Differentiated human airway organoids to assess infectivity of emerging influenza virus

Zhou, Jie; Li, Cun; Sachs, Norman; Chiu, Man Chun; Wong, Bosco Ho-Yin; Chu, Hin; Poon, Vincent Kwok-Man; Wang, Dong; Zhao, Xiaoyu; Wen, Lei; Song, Wenjun; Yuan, Shuofeng; Wong, Kenneth Kak-Yuen; Chan, Jasper Fuk-Woo; To, Kelvin Kai-Wang; Chen, Honglin; Clevers, Hans; Yuen, Kwok-Yung

In this study, we describe the PD of human ASC-derived AO culture for investigating a major animal and human pathogen, influenza virus. In particular, our differentiation conditions increase the numbers of ciliated cells (Fig. 2) , the major cell type in the human airway epithelium. The PD medium induces ciliated cell numbers to a nearly physiological level, with synchronously beating cilia readily discernible in every organoid (Movies S2 and S3). In addition, the expression levels of serine proteases (Fig. 3) , known to be important for productive viral infection, were elevated after PD. Among the up-regulated HA-activating serine proteases, the dramatically increased expression of HAT in the differentiated AOs can very likely be attributed to the increased abundance of ciliated cells, since ciliated cells are the main source of HAT in the human respiratory tract (23) . Thus, the differentiated AOs can simulate the human airway epithelium morphologically and functionally. As a further improvement, we developed 2D PD airway monolayers with an intact epithelial barrier to allow exclusively apical exposure to viruses (Fig. 5A and SI Appendix, Fig. S1 ), the natural mode of IAV infection in the human respiratory tract. We then utilized two pairs of viruses with known infectivity to demonstrate, as a proof of concept, that these organoids indeed show significantly higher susceptibility to the human-infective viruses than to the poorly human-infective viruses. These 3D and 2D differentiated AOs support the active replication of human-infective H7N9/Ah and H1N1pdm. In contrast, the H7N2 virus, which has been temporally and spatially cocirculating with H7N9 viruses in domestic poultry and contains internal genes similar to those in H7N9 viruses, replicated much less efficiently in both models. Similarly, the swine H1N1 isolate showed a substantially lower growth capacity than its counterpart, human-adapted H1N1pdm (Fig. 5B) .