Bóna Márta, Kiss István, Dénes Lilla, Szilasi Anna, Mándoki Míra
Department of Animal Hygiene, Herd Health and Mobile Clinic, University of Veterinary Medicine, István utca 2., 1078 Budapest, Hungary.
Ceva-Phylaxia Co. Ltd., Szállás utca 5., 1107 Budapest, Hungary.
Animals (Basel). 2023 Mar 14;13(6):1052. doi: 10.3390/ani13061052.
The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin-biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30-50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.
低致病性禽流感病毒（LPAIV）的H9N2亚型是一种广泛存在的家禽病原体，也可感染人类。病毒感染的特征描述是一个复杂的过程，涉及临床、病理和病毒学调查。本研究的目的是调整和优化一种针对LPAIV开发的免疫组织化学（IHC）技术，专门用于检测感染组织中的H9N2病毒抗原。将21日龄的肉鸡通过不同感染途径（即鼻内-气管内和静脉内）接种三种不同的H9N2病毒株，或与传染性支气管炎病毒（IBV）共同感染，并在感染后观察11天。对建议的免疫组织化学方案进行了修改：（i）用3-氨基-9-乙基咔唑（AEC）替代二氨基联苯胺（DAB）作为显色剂；（ii）使用单克隆一抗和过氧化物酶标记的抗小鼠二抗的间接两步免疫反应，而不是抗生物素蛋白-生物素复合物。禽流感病毒抗原在受影响细胞的细胞核中呈现红色沉淀，但也可在细胞质中识别。在受攻击鸡的气管、气囊和肺中观察到轻度充血和淤血，少数病例在分叉处发现纤维蛋白渗出物。对照组未发现大体病理或免疫组织化学病变。使用优化后的方案和相关评分系统，结果表明，无论接种途径如何，所测试的H9N2毒株均表现出呼吸道和泌尿道嗜性。在第5天，30%-50%的样本在呼吸道和肾脏中检测到病毒抗原。在第11天，未观察到免疫组织化学信号，表明缺乏病毒复制。不同H9N2病毒株之间在病毒抗原表达上存在轻微差异，但与高致病性禽流感（HPAI）不同，在脑和胰腺中未检测到病毒抗原。因此，免疫组织化学可被视为表征H9N2低致病性禽流感病毒感染的工具集中一种信息丰富的可视化补充方法。
