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Background: A novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, to cause a respiratory disease (COVID-19) of varying severity in Wuhan China, subsequently spreading to other parts of China and beyond. Methods: We infected ex vivo explant cultures of the human conjunctiva, bronchus and lung, and in vitro cultures of primary human alveolar epithelial cells and macrophages with SARS-CoV-2, and assessed viral tropism, replication competence and innate immune responses, in comparison with SARS-CoV, MERS-CoV, and the 2009 pandemic influenza H1N1 (pdmH1N1) virus. Findings: SARS-CoV-2 infected ciliated, mucus secreting and club cells of bronchial epithelium, spindled morphologically type I pneumocytes in the lung, and the conjunctival mucosa. Virus replication competence of SARS-CoV-2 in the bronchus was higher than that of SARS-CoV but lower than pdmH1N1. SARS-CoV-2 replication was comparable with SARS-CoV and pdmH1N1 in the lung but was lower than MERS-CoV. SARS-CoV-2 virus was a less potent inducer of pro-inflammatory cytokines compared with H5N1 and MERS-CoV. Influenza virus infection of alveolar epithelial cells increased ACE2 expression. Interpretation: The conjunctival epithelium and the conducting airways appear to be potential portals of infection of SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated comparably in the alveolar epithelium explaining the progression of infection to a primary viral pneumonia. Funding Statement: US National Institute of Allergy and Infectious Diseases (NIAID) under Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract no. HHSN272201400006C and the Theme Based Research Scheme (Ref: T11-705/14N), Hong Kong Special Administrative Region. Declaration of Interests: There is no conflict of interest for all authors. Ethics Approval Statement: All experiments were carried out in a Bio-safety level 3 (BSL-3) facility. Informed consent was obtained from all subjects and approval was granted by the Institutional Review Board (IRB) of the University of Hong Kong and the Hospital Authority (Hong Kong West) (approval no: UW 20-167).

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has emerged as a coronavirus infection of humans in the past 5 years. Though confined to certain geographical regions of the world, infection has been associated with a case fatality rate of 35%, and this mortality may be higher in ventilated patients. As there are few readily available animal models that accurately mimic human disease, it has been a challenge to ethically determine what optimum treatment strategies can be used for this disease. We used in-vitro and human ex-vivo explant cultures to investigate the effect of two immunomodulatory agents, interferon alpha and cyclosporine, singly and in combination, on MERS-CoV replication. In both culture systems the combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. PCR SuperArray analysis showed that the reduction of virus replication was associated with a greater induction of interferon stimulated genes. As these therapeutic agents are already licensed for clinical use, it may be relevant to investigate their use for therapy of human MERS-CoV infection., Highlights • The effect of interferon-α and/or cyclosporine on MERS-CoV replication was evaluated with a human ex-vivo culture model. • All treatments were able to reduce MERS-CoV replication. • The combined treatment was more effective than either agent used alone in reducing MERS-CoV replication. • The effect of the combined treatment group was associated with a greater induction of interferon stimulated genes.

Despite causing regular seasonal epidemics with substantial morbidity, mortality and socioeconomic burden, there is still a lack of research into influenza B viruses (IBVs). In this study, we provide for the first time a systematic investigation on the tropism, replication kinetics and pathogenesis of IBVs in the human respiratory tract.Physiologically relevantex vivoexplant cultures of human bronchus and lung, human airway organoids, andin vitrocultures of differentiated primary human bronchial epithelial cells and type-I-like alveolar epithelial cells were used to study the cellular and tissue tropism, replication competence and induced innate immune response of 16 IBV strains isolated from 1940 to 2012 in comparison with human seasonal influenza A viruses (IAVs), H1N1 and H3N2. IBVs from the diverged Yamagata- and Victoria-like lineages and the earlier undiverged period were included.The majority of IBVs replicated productively in human bronchus and lung with similar competence to seasonal IAVs. IBVs infected a variety of cell types, including ciliated cells, club cells, goblet cells and basal cells, in human airway organoids. Like seasonal IAVs, IBVs are low inducers of pro-inflammatory cytokines and chemokines. Most results suggested a higher preference for the conducting airway than the lower lung and strain-specific rather than lineage-specific pathogenicity of IBVs.Our results highlighted the non-negligible virulence of IBVs which require more attention and further investigation to alleviate the disease burden, especially when treatment options are limited.

Novel avian H7N9 virus emerged in China in 2013 resulting in a case fatality rate of around 39% and continues to pose zoonotic and pandemic risk. Amino acid substitutions in PB2 protein were shown to influence the pathogenicity and transmissibility of H7N9 following experimental infection of ferrets and mice. In this study, we evaluated the role of amino acid substitution PB2-627K or compensatory changes at PB2-591K and PB2-701N, on the tropism and replication competence of H7N9 viruses for human and swine respiratory tracts using ex vivo organ explant cultures. Recombinant viruses of A/Shanghai/2/2013 (rgH7N9) and its mutants with PB2-K627E, PB2-K627E + Q591K and PB2-K627E + D701N were generated by plasmid-based reverse genetics. PB2-E627K was essential for efficient replication of rgH7N9 in ex vivo cultures of human and swine respiratory tracts. Mutant rgPB2-K627E + D701N replicated better than rgPB2-K627E in human lung but not as well as rgH7N9 virus. The rgPB2-K627E mutant failed to replicate in human type I-like pneumocytes (ATI) and peripheral blood monocyte-derived macrophages (PMϕ) at 37 °C while the compensatory mutant rgPB2-K627E + Q591K and rgPB2-K627E + D701N had partly restored replication competence in PMϕ. Our results demonstrate that PB2-E627K was important for efficient replication of influenza H7N9 in both human and swine respiratory tracts.

Highly pathogenic H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals remain the primary choice for treating H5N1 diseases and have their limitations. Encouraging findings highlight the beneficial effects of combined treatment of host targeting agents with immune-modulatory activities. This study evaluated the undefined roles of sterol metabolic pathway in viral replication and cytokine induction by H5N1 virus in human alveolar epithelial cells. The suppression of the sterol biosynthesis by Simvastatin in human alveolar epithelial cells led to reduction of virus replication and cytokine production by H5N1 virus. We further dissected the antiviral role of different regulators of the sterol metabolism, we showed that Zometa, FPT inhibitor III, but not GGTI-2133 had anti-viral activities against both H5N1 and H1N1 viruses. More importantly, FPT inhibitor III treatment significantly suppressed cytokine production by H5N1 virus infected alveolar epithelial cells. Since both viral replication itself and the effects of viral hyper-induction of cytokines contribute to the immunopathology of severe H5N1 disease, our findings highlights the therapeutic potential of FPT inhibitor III for severe human H5N1 diseases. Furthermore, our study is the first to dissect the roles of different steps in the sterol metabolic pathway in H5N1 virus replication and cytokine production.