Development and Characterization of Chicken Lung Organoids for Future In Vitro Modeling of Avian Influenza Virus-Host Cell Interaction [scRNA-seq]

High pathogenicity avian influenza viruses pose a growing threat to poultry, livestock, wildlife, and humans as they undergo accelerated expansion of host and geographical ranges. Since 2020, these viruses have driven a panzootic characterized by extensive viral diversification and spillover into species previously considered to be resistant. There is currently a lack of physiologically relevant in vitro models that can be used to screen the rapidly changing viral landscape. To address this need, we describe the first chicken lung organoids derived from adult stem cells of specific pathogen free White Leghorns. We analyze their gene expression with bulk RNA sequencing, confirm their cellular heterogeneity via single-nuclei RNA sequencing, and provide basic morphological characterization using hematoxylin and eosin (H&E) staining, immunohistochemistry, immunofluorescence, and transmission electron microscopy. The results indicate that the organoids contained several cell types, including non-ciliated columnar, cuboidal, squamous, and mucin-producing cells, representative of different regions of the avian respiratory system. Furthermore, expression of genes relevant to influenza A virus replication appeared to be conserved across different samples. These organoids have the potential to effectively and efficiently model viral infection of the chicken lung, enabling the investigation of viral pathogenesis and evolutionary potential, virus-host interactions, and discovery of targets for antiviral treatments. Overall design: RNA-seq profiling of three SPF White Leghorn chicken organoids and corresponding tissues.

高致病性禽流感病毒（High Pathogenicity Avian Influenza Viruses, HPAIVs）的宿主与地理范围加速扩张，对家禽、家畜、野生动物及人类构成日益严峻的威胁。自2020年以来，此类病毒引发了一场以病毒广泛变异、并溢出至此前被认为具有抗性的物种为特征的动物大流行。目前尚缺乏能够适配快速演化的病毒谱系、且具有生理相关性的体外模型用于相关筛选。为解决这一需求，本研究首次报道了源自无特定病原体（Specific Pathogen Free, SPF）白来航鸡成体干细胞的鸡肺类器官。本研究通过批量RNA测序分析其基因表达谱，借助单细胞核RNA测序验证其细胞异质性，并采用苏木精-伊红（Hematoxylin and Eosin, H&E）染色、免疫组织化学、免疫荧光及透射电子显微镜开展基础形态学表征。结果显示，该类器官包含多种细胞类型，包括非纤毛柱状细胞、立方细胞、鳞状细胞及产黏蛋白细胞，可模拟禽类呼吸系统不同区域的细胞组成。此外，与甲型流感病毒复制相关的基因在不同样本中均呈现保守表达。此类鸡肺类器官可有效模拟鸡肺部的病毒感染过程，为研究病毒致病机制与演化潜力、病毒-宿主互作以及筛选抗病毒治疗靶点提供有力工具。整体实验设计：对3株SPF白来航鸡肺类器官及其对应组织开展RNA测序转录组分析。