Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host range determinant since it mediates virus binding to host-specific cellular receptors1–3. Here, we therefore assessed the molecular changes in HA that would allow an H5 HA-possessing virus to transmit among mammals. We identified a reassortant virus with H5 HA possessing four mutations in a 2009 pandemic H1N1 virus backbone capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but it was not highly pathogenic and did not cause mortality. These results suggest that H5 HA can convert to an HA that supports efficient viral transmission in mammals. However, we do not know whether the four mutations in the H5 HA identified in this study would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral genes may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need for pandemic preparedness for H5 HA-possessing viruses and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production, and distribution of effective countermeasures.
Stem cells undergo dynamic changes in response to injury to regenerate lost cells. However, the identity of transitional states and the mechanisms that drive their trajectories remain understudied. Using lung organoids, multiple in vivo repair models, single-cell transcriptomics, and lineage tracing, we find that alveolar type-2 epithelial cells undergoing differentiation into type-1 cells acquire pre-alveolar type-1 transitional cell state (PATS) en route to terminal maturation. Transitional cells undergo extensive stretching during differentiation, making them vulnerable to DNA damage. PATS show an enrichment of TP53, TGFβ, DNA-damage response signalling, and cellular senescence. Gain and loss of function and genomic binding assays revealed a direct transcriptional control of PATS by TP53 signalling. Notably, PATS-like cells were accumulated in human fibrotic lungs, suggesting persistence of the transitional state in fibrosis. Our study thus
Summary Avian influenza A viruses rarely infect humans, but if they do and transmit among them, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern due to the appreciable case fatality rate associated with these infections (>25%), potential instances of human-to-human transmission1, and the lack of pre-existing immunity among humans to viruses of this subtype. Here, we therefore characterized two early human A(H7N9) isolates, A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9; hereafter referred to as Anhui/1 and Shanghai/1, respectively). In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011; H7N9; Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/04/2009; H1N1; CA04). Anhui/1, Shanghai/1, and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates (NHPs), Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs upon intranasal inoculation. Most critically, Anhui/1 transmitted via respiratory droplets in one of three pairs of ferrets. Glycan arrays demonstrated that Anhui/1, Shanghai/1, and A/Hangzhou/1/2013 (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was less sensitive than a pandemic 2009 H1N1 virus to neuraminidase inhibitors, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets, and NHPs and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.
Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of COVID-19 pathogenesis. We report a feeder-free, scalable, chemically-defined, and modular alveolosphere culture system for propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the SARS-CoV-2 receptor ACE2 and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirrors features of COVID-19 lungs, including emergence of interferon mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with interferons recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres thus provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases.
Lungs are composed of a system of highly branched tubes that bring air into the alveoli, where gas exchange takes place. The proximal and distal regions of the lung contain epithelial cells specialized for different functions: basal, secretory and ciliated cells in the conducting airways and type II and type I cells lining the alveoli. Basal, secretory and type II cells can be grown in three-dimensional culture, with or without supporting stromal cells, and under these conditions they give rise to self-organizing structures known as organoids. This Review summarizes the different methods for generating organoids from cells isolated from human and mouse lungs, and compares their final structure and cellular composition with that of the airways or alveoli of the adult lung. We also discuss the potential and limitations of organoids for addressing outstanding questions in lung biology and for developing new drugs for disorders such as cystic fibrosis and asthma.
SUMMARY Host factors required for viral replication are ideal drug targets because they are less likely than viral proteins to mutate under drug-mediated selective pressure. Although genome-wide screens have identified host proteins involved in influenza virus replication, limited mechanistic understanding of how these factors affect influenza has hindered potential drug development. We conducted a systematic analysis to identify and validate host factors that associate with influenza virus proteins and affect viral replication. After identifying over one thousand host factors that co-immunoprecipitate with specific viral proteins, we generated a network of virus-host protein interactions based on the stage of the viral lifecycle affected upon host factor down-regulation. Using compounds that inhibit these host factors, we validated several proteins, notably Golgi-specific brefeldin A resistant guanine nucleotide exchange factor (GBF1) and JAK1, as potential antiviral drug targets. Thus, virus-host interactome screens are powerful strategies to identify targetable host factors and guide antiviral drug development.
Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.
Summary Inflammatory responses are known to facilitate tissue recovery following injury. However, the precise mechanisms that enhance lung alveolar regeneration remain unclear. Here, using an organoid-based screening assay, we find that interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) enhance the proliferation of AEC2s while maintaining their differentiation capacity. Furthermore, we find that expression of IL-1β and TNFα are induced in the AEC2 niche following influenza-induced injury in vivo , and lineage tracing analysis revealed that surviving AEC2s around the damaged area contribute to alveolar regeneration. Through genetic and pharmacological modulation of multiple components of the IL-1-nuclear factor κB (NF-κB) signaling axis, we show that cell-intrinsic as well as stromal mediated IL-1 signaling are essential for AEC2 mediated lung regeneration. Taken together, we propose that the IL-1/TNFα-NF-κB signaling axis functions as a component of an inflammation-associated niche to regulate proliferation of surviving AEC2s and promote lung regeneration.
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