Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1β. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-β) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.