Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection.

The extent of lung regeneration following catastrophic damage and the potential role of adult stem cells in such a process remains obscure. Sublethal infection of mice with an H1N1 influenza virus related to that of the 1918 pandemic triggers massive airway damage followed by apparent regeneration. We show here that p63-expressing stem cells in the bronchiolar epithelium undergo rapid proliferation after infection and radiate to interbronchiolar regions of alveolar ablation. Once there, these cells assemble into discrete, Krt5+ pods and initiate expression of markers typical of alveoli. Gene expression profiles of these pods suggest that they are intermediates in the reconstitution of the alveolar-capillary network eradicated by viral infection. The dynamics of this p63-expressing stem cell in lung regeneration mirrors our parallel finding that defined pedigrees of human distal airway stem cells assemble alveoli-like structures in vitro and suggests new therapeutic avenues to acute and chronic airway disease.

Influence of glycan structure on the colonization of Streptococcus pneumoniae on human respiratory epithelial cells.

Virtually all living cells are encased in glycans. They perform key cellular functions such as immunomodulation and cell-cell recognition. Yet, how their composition and configuration affect their functions remains enigmatic. Here, we constructed isogenic capsule-switch mutants harboring 84 types of capsular polysaccharides (CPSs) in Streptococcus pneumoniae. This collection enables us to systematically measure the affinity of structurally related CPSs to primary human nasal and bronchial epithelial cells. Contrary to the paradigm, the surface charge does not appreciably affect epithelial cell binding. Factors that affect adhesion to respiratory cells include the number of rhamnose residues and the presence of human-like glycomotifs in CPS. Besides, pneumococcal colonization stimulated the production of interleukin 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractantprotein-1 (MCP-1) in nasal epithelial cells, which also appears to be dependent on the serotype. Together, our results reveal glycomotifs of surface polysaccharides that are likely to be important for colonization and survival in the human airway.

Enterovirus-A71 exploits RAB11 to recruit chaperones for virus morphogenesis.

BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.

Differential effects of microRNAs miR-21, miR-99 and miR-145 on lung regeneration and inflammation during recovery from influenza pneumonia.

In a mouse model of influenza pneumonia, we previously documented that proliferating alveolar type II (AT2) cells are the major stem cells involved in early lung recovery. Profiling of microRNAs revealed significant dysregulation of specific ones, including miR-21 and miR-99a. Moreover, miR-145 is known to exhibit antagonism to miR-21. This follow-up study investigated the roles of microRNAs miR-21, miR-99a, and miR-145 in the murine pulmonary regenerative process and inflammation during influenza pneumonia. Inhibition of miR-21 resulted in severe morbidity, and in significantly decreased proliferating AT2 cells due to impaired transition from innate to adaptive immune responses. Knockdown of miR-99a culminated in moderate morbidity, with a significant increase in proliferating AT2 cells that may be linked to PTEN downregulation. In contrast, miR-145 antagonism did not impact morbidity nor the proliferating AT2 cell population, and was associated with downregulation of TNF-alpha, IL1-beta, YM1, and LY6G. Hence, a complex interplay exists between expression of specific miRNAs, lung regeneration, and inflammation during recovery from influenza pneumonia. Inhibition of miR-21 and miR-99a (but not miR-145) can lead to deleterious cellular and molecular effects on pulmonary repair and inflammatory processes during influenza pneumonia.

The Suppressor of Cytokine Signalling family of proteins and their potential impact on COVID-19 disease progression.

The family of Suppressor of Cytokine Signalling (SOCS) proteins plays pivotal roles in cytokine and immune regulation. Despite their key roles, little attention has been given to the SOCS family as compared to other feedback regulators. To date, SOCS proteins have been found to be exploited by viruses such as herpes simplex virus (HSV), hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus, respiratory syncytial virus (RSV), Ebola virus, influenza A virus (IAV) and SARS-CoV, just to name a few. The hijacking and subsequent upregulation of the SOCS proteins upon viral infection, suppress the associated JAK-STAT signalling activities, thereby reducing the host antiviral response and promoting viral replication. Two SOCS protein family members, SOCS1 and SOCS3 are well-studied and their roles in the JAK-STAT signalling pathway are defined as attenuating interferon (IFN) signalling upon viral infection. The upregulation of SOCS protein by SARS-CoV during the early stages of infection implies strong similarity with SARS-CoV-2, given their closely related genomic organisation. Thus, this review aims to outline the plausibility of SOCS protein inhibitors as a potential therapeutic regimen for COVID-19 patients. We also discuss the antagonists against SOCS protein to offer an overview on the previous 'successes' of SOCS protein inhibition in various viral infections that may portray possible clues for COVID-19 disease management.

The Association of Helicobacter pylori Biofilm with Enterovirus 71 Prolongs Viral Viability and Survival.

The transition time during which a virus leaves its host and infects the next susceptible host is critical for virus survival. Enterovirus 71 (EV71) is stable in aqueous environments, but its molecular interactions with bacteria and their biofilms are not well-established. Helicobacter pylori is a highly successful gut bacterial pathogen, with its capacity to form biofilms being linked to its transmission. Given that both are gut-associated microbes, we hypothesized that biofilms formed by H. pylori may play a significant role in the survival of EV71 in the external environment. In this study, we examine the interactions of EV71 with the preformed biofilm of H. pylori to mimic its natