传染性支气管炎病毒对鸡免疫反应及重组禽β-防御素11和12抗病毒作用的研究

doi:10.16431/j.cnki.1671-7236.2023.01.025

摘要/Abstract

摘要： 【目的】探究呼吸型传染性支气管炎病毒（Infectious bronchitis virus，IBV）对蛋鸡免疫反应的影响及重组禽β-防御素（avian β-defensins，AvBDs）的抗病毒作用。【方法】试验将70只7日龄SPF白来航鸡随机分为2组，每组35只。攻毒组通过滴鼻点眼途径接种呼吸型IBV强毒（CK/CH/LSD/05I），对照组以相同途径接种等剂量灭菌PBS。定时观察临床症状14 d并记录，在攻毒后12 h、36 h、3 d、7 d和14 d 2组各随机选取5只鸡心脏采血处死，检测血清抗体水平并收集部分气管和肾脏进行组织病理学检测。采集法氏囊、脾脏、肾脏和气管，提取RNA，运用实时荧光定量PCR方法检测组织中IBV的病毒载量、Toll样受体（Toll-like receptors，TLRs）及信号转导分子、AvBDs基因表达量变化。将重组AvBD11和AvBD12转染鸡胚肾细胞（chicken embryo kidney，CEK）并在病毒感染细胞后6、12、24、36和48 h收取细胞上清和细胞用于提取RNA，通过实时荧光定量PCR方法检测病毒载量。【结果】感染呼吸型IBV强毒后，鸡出现轻微呼吸道症状，剖检无明显病理变化。在感染14 d后，鸡血清抗体水平转阳且阳性率为100%。实时荧光定量PCR结果显示，对照组均未检测到病毒，攻毒组仅在感染14 d后的气管中检测到病毒且病毒载量低。与对照组相比，攻毒组脾脏中TLR1、TLR5、AvBD9和AvBD12基因表达量在感染后7 d均显著升高（P<0.05），攻毒组法氏囊中TLR1、TLR2、TLR3、AvBD5和AvBD9基因表达量及气管中核转录因子-κB p65（NF-κB p65）、NF-κB c-Rel、AvBD5、AvBD9、AvBD11和AvBD13基因表达量在感染14 d后均显著升高（P<0.05）。体外抗病毒结果显示，与转染空载体组相比，转染AvBD11的CEK细胞在感染24和48 h后病毒载量显著下调（P<0.05），细胞上清的病毒载量在感染48 h后显著下调（P<0.05）；转染AvBD12的CEK细胞在感染12和24 h后病毒载量显著下调（P<0.05），细胞上清的病毒载量在感染12和48 h后显著下调（P<0.05），说明重组AvBD11和AvBD12在CEK细胞中具有抗IBV活性。【结论】呼吸型IBV强毒感染机体后增强了相关受体的基因表达和信号转导，上调了AvBDs基因表达，加强了机体免疫反应。重组AvBD11和AvBD12具有抗病毒作用，为无抗饲料的配制及研究提供了新思路。

关键词: 传染性支气管炎病毒(IBV); 鸡; Toll样受体; 禽β-防御素; 抗病毒作用

Abstract: 【Objective】 This experiment was conducted to study the host immune response to respiratory Infectious bronchitis virus virulent strain infection in layer and discover the antiviral effect of recombinant avian β-defensin (AvBDs).【Method】 70 7-day old White Leghorn chickens were randomly devided into two groups, thirty-five chickens in each group respectively.The challenge group was inoculated with respiratory IBV virulent strain (CK/CH/LSD/05I) by a combined intraocular and intranasal route, while the control group was inoculated with PBS by the same way.The clinical symptoms were regularly observed for 14 d and recorded.Five chickens from each group were randomly selected to kill by blood sampling at 12 h, 36 h, 3 d, 7 d and 14 d after the infection.Blood samples were collected to detect the level of serum antibody.Part of trachea and kidney were collected for histopathological examination.RNA was extracted from bursa of Fabricius, spleen, kidney and trachea, which was used to detect the viral load of IBV, the expression of Toll-like receptor (TLRs) and signal transduction molecules, AvBDs by Real-time quantitative PCR.Recombinant AvBD11 and AvBD12 were transfected into chicken embryo kidney (CEK) cells.The supernatant and cells were collected at 6, 12, 24, 36 and 48 h after the infection.RNA was extracted for detecting the viral load by Real-time quantitative PCR.【Result】 After being infected with respiratory IBV virulent strain, chickens showed slight respiratory symptoms, and there was no obvious pathological change in autopsy.The antibody level of the infection group turned positive at 14 d post infection, and the positive rate of respiratory IBV virulent strain group was 100%.The results of Real-time quantitative PCR detection showed that no virus was detected in control group, IBV antigen was only detected in the trachea at 14 d post infection in challenge group and the virus load was very low.Compared with control group, the expression of TLR1, TLR5, AvBD9 and AvBD12 genes in spleen were significantly up-regulated at 7 d post infection with respiratory IBV virulent strain (P<0.05).In challenge group, the expression of TLR1, TLR2, TLR3, AvBD5 and AvBD9 genes in bursa of Fabricius and the expression of nuclear factor-κB p65 (NF-κB p65), NF-κB c-Rel, AvBD5, AvBD9, AvBD11 and AvBD13 genes in trachea were significantly increased at 14 d after the infection (P<0.05).The results of recombinant defensin antiviral experiment showed that compared with the empty vector group, the viral load of CEK cells transfected with AvBD11 was significantly down-regulated at 24 and 48 h after the infection (P<0.05), and the viral load in the supernatant of CEK cells transfected with AvBD11 was significantly down-regulated at 48 h post infection (P<0.05).Compared with the transfected empty vector group, the viral load of CEK cells transfected with AvBD12 was significantly down-regulated at 12 and 24 h after the infection (P<0.05), and the viral load in the supernatant of CEK cells transfected with AvBD12 was significantly down-regulated at 12 and 48 h post infection (P<0.05).It showed that recombinant AvBD11 and AvBD12 had anti-IBV activity in CEK cells.【Conclusion】 The infection of the respiratory IBV virulent strain enhanced the gene expression of relative receptor and signal transduction molecules, upregulated the gene expression of defensins, and strengthened the body's natural immune response.The recombinant defensins AvBD11 and AvBD12 had antiviral effects, which provided a new idea for the preparation and research of non-antiviral feed.

Key words: Infectious bronchitis virus (IBV); chicken; Toll-like receptor; avian β-defensin; antiviral effect

中图分类号:

S852.4

谢晶晶, 任梦婷, 范佳慧, 焦亚茹, 韩宗玺, 马得莹. 传染性支气管炎病毒对鸡免疫反应及重组禽β-防御素11和12抗病毒作用的研究[J]. 中国畜牧兽医, 2023, 50(1): 246-259.

XIE Jingjing, REN Mengting, FAN Jiahui, JIAO Yaru, HAN Zongxi, MA Deying. Study on Immune Response of Infectious Bronchitis Virus to Chickens and Antiviral Effect of Recombinant Avian Beta-defensins 11 and 12[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 246-259.

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