Molecular characterisation of influenza B virus from the 2017/18 season in primary models of the human lung reveals improved adaptation 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.

2017/2018年流感季以异常高发的重症感染与住院病例为典型特征，本季的主导流行毒株并非甲型流感病毒（Influenza A Virus），而是乙型流感病毒（Influenza B Virus, IBV）山形谱系（Yamagata lineage）毒株。尽管此次IBV/Yam毒株的主导地位与疫苗匹配度不符（vaccine mismatch）相关，但有研究推测病毒固有特性（virus intrinsic features）可能对感染的临床严重程度（clinical severity）存在贡献。本研究对2018年3例重症流感患者体内分离的IBV毒株开展了分子与表型特征分析（molecular and phenotypic characterization），并将这3株2018年的IBV分离株与2016年的IBV/Yam分离株作为对照，采用复杂度逐级提升的实验模型（experimental model）进行验证，包括人肺外植体（human lung explant）、肺类器官（lung organoid）及肺泡巨噬细胞（alveolar macrophage）。病毒基因组测序（viral genome sequencing）结果显示，除核蛋白（nucleoprotein, NP）、基质蛋白1（matrix protein 1, M1）与核输出蛋白（nuclear export protein, NEP）的编码基因外，其余所有病毒基因均存在进化支（clade）特异性与分离株（isolate）特异性突变。不同细胞系（cell line）中的复制动力学（replication kinetics）比较实验进一步证实，2018年的IBV分离株具备更优的复制适配性（replication fitness）、更高的温度耐受性，且进化出了免疫逃逸机制（immune evasion mechanism）。最为关键的是，感染后人肺外植体的免疫组织化学分析（immunohistochemistry）结果显示，病毒的细胞嗜性（cell tropism）发生了显著改变，感染范围从肺泡Ⅱ型上皮细胞（alveolar type 2, AT2）扩展至肺泡Ⅰ型上皮细胞（alveolar type 1, AT1）与巨噬细胞。最终，感染后人肺外植体的转录组学分析（transcriptomics）表明，2018年的IBV分离株可显著下调Ⅰ型与Ⅲ型干扰素（Interferon, IFN）的表达水平，佐证了其存在额外的免疫逃逸机制。本研究结果证实，2017/2018年流感季的重症高发不仅源于疫苗匹配度不符，同时也因流行的IBV毒株对人类下呼吸道（lower respiratory tract）环境的适应性增强而进一步加剧。