Human Airway Epithelial Cultures Research Articles

The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus.

The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.

Reversible rearrangement of the cellular cytoskeleton: A key to the broad-spectrum antiviral activity of novel amphiphilic polymers.

The battle against emerging viral infections has been uneven, as there is currently no broad-spectrum drug available to contain the spread of novel pathogens throughout the population. Consequently, the pandemic outbreak that occurred in early 2020 laid bare the almost empty state of the pandemic box. Therefore, the development of novel treatments with broad specificity has become a paramount concern in this post-pandemic era. Here, we propose copolymers of poly (sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and poly (sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn) and random (P(AMPSm-co-AaUn)) that show efficacy against a broad range of alpha and betacoronaviruses. Owing to their intricate architecture, these polymers exhibit a highly distinctive mode of action, modulating nano-mechanical properties of cells and thereby influencing viral replication. Through the employment of confocal and atomic force microscopy techniques, we discerned perturbations in actin and vimentin filaments, which correlated with modification of cellular elasticity and reduction of glycocalyx layer. Intriguingly, this process was reversible upon polymer removal from the cells. To ascertain the applicability of our findings, we assessed the efficacy and underlying mechanism of the inhibitors using fully differentiated human airway epithelial cultures, wherein near-complete abrogation of viral replication was documented. Given their mode of action, these polymers can be classified as biologically active nanomaterials that exploit a highly conserved molecular target-cellular plasticity-proffering the potential for truly broad-spectrum activity while concurrently for drug resistance development is minimal.

Viral Interference During Influenza A-SARS-CoV-2 Coinfection of the Human Airway Epithelium and Reversal by Oseltamivir.

To gain insight into interactions among respiratory viruses, we modeled influenza A virus (IAV)-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coinfections using differentiated human airway epithelial cultures. Replicating IAV induced a more robust interferon response than SARS-CoV-2 and suppressed SARS-CoV-2 replication in both sequential and simultaneous infections, whereas SARS-CoV-2 did not enhance host cell defense during influenza infection or suppress IAV replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the host antiviral response and restored SARS-CoV-2 replication. These results demonstrate how perturbations in one viral infection can impact its effect on a coinfecting virus.

A comparative study of in vitro air–liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

BackgroundThe airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air–liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized.MethodsBCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens.ResultsWe confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus.ConclusionsTaken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Influence of cell type specific infectivity and tissue composition on SARS-CoV-2 infection dynamics within human airway epithelium.

Human airway epithelium (HAE) represents the primary site of viral infection for SARS-CoV-2. Comprising different cell populations, a lot of research has been aimed at deciphering the major cell types and infection dynamics that determine disease progression and severity. However, the cell type-specific replication kinetics, as well as the contribution of cellular composition of the respiratory epithelium to infection and pathology are still not fully understood. Although experimental advances, including Air-liquid interface (ALI) cultures of reconstituted pseudostratified HAE, as well as lung organoid systems, allow the observation of infection dynamics under physiological conditions in unprecedented level of detail, disentangling and quantifying the contribution of individual processes and cells to these dynamics remains challenging. Here, we present how a combination of experimental data and mathematical modelling can be used to infer and address the influence of cell type specific infectivity and tissue composition on SARS-CoV-2 infection dynamics. Using a stepwise approach that integrates various experimental data on HAE culture systems with regard to tissue differentiation and infection dynamics, we develop an individual cell-based model that enables investigation of infection and regeneration dynamics within pseudostratified HAE. In addition, we present a novel method to quantify tissue integrity based on image data related to the standard measures of transepithelial electrical resistance measurements. Our analysis provides a first aim of quantitatively assessing cell type specific infection kinetics and shows how tissue composition and changes in regeneration capacity, as e.g. in smokers, can influence disease progression and pathology. Furthermore, we identified key measurements that still need to be assessed in order to improve inference of cell type specific infection kinetics and disease progression. Our approach provides a method that, in combination with additional experimental data, can be used to disentangle the complex dynamics of viral infection and immunity within human airway epithelial culture systems.

