBV[J].Progress in Veterinary Medicine,2016,37(3):19-24.(in Chinese)
[28] 白思宇,杨倩,仇华吉.干扰素刺激基因的抗病毒机制[J].微生物学报,2018,58(3):361-371. BAI S Y,YANG Q,QIU H J.Antiviral mechanisms of interferon-stimulated genes[J].Acta Microbiologica Sinica,2018,58(3):361-371.(in Chinese)
[29] 沈文康,谢芝勋,王盛,等.禽呼肠病毒在鸡胚成纤维细胞系中的增殖规律[J].动物医学进展,2018,39(1):30-33. SEHEN W K,XIE Z X,WANG S,et al.Proliferation of Avian reovirus in chicken embryo fibroblast cells[J].Progress in Veterinary Medicine,2018,39(1):30-33.(in Chinese)
[30] 黄莉,谢芝勋,蓝元喜,等.禽呼肠孤病毒感染对SPF鸡外周血淋巴细胞中天然免疫相关基因转录的影响[J].畜牧兽医学报,2017,48(1):116-123. HUANG L,XIE Z X,LAN Y X,et al.Effects of Avian reovirus infection on transcription of innate immune genes in peripheral blood lymphocytes of SPF chickens[J].Acta Veterinaria et Zootechnica Sinica,2017,48(1):116-123.(in Chinese)