H9N2亚型禽流感病毒感染鸡呼吸道应答模式研究

doi:10.16431/j.cnki.1671-7236.2024.09.025

摘要/Abstract

摘要： 【目的】通过建立H9N2亚型禽流感病毒(AIV)感染鸡上呼吸道炎症模型，探索其感染鸡上呼吸道引起的炎症反应。【方法】将H9N2亚型AIV流行毒株GD10142，按照100 μL/只(10⁸ EID₅₀/100 μL)的剂量通过滴鼻点眼方法感染SPF鸡，并在感染后观察鸡的临床特征，采集感染后0、1、3、5、7、9 d的咽拭子和血清样品；感染后5和9 d剖检观察鸡的呼吸道和肺脏病理变化，收集气管灌洗液、气管和肺脏组织；通过实时荧光定量PCR检测咽拭子、气管灌洗液、气管和肺脏组织中的病毒载量；应用ELISA方法检测感染后血清总IgG和H9N2亚型AIV特异性IgG的变化规律，通过血清中和试验分析血清中和抗体的活性；通过ELISA方法检测血清和气管灌洗液中炎症相关细胞因子白细胞介素6(IL-6)、IL-10、IL-1β、肿瘤坏死因子α(TNF-α)和核转录因子κB(NF-κB)的水平。【结果】 H9N2亚型AIV感染后鸡没有出现典型的临床特征，但感染后1～5 d在咽拭子中可检测到病毒核酸，且感染3 d后病毒载量最高，感染5 d后检测不到病毒核酸；感染5 d时，在气管灌洗液、气管和肺脏检测到病毒核酸，其中前两者载量高于肺脏；血清总IgG和H9N2亚型AIV特异性IgG在感染后3 d增多，感染后5～9 d维持较高水平，且具有较强的病毒中和活性；血清IL-10、IL-1β、TNF-α和NF-κB也在感染后3 d开始呈上升趋势；但气管灌洗液中IL-6、IL-10、IL-1β、TNF-α和NF-κB含量很低，且感染前后无明显变化。【结论】本研究成功建立了H9N2亚型AIV感染鸡呼吸道模型，确定上呼吸道是H9N2亚型AIV主要感染和增殖部位，H9N2亚型AIV感染诱导机体产生促炎和抑炎细胞因子，可能是感染动物维持免疫稳态的重要因素。本研究为深入探索H9N2亚型AIV的致病机制和宿主防御机制奠定了基础。

关键词: H9N2亚型禽流感病毒; 呼吸道感染; 免疫应答; 模型评价

Abstract: 【Objective】 This study was conducted to establish a model of upper respiratory tract inflammation in chickens infected with H9N2 subtype Avian influenza virus (AIV) and explore the inflammatory response caused by its infection. 【Method】 H9N2 subtype AIV epidemic strain GD10142 was infected with SPF chickens by nose and eye drops at a dose of 100 μL/bird (10⁸ EID₅₀/100 μL).The clinical characteristics of chickens were observed after infection, and throat swabs and serum samples were collected at 0, 1, 3, 5, 7 and 9 days after infection.The pathological changes of respiratory tract and lung were observed 5 and 9 days after infection, and tracheal lavage fluid, trachea and lung tissues were collected.The viral load in throat swab, trachea lavage fluid, trachea and lung tissue was detected by Real-time quantitative PCR.The changes of serum total IgG and H9N2 subtype AIV-specific IgG were detected by ELISA, and the neutralization activity of antiserum was analyzed by serum neutralization test.The levels of inflammation-related cytokines interleukin-6 (IL-6), IL-10, IL-1β, tumor necrosis factor α (TNF-α) and nuclear transcription factor κB (NF-κB) in serum and trachea lavage fluid were detected by ELISA. 【Result】 After infection with H9N2 subtype AIV, chickens did not show typical clinical features, but viral nucleic acid could be detected in throat swabs 1-5 days after infection, and the viral load was highest 3 days after infection, and no viral nucleic acid could be detected 5 days after infection.After 5 days of infection, viral nucleic acid was detected in trachea lavage fluid, trachea and lung, and the first two viral nucleic acid loads were higher than those in lung.The total serum IgG and H9N2 subtype AIV-specific IgG increased 3 days after infection, maintained at high levels 5-9 days after infection, and exhibited strong virus neutralizing activity.IL-10, IL-1β, TNF-α and NF-κB in serum also showed an upward trend starting from 3 days after infection, however, the contens of IL-6, IL-10, IL-1β, TNF-α and NF-κB in tracheal lavage fluid were very low, and there was no significant change before and after infection. 【Conclusion】 In this study, the respiratory tract model of chickens infected with H9N2 subtype AIV was successfully established, and the upper respiratory tract was determined to be the main site of infection and proliferation of H9N2 subtype AIV.The infection of H9N2 subtype AIV induced the production of pro-inflammatory and anti-inflammatory cytokines, which might be an important factor in the maintenance of immune homeostasis in infected animals.This study laid a foundation for further exploring the pathogenic mechanism and host defense mechanism of H9N2 subtype AIV.

Key words: H9N2 subtype Avian influenza virus; respiratory tract infection; immune response; model evaluation

中图分类号:

S852.65

池周颖, 李天旭, 吴亚鑫, 瞿孝云, 李思婕, 孙敏华, 张建峰, 廖明, 杜寿文. H9N2亚型禽流感病毒感染鸡呼吸道应答模式研究[J]. 中国畜牧兽医, 2024, 51(9): 3970-3979.

CHI Zhouying, LI Tianxu, WU Yaxin, QU Xiaoyun, LI Sijie, SUN Minhua, ZHANG Jianfeng, LIAO Ming, DU Shouwen. Exploration of Respiratory Tract Response Patterns in Chickens Infected with H9N2 Subtype Avian Influenza Virus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3970-3979.

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