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

Proteolytic cleavage of viral glycoprotein HA is essential for IAV to acquire infectivity, since only the cleaved HA molecule mediates the membrane fusion between the virus and the host cell, a process required for the initiation of infection. HA proteins of low-pathogenic avian IAVs and human IAVs carry a single basic amino acid arginine at the cleavage site (6, 7), recognized by trypsin-like serine proteases. Productive infection of Significance Influenza virus infection represents a major threat to public health worldwide. There is no biologically relevant, reproducible, and readily available in vitro model for predicting the infectivity of influenza viruses in humans. Based on the longterm expanding 3D human airway organoids, we developed proximal differentiation and further established a 2D monolayer culture of airway organoids. The resultant 3D and 2D proximal differentiated airway organoids can morphologically and functionally simulate human airway epithelium and as a proof of concept can discriminate human-infective influenza viruses from poorly human-infective viruses. Thus, the proximal differentiated airway organoids can be utilized to predict the infectivity of influenza viruses and, more broadly, provide a universal platform for studying the biology and pathology of the human airway. these viruses in the human airway thus requires serine proteases such as TMPRSS2, TMPRSS4, HAT, and others (8) . However, HA proteins of highly pathogenic avian viruses, such as H5N1, contain a polybasic cleavage site which is activated by ubiquitously expressed proteases. Current in vitro models for studying influenza infection in the human respiratory tract involve shortterm cultures of human lung explants and primary airway epithelial cells. Human lung explants are not readily available on a routine basis. In addition, the rapid deterioration of primary tissue is a major problem in infection experiments, since there is no protocol to maintain tissue viability in vitro. Under air-liquid interface conditions, basal cells isolated from human airway can polarize and undergo mucociliary differentiation. However, this capacity is lost within two or three passages (9) . Collectively, these primary tissues and cells hardly constitute a convenient, reproducible model to study human respiratory pathogens. Although various cell lines, e.g., A549 and Madin-Darby canine kidney (MDCK) cells, have been commonly used to propagate influenza viruses and to study their virology, they poorly recapitulate the histology of human airway epithelium. In addition, due to the low serine protease activity, most cell lines do not support the growth of the influenza viruses with a monobasic HA cleavage site unless the culture medium is supplemented with an exogenous serine protease, trypsin treated with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). Thus, a biologically relevant, reproducible, and readily available in vitro model remains urgently needed for studying the biology and pathology of the human respiratory tract.