不同来源传染性支气管炎病毒诱导SPF鸡发病的免疫机制研究

doi:10.16431/j.cnki.1671-7236.2021.12.031

摘要/Abstract

摘要： 试验旨在探讨不同来源的传染性支气管炎病毒(Infectious bronchitis virus,IBV)诱导SPF鸡发病的免疫机制。选用140只1日龄SPF白来航鸡,随机分为4组,3组攻毒组通过滴鼻点眼途径分别接种鸡源IBV强毒株、鸡源IBV弱毒株和野鸡源IBV毒株3个毒株,对照组以同种方式接种等量灭菌的磷酸盐缓冲液。在感染后12 h、36 h、72 h、7 d和14 d,每组随机选取5只进行剖检,并分别采集法氏囊、肾脏和气管组织,剩余鸡用于观察临床症状、发病及死亡情况。应用实时荧光定量PCR检测攻毒后不同时间点采集的各组织中IBV的病毒载量、Toll样受体(Toll-like receptors,TLRs)及部分细胞因子(白细胞介素(interleukin,IL)和干扰素(interferon,IFN))表达量的变化。结果显示,感染不同来源IBV毒株之后仅鸡源IBV强毒株感染组SPF鸡出现抑郁、翅膀下垂、甩头等典型的临床症状,且在感染后5~10 d共有7只死亡,死亡率为20%。病理剖检发现,感染鸡源IBV强毒株的鸡肾脏肿大、尿酸盐沉积和有花斑样病变,而感染野鸡源IBV毒株、鸡源IBV弱毒株和对照组的鸡无明显的眼观病变。实时荧光定量PCR结果显示,在鸡源IBV强毒株组的法氏囊、肾脏和气管3个组织中均检测到病毒。对照组和野鸡源IBV毒株组中均未检测到病毒,鸡源IBV弱毒株组只在部分组织中检测到病毒。在感染后72 h,鸡源IBV强毒株组与其他各组相比,TLR1、TLR2、TLR3、TLR5、TLR7和TLR15基因在法氏囊中的表达量均显著升高(P<0.05),IL-6和IFN-β参与更强烈的抗病毒免疫反应;在感染后7 d,鸡源IBV弱毒株组与其他各组相比,肾脏中TLR2、TLR3、TLR15、TLR21、IL-6和IL-18基因表达量均显著升高(P<0.05)。野鸡源IBV感染后36 h法氏囊组织中IFN-γ基因表达量显著上调(P<0.05)。综上所述,3个IBV毒株中仅鸡源IBV强毒感染引起SPF鸡典型临床发病症状与可视组织病变,且可提高SPF鸡组织中免疫相关因子的基因表达量。本研究结果揭示,不同来源的IBV对SPF鸡的不同致病性与其感染诱导的免疫反应不同有关。

关键词: 传染性支气管炎病毒(IBV); 免疫机制; Toll样受体; 细胞因子

Abstract: This experiment was conducted to investigate the host innate immune response to different origins of Infectious bronchitis virus (IBV) infection in SPF chicken.One hundred and forty one-day-old SPF White Leghorn chickens were randomly divided into four groups, including three challenge groups and one control group.The challenge groups were inoculated 3 IBVs (including chicken originated IBV virulent strain, chicken originated IBV attenuated strain, and pheasant originated IBV strain) by intranasal eye drops, and the control group was mock inoculated with phosphate-buffered saline (PBS) by the same route.From each of these infected groups five chickens were humanely sacrificed to collected their bursa of Fabricius, kidney and trachea at 12 hour post infection (hpi), 36 hpi, 72 hpi, 7 day post infection (dpi) and 14 dpi.The remaining chickens in each group were continuously observed for clinical symptoms, morbidity and mortality before being killed.The expression of IBV viral load, Toll-like receptors (TLRs) and some cytokines of interleukin (IL) and interferon(IFN) were evaluated by Real-time quantitative PCR.The results showed that SPF chickens infected with chicken origined IBV virulent strain developed clinical sympotoms, the infected SPF chickens showed depression, wing ptosis and other classical symptoms.A total of 7 chickens died during the period of infection (5-10 d), the mortality was 20%.In contrast, for chickens infected with pheasant origined IBV strain, chicken origined IBV attenuated strain and control group, no respiratory clinical signs and no gross lesions were observed during the experimental period.Chickens infected with chicken origined IBV virulent strain demonstrate enlarged kidneys, urate deposits and variegated lesions.Viral loads were detected in bursa of Fabricius, spleen, kidney and trachea of chicken infected with chicken originated IBV virulent.In addition, viral loads were detected in some tissues of the chicken originated IBV attenuated group, but were not detected in the tissues of control and pheasant origined IBV group.The expressions of TLR1, TLR2, TLR3, TLR5, TLR7 and TLR15 genes in bursa of Fabricius were dramatically up-regulated in chicken originated IBV virulent strain group at 72 hpi compared with other groups (P<0.05), IL-6 and IFN-β were released to participate in stronger antiviral immune response at 72 hpi.In chicken originated IBV attenuated strain challenged group, the expressions of TLR2, TLR3, TLR15, TLR21, IL-6 and IL-18 genes in kidney were significantly increased at 7 dpi (P<0.05).The expression of IFN-γ gene in bursa of Fabricius was significantly up-regulated at 36 hpi after infection with pheasant originated IBV strain (P<0.05).In summary, chicken originated IBV virulent strain was highly pathogenic to SPF chicken, the chicken origined IBV virulent strain could dramatically up-regulated the expression of immune-related cytokines in SPF chicken.The above results revealed that the different pathogenicity of IBV virus from different origins to SPF chickens was related to the different immune response.