Influenza virus infection modulates expression of the anti-inflammatory mucin 1 in both epithelial and immune cells

Abstract Cell-associated mucin 1(MUC1/Muc1) is a component of the mucus barrier in the respiratory tract that acts as a physical barrier and signaling molecule. Our prior work shows that MUC1 expression is increased during influenza virus (IAV) infection in human airway epithelial (HAE) cultures in vitro. Here we further detail MUC1 expression patterns and report that IAV-induced cytokines, including type I interferons (IFNs), trigger the expression of a low-molecular-weight form of MUC1 which localizes to the cytoplasm and nucleus, and is expressed in different epithelial cell types. Further, IFN-driven changes in the cellular composition of HAE toward a more secretory phenotype require MUC1, suggesting that MUC1 plays an important role in shaping the epithelial response during viral infection. Using a mouse model of IAV infection, we confirmed the upregulation of Muc1 throughout the epithelium in vivoand observed altered Muc1 levels in lung immune cells. Specifically, Muc1 was upregulated in interstitial macrophages but downregulated in antigen-presenting cells (APCs) including alveolar macrophages and dendritic cells as early as 2 days post-infection (dpi). By 5 dpi, Muc1 was significantly upregulated in various T cell subsets and their effector counterparts including CD4 +, CD8 +, and double-negative effector T cells. We also found a negative correlation between Muc1 levels in APCs and the expansion of effector T cell populations, supporting the putative immunoregulatory roles of MUC1/Muc1. Our ongoing work further explores the impact of Muc1 expression in specific cellular subsets on the immune response and IAV pathogenesis. This work was supported by the National Heart, Lung, and Blood Institute (R01HL151840, to MAS)

Unraveling the cell death induced by SARS-CoV-2 in lung epithelial cells

Abstract The COVID-19 pandemic has continued to affect people’s lives worldwide, and SARS-CoV-2, the pathogen that causes the pandemic, has infected over 100 million people and claimed over 10 million lives globally. There is strong evidence that lung damage contributes to the inflammatory responses against SARS-CoV-2, and that diverse forms of cell death occur in myeloid cells upon infection. However, less is known about the cell death pathways activated in the primary cell target of SARS-CoV-2, lung epithelial cells. Previously, we showed that conventional pyroptosis was activated in bystander myeloid cells. Using a human epithelial cell line with the ACE2 receptor and primary human airway epithelial culture, we analyzed the impact of SARS-CoV-2 infection on cell death. The latter is grown at an air-liquid interface (ALI) and is used to model airway pseudostratified epithelium. SARS-CoV-2 infection of these cells triggers lytic cell death. Pathway analysis shows that biomarkers of necroptosis, apoptosis, and pyroptosis are activated and increase with time, but some forms of cell death are limited predominantly to cells that are co-stained with SARS-CoV-2 viral antigens while others are not. RT-PCR and single-cell RNAseq analysis revealed that cell death genes increase with high viral titer and severity of disease in patients. R56 AI158314, R01 AI158314, AI029564, AI141333, CA232109

Global loss of cellular m6A RNA methylation following infection with different SARS-CoV-2 variants.

Insights into host-virus interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N 6-Methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during stress response. Gene expression profiles observed postinfection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants causes a loss of m6A in cellular RNAs, whereas m6A is detected abundantly in viral RNA. METTL3, the m6A methyltransferase, shows an unusual cytoplasmic localization postinfection. The B.1.351 variant has a less-pronounced effect on METTL3 localization and loss of m6A than did the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2-infected cells. Further, transcripts with m6A modification are preferentially down-regulated postinfection. Inhibition of the export protein XPO1 results in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which is compromised by SARS-CoV-2 infection, is restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.

R-Propranolol Has Broad-Spectrum Anti-Coronavirus Activity and Suppresses Factors Involved in Pathogenic Angiogenesis.

The SARS-CoV-2 pandemic highlighted the need for broad-spectrum antivirals to increase our preparedness. Patients often require treatment by the time that blocking virus replication is less effective. Therefore, therapy should not only aim to inhibit the virus, but also to suppress pathogenic host responses, e.g., leading to microvascular changes and pulmonary damage. Clinical studies have previously linked SARS-CoV-2 infection to pathogenic intussusceptive angiogenesis in the lungs, involving the upregulation of angiogenic factors such as ANGPTL4. The β-blocker propranolol is used to suppress aberrant ANGPTL4 expression in the treatment of hemangiomas. Therefore, we investigated the effect of propranolol on SARS-CoV-2 infection and the expression of ANGPTL4. SARS-CoV-2 upregulated ANGPTL4 in endothelial and other cells, which could be suppressed with R-propranolol. The compound also inhibited the replication of SARS-CoV-2 in Vero-E6 cells and reduced the viral load by up to ~2 logs in various cell lines and primary human airway epithelial cultures. R-propranolol was as effective as S-propranolol but lacks the latter's undesired β-blocker activity. R-propranolol also inhibited SARS-CoV and MERS-CoV. It inhibited a post-entry step of the replication cycle, likely via host factors. The broad-spectrum antiviral effect and suppression of factors involved in pathogenic angiogenesis make R-propranolol an interesting molecule to further explore for the treatment of coronavirus infections.

