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Differentiated human airway organoids to assess infectivity of emerging influenza virus.

ABSTRACT: Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The human-infective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human.

SUBMITTER: Zhou J

PROVIDER: S-EPMC6042130 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Differentiated human airway organoids to assess infectivity of emerging influenza virus.

Zhou Jie J Li Cun C Sachs Norman N Chiu Man Chun MC Wong Bosco Ho-Yin BH Chu Hin H Poon Vincent Kwok-Man VK Wang Dong D Zhao Xiaoyu X Wen Lei L Song Wenjun W Yuan Shuofeng S Wong Kenneth Kak-Yuen KK Chan Jasper Fuk-Woo JF To Kelvin Kai-Wang KK Chen Honglin H Clevers Hans H Yuen Kwok-Yung KY

Proceedings of the National Academy of Sciences of the United States of America 20180611 26

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differen ...[more]

PMID: 29891677

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Project description:The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.

Project description:Pandemic H1N1 (pH1N1) influenza virus emerged from swine in 2009 with an adequate capability to infect and transmit between people. In subsequent years, it has circulated as a seasonal virus and evolved further human-adapting mutations. Mutations in the hemagglutinin (HA) stalk that increase pH stability have been associated with human adaptation and airborne transmission of pH1N1 virus. Yet, our understanding of how pH stability impacts virus-host interactions is incomplete. Here, using recombinant viruses with point mutations that alter the pH stability of pH1N1 HA, we found distinct effects on virus phenotypes in different experimental models. Increased pH sensitivity enabled viruses to uncoat in endosomes more efficiently, manifesting as increased replication rate in typical continuous cell cultures under single-cycle conditions. A more acid-labile HA also conferred a small reduction in sensitivity to antiviral therapeutics that act at the pH-sensitive HA fusion step. Conversely, in primary human airway epithelium cultured at the air-liquid interface, increased pH sensitivity attenuated multicycle viral replication by compromising virus survival in the extracellular microenvironment. In a mouse model of influenza pathogenicity, there was an optimum HA activation pH, and viruses with either more- or less-pH-stable HA were less virulent. Opposing pressures inside and outside the host cell that determine pH stability may influence zoonotic potential. The distinct effects that changes in pH stability exert on viral phenotypes underscore the importance of using the most appropriate systems for assessing virus titer and fitness, which has implications for vaccine manufacture, antiviral drug development, and pandemic risk assessment.IMPORTANCE The pH stability of the hemagglutinin surface protein varies between different influenza strains and subtypes and can affect the virus' ability to replicate and transmit. Here, we demonstrate a delicate balance that the virus strikes within and without the target cell. We show that a pH-stable hemagglutinin enables a human influenza virus to replicate more effectively in human airway cells and mouse lungs by facilitating virus survival in the extracellular environment of the upper respiratory tract. Conversely, after entering target cells, being more pH stable confers a relative disadvantage, resulting in less efficient delivery of the viral genome to the host cell nucleus. Since the balance we describe will be affected differently in different host environments, it may restrict a virus' ability to cross species. In addition, our findings imply that different influenza viruses may show variation in how well they are controlled by antiviral strategies targeting pH-dependent steps in the virus replication cycle.

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Differentiated human airway organoids to assess infectivity of emerging influenza virus.

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Differentiated human airway organoids to assess infectivity of emerging influenza virus.

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