Key words: Infectious bronchitis virus (IBV); immune mechanism; Toll-like receptor; cytokines

中图分类号:

S852.65⁺7

马占邦, 许丽文, 王芳芳, 张莉莉, 侯雨彤, 韩宗玺, 马得莹. 不同来源传染性支气管炎病毒诱导SPF鸡发病的免疫机制研究[J]. 中国畜牧兽医, 2021, 48(12): 4628-4640.

MA Zhanbang, XU Liwen, WANG Fangfang, ZHANG Lili, HOU Yutong, HAN Zongxi, MA Deying. Effects of Different Origins of Infectious Bronchitis Virus on Immune Function in SPF Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4628-4640.

参考文献

[1] 黄梦姣,张芸,薛春宜,等.应对日益严峻的挑战:中国禽传染性支气管炎研究[J].微生物学通报,2019,46(7):1837-1849. HUANG M J,ZHANG Y,XUE C Y,et al.To meet the growing challenge:Research of avian infectious bronchitis in China[J].Microbiology,2019,46(7):1837-1849.(in Chinese)
[2] ABDOLLAHI H,REZAEI-TAVIRANI M,GHALYANCHILANGEROUDI A,et al.Coronavirus:Proteomics analysis of chicken kidney tissue infected with variant 2 (IS-1494)-like Avian infectious bronchitis virus[J].Archives of Virology,2021,166(1):101-113.
[3] CAVANAGH D.Coronavirus Avian infectious bronchitis virus[J].Veterinary Research,2007,38(2):281-297.
[4] GUO H,HUANG M,YUAN Q,et al.The important role of lipid raft-mediated attachment in the infection of cultured cells by Coronavirus Infectious bronchitis virus Beaudette strain[J].PLoS One,2017,12(1):e0170123.
[5] ELLIS S,KEEP S,BRITTON P,et al.Recombinant Infectious bronchitis viruses expressing chimeric spike glycoproteins induce partial protective immunity against homologous challenge despite limited replication in vivo[J].Journal of Virology,2018,92(23):e01473-18.
[6] BOLTZ D,NAKAI M,BAHRA J.Avian infectious bronchitis virus:A possible cause of reduced fertility in the rooster[J].Avian Diseases,2004,48(4):909-915.
[7] IGNJATOVIĆ J,SAPATS S.Avian infectious bronchitis virus[J].Revue Scientifique et Technique,2000,19(2):493-508.
[8] CHHABRA R,BALL C,CHANTREY J,et al.Differential innate immune responses induced by classical and variant Infectious bronchitis viruses in specific pathogen free chicks[J].Developmental and Comparative Immunology,2018,87:16-23.
[9] PERRONE L A,PLOWDEN J K,GARCÍA-SASTRE A,et al.H5N1 and 1918 pandemic Influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice[J].PLoS Pathogens,2008,4(8):e1000115.
[10] TAKEUCHI O,AKIRA S.Pattern recognition receptors and inflammation[J].Cell,2010,140(6):805-820.
[11] CHEN S,CHENG A,WANG M.Innate sensing of viruses by pattern recognition receptors in birds[J].Veterinary Research,2013,44(1):82.
[12] GUO X,ROSA A,CHEN D G,et al.Molecular mechanisms of primary and secondary mucosal immunity using Avian infectious bronchitis virus as a model system[J].Veterinary Immunology and Immunopathology,2008,121(3-4):332-343.
[13] WANG X,ROSA A,OLIVERIRA H,et al.Transcriptome of local innate and adaptive immunity during early phase of infectious bronchitis viral infection[J].Viral Immunology,2006,19(4):768-774.
[14] LOPEZ-CASTEJON G,BROUGH D.Understanding the mechanism of IL-1β secretion[J].Cytokine and Growth Factor Reviews,2011,22(4):189-195.
[15] AMARASINGHE A,ABDUL-CADER M S,ALMATROUK Z,et al.Induction of innate host responses characterized by production of interleukin (IL)-1β and recruitment of macrophages to the respiratory tract of chickens following infection with Infectious bronchitis virus (IBV)[J].Veterinary Microbiology,2018,215:1-10.