Vaping additives cannabinoid oil and vitamin E acetate adhere to and damage the human airway epithelium.

E-cigarette, or vaping product use-associated lung injury (EVALI), is a severe respiratory disorder that caused a sudden outbreak of hospitalized young people in 2019. Using cannabis oil containing vaping products, including vitamin E acetate contaminants, was found to be strongly associated with EVALI. However, the underlying tissue impacts of the condition are still largely unknown. Here, we focused on the vehicle cannabinoid oil (CBD oil) and contaminant vitamin E acetate (VEA) effects on airway epithelial cells. Primary human bronchial epithelial (HBE) cultures were exposed to e-liquid aerosols that contained CBD oil and VEA in combination or the common e-liquid components PG/VG with and without nicotine. Cell viability analysis indicated dramatically increased cell death counts after 3 days of CBD exposure, and this effect was even higher after CBD + VEA exposure. Microscopic examination of the cultures revealed cannabinoid and VEA depositions on the epithelial surfaces and cannabinoid accumulation in exposed cells, followed by cell death. These observations were supported by proteomic analysis of the cell secretions that exhibited increases in known markers of airway epithelial toxicity, such as xenobiotic enzymes, factors related to oxidative stress response, and cell death indicators. Overall, our study provides insights into the association between cannabinoid oil and vitamin E acetate vaping and lung injury. Collectively, our results suggest that the adherent accumulation of CBD oil on airway surfaces and the cellular uptake of both CBD oil- and VEA-containing condensates cause elevated metabolic stress, leading to increased cell death rates in human airway epithelial cultures.

MUC5B mobilizes and MUC5AC spatially aligns mucociliary transport on human airway epithelium.

Secreted mucus is a frontline defense against respiratory infection, enabling the capture and swift removal of infectious or irritating agents from the lungs. Airway mucus is composed of two mucins: mucin 5B (MUC5B) and 5AC (MUC5AC). Together, they form a hydrogel that can be actively transported by cilia along the airway surface. In chronic respiratory diseases, abnormal expression of these mucins is directly implicated in dysfunctional mucus clearance. Yet, the role of each mucin in supporting normal mucus transport remains unclear. Here, we generate human airway epithelial tissue cultures deficient in either MUC5B or MUC5AC to understand their individual contributions to mucus transport. We find that MUC5B and MUC5AC deficiency results in impaired and discoordinated mucociliary transport, respectively, demonstrating the importance of each mucin to airway clearance.

In vitro testing of salt coating of fabrics as a potential antiviral agent in reusable face masks

During the coronavirus disease (COVID-19) pandemic, wearing face masks in public spaces became mandatory in most countries. The risk of self-contamination when handling face masks, which was one of the earliest concerns, can be mitigated by adding antiviral coatings to the masks. In the present study, we evaluated the antiviral effectiveness of sodium chloride deposited on a fabric suitable for the manufacturing of reusable cloth masks using techniques adapted to the home environment. We tested eight coating conditions, involving both spraying and dipping methods and three salt dilutions. Influenza A H3N2 virus particles were incubated directly on the salt-coated materials, collected, and added to human 3D airway epithelial cultures. Live virus replication in the epithelia was quantified over time in collected apical washes. Relative to the non-coated material, salt deposits at or above 4.3 mg/cm2 markedly reduced viral replication. However, even for larger quantities of salt, the effectiveness of the coating remained dependent on the crystal size and distribution, which in turn depended on the coating technique. These findings confirm the suitability of salt coating as antiviral protection on cloth masks, but also emphasize that particular attention should be paid to the coating protocol when developing consumer solutions.

643 Delivery of gp64-pseudotyped lentivirus carrying codon-optimized cystic fibrosis transmembrane conductance regulator provides better functional restoration in human cystic fibrosis airway epithelial cultures

643 Delivery of gp64-pseudotyped lentivirus carrying codon-optimized cystic fibrosis transmembrane conductance regulator provides better functional restoration in human cystic fibrosis airway epithelial cultures

Enterovirus D68 Infection in Human Primary Airway and Brain Organoids: No Additional Role for Heparan Sulfate Binding for Neurotropism.

