Molecular characterisation of influenza B virus from 2017/18 season in primary models of human lung reveals improved adaption to the lower respiratory tract

The 2017/18 influenza season was characterized by unusual high numbers of severe infections and hospitalizations. Instead of influenza A viruses, this season was dominated by infections with influenza B viruses of the Yamagata lineage. While this IBV/Yam dominance was associated with a vaccine mismatch, a contribution of virus intrinsic features to the clinical severity of the infections was speculated. Here, we performed a molecular and phenotypic characterization of three IBV isolates from patients with severe flu symptoms in 2018 and compared it to an IBV/Yam isolate from 2016 using experimental models of increasing complexity, including human lung explants, lung organoids, and alveolar macrophages. Viral genome sequencing revealed the presence of clade but also isolate specific mutations in all viral genes, except NP, M1, and NEP. Comparative replication kinetics in different cell lines provided further evidence for improved replication fitness, tolerance towards higher temperatures, and the development of immune evasion mechanisms by the 2018 IBV isolates. Most importantly, immunohistochemistry of infected human lung explants revealed an impressively altered cell tropism, extending from AT2 to AT1 cells and macrophages. Finally, transcriptomics of infected human lung explants demonstrated significantly reduced amounts of type I and type III IFNs by the 2018 IBV isolate, supporting the existence of additional immune evasion mechanisms. Our results show that the severeness of the 2017/18 Flu season was not only the result of a vaccine mismatch but was also facilitated by improved adaptation of the circulating IBV strains to the environment of the human lower respiratory tract.

2017/18流感季的显著特征是重症感染与住院病例数量异常偏高。与以往甲型流感病毒（influenza A viruses）主导的情况不同，本季流行的主要是山形谱系（Yamagata lineage）的乙型流感病毒（influenza B viruses）。尽管乙型流感病毒山形谱系（IBV/Yam）的主导地位与疫苗不匹配相关，但有推测认为病毒的固有特征亦对感染的临床严重性有所贡献。在此，我们对2018年重症流感患者来源的三株乙型流感病毒分离株进行了分子与表型特征分析，并利用复杂度递增的实验模型（包括人肺组织外植体（human lung explants）、肺类器官（lung organoids）及肺泡巨噬细胞（alveolar macrophages）），将其与2016年的一株乙型流感病毒山形谱系分离株进行比较。病毒基因组测序（viral genome sequencing）结果显示，除NP、M1及NEP基因外，所有病毒基因中均存在谱系特异性及分离株特异性突变。不同细胞系中的复制动力学（replication kinetics）比较分析为2018年乙型流感病毒分离株具有更强的复制适应性、更高的温度耐受性及免疫逃逸机制（immune evasion mechanisms）的形成提供了进一步证据。尤为重要的是，对感染后人肺组织外植体的免疫组织化学（immunohistochemistry）分析显示，病毒的细胞嗜性发生显著改变，从II型肺泡上皮细胞（AT2 cells）扩展至I型肺泡上皮细胞（AT1 cells）及巨噬细胞。最后，对感染后人肺组织外植体的转录组学（transcriptomics）分析表明，2018年乙型流感病毒分离株感染后，I型和III型干扰素（IFNs）的表达量显著降低，这为额外免疫逃逸机制的存在提供了支持。我们的研究结果表明，2017/18流感季的严重性不仅源于疫苗不匹配，还与流行的乙型流感病毒株对人类下呼吸道环境适应性的增强有关。