[16] DHUPKAR P,GORDON N.Interleukin-2:Old and new approaches to enhance immune-therapeutic efficacy[J].Advances in Experimental Medicine and Biology,2017,995:33-51.
[17] LI Z,WANG H,CHEN Y,et al.Interleukin-18 protects mice from Enterovirus 71 infection[J].Cytokine,2017,96:132-137.
[18] LIN F,YOUNG H.Interferons:Success in anti-viral immunotherapy[J].Cytokine and Growth Factor Reviews,2014,25(4):369-376.
[19] WOODS R,WESLEY R.Seroconversion of pigs in contact with dogs exposed to Canine coronavirus[J].Canadian Journal of Veterinary Research,1992,56:78-80.
[20] CAVANAGH D,MAWDITT K,WELCHMAN D,et al.Coronaviruses from pheasants (Phasianus colchicus) are genetically closely related to Coronaviruses of domestic fowl (Infectious bronchitis virus) and turkeys[J].Avian Pathology,2002,31:181-193.
[21] HAN Z,XU X,LI H,et al.Replication and vaccine protection of multiple Infectious bronchitis virus strains in pheasants (Phasianus colchicus)[J].Infection Genetics and Evolution,2021,93:104980.
[22] XU L,REN M,SHENG J,et al.Genetic and biological characteristics of four novel recombinant Avian infectious bronchitis viruses isolated in China[J].Virus Research,2019,263:87-97.
[23] DAI J,GU L,SU Y,et al.Inhibition of curcumin on Influenza A virus infection and influenzal pneumonia via oxidative stress,TLR2/4,p38/JNK MAPK and NF-kappaB pathways[J].International Immunopharma-cology,2018,54:177-187.
[24] 陈玉秋.鸭源新城疫强毒株感染番鸭后对其免疫相关基因影响的研究[D].哈尔滨:东北农业大学,2017. CHEN Y Q.The effects on related genes of immune response to a duck originated Newcastle disease virulent strain infection in muscovy duck[D].Harbin:Northeast Agricultural University,2017.(in Chinese)
[25] LI Y,GAO Y,CUI T,et al.Retinoic acid facilitates Toll-like receptor 4 expression to improve intestinal barrier function through retinoic acid receptor beta[J].Cellular Physiology and Biochemistry,2017,42(4):1390-1406.
[26] CONG F,LIU X,HAN Z,et al.Transcriptome analysis of chicken kidney tissues following Coronavirus Avian infectious bronchitis virus infection[J].BMC Genomics,2013,14(1):743.
[27] KAMEKA A,HADDADI S,KIM D,et al.Induction of innate immune response following Infectious bronchitis corona virus infection in the respiratory tract of chickens[J].Virology,2014,450-451:114-121.
[28] ZHU J,XU S,LI X,et al.Infectious bronchitis virus inhibits activation of the TLR7 pathway,but not the TLR3 pathway[J].Archives of Virology,2020,165(9):2037-2043.
[29] ABDEL-MAGEED A,ISOBE N,YOSHIMURA Y.Effects of different TLR ligands on the expression of proinflammatory cytokines and avian beta-defensins in the uterine and vaginal tissues of laying hens[J].Veterinary Immunology and Immunopathology,2014,162(3-4):132-141.
[30] RAHMAN M,EO S.Prospects and challenges of using chicken cytokines in disease prevention[J].Vaccine,2012,30(50):7165-7173.
[31] KARPALA A,LOWENTHAL J,BEAN A.Activation of the TLR3 pathway regulates IFNbeta production in chickens[J].Developmental and Comparative Immunology,2008,32(4):435-444.
[32] PEI J,SEKELLICK M,MARCUS P,et al.Chicken interferon type Ⅰ inhibits Infectious bronchitis virus replication and associated respiratory illness[J].Journal of Interferon and Cytokine Research,2001,21(12):1071-1079.
[33] SUAREZ D,SCHULTZ-CHERRY S.Immunology of Avian influenza virus:A review[J].Development and Comparative Immunology,2000,24(2-3):269-283.