ABSTRACTEnterovirus D68 (EV-D68) is an RNA virus that can cause outbreaks of acute flaccid paralysis (AFP), a polio-like disease. Before 2010, EV-D68 was a rare pathogen associated with mild respiratory symptoms, but the recent EV-D68 related increase in severe respiratory illness and outbreaks of AFP is not yet understood. An explanation for the rise in severe disease is that it may be due to changes in the viral genome resulting in neurotropism. In this regard, in addition to sialic acid, binding to heparan sulfate proteoglycans (HSPGs) has been identified as a feature for viral entry of some EV-D68 strains in cell lines. Studies in human primary organotypic cultures that recapitulate human physiology will address the relevance of these HSPG-binding mutations for EV-D68 infection in vivo. Therefore, in this work, we studied the replication and neurotropism of previously determined sialic acid-dependent and HSPG-dependent strains using primary human airway epithelial (HAE) cultures and induced human pluripotent stem cell (iPSC)-derived brain organoids. All three strains (B2/2042, B2/947, and A1/1348) used in this study infected HAE cultures and human brain organoids (shown for the first time). Receptor-blocking experiments in both cultures confirm that B2/2042 infection is solely dependent on sialic acid, while B2/947 and A1/1348 (HSPG to a lesser extent) binds to sialic acid and HSPG for cell entry. Our data suggest that HSPG-binding can be used by EV-D68 for entry in human physiological models but offers no advantage for EV-D68 infection of brain cells.IMPORTANCE Recent outbreaks of enterovirus D68, a nonpolio enterovirus, is associated with a serious neurological condition in young children, acute flaccid myelitis (AFM). As there is no antiviral treatment or vaccine available for EV-D68 it is important to better understand how EV-D68 causes AFM and why only recent outbreaks are associated with AFM. We investigated if a change in receptor usage of EV-D68 increases the virulence of EV-D68 in the airway or the central nervous system and thus could explain the increase in AFM cases. We studied this using physiologically relevant human airway epithelium and cerebral organoid cultures that are physiologically relevant human models. Our data suggest that heparan sulfate proteoglycans can be used by EV-D68 as an additional entry receptor in human physiological models but offers no advantage for EV-D68 infection of brain cells, and our data show the potential of these 46 innovative models for virology.

Immunofluorescence-Mediated Detection of Respiratory Virus Infections in Human Airway Epithelial Cultures.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard‐to‐grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well‐differentiated human airway epithelial cell cultures at air‐liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses—specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—and provide a protocol for the detection of double‐stranded RNA or viral antigen–positive cells by immunofluorescence microscopy. These techniques, together with a post‐imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human airway epithelial cultures at air‐liquid interface (HAE‐ALI) Basic Protocol 2: Viral inoculation of HAE‐ALI Basic Protocol 3: Immunofluorescence (IF)‐based detection of infected cells in HAE‐ALI

Neutrophil Extracellular Traps Do Not Induce Injury and Inflammation in Well-Differentiated RSV-Infected Airway Epithelium.

Respiratory syncytial virus (RSV) lower respiratory tract infection (LRTI) causes a major burden of disease. The host response in RSV-LRTI is characterized by airway epithelial injury, inflammation and neutrophil influx, with the formation of neutrophil extracellular traps (NETs). However, the precise role of NETs in the pathophysiology of RSV-LRTI remains to be elucidated. Here, we used well-differentiated human airway epithelial cultures (HAE) of a pediatric and adult donor to study whether NETs cause airway epithelial injury and inflammation in the setting of RSV infection. The exposure of uninfected and RSV-infected HAE cultures to NETs, as produced by stimulation of neutrophils by a low dose of phorbol 12-myristate 13-acetate (PMA), did not induce or aggravate cell injury or inflammation. RSV infection of HAE cultures caused release of pro-inflammatory cytokines such as IL-6 and RANTES in both adult and pediatric cultures, but the differential gene expression for regulated cell death differed between culture donors. In this in vitro airway epithelial model, NETs in the setting of RSV infection did not cause or aggravate epithelial injury or inflammation.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models—the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

584: Delivery of SP-101 restores CFTR function in human CF airway epithelial cultures and drives hCFTRΔR transgene expression in the airways of ferrets

584: Delivery of SP-101 restores CFTR function in human CF airway epithelial cultures and drives hCFTRΔR transgene expression in the airways of ferrets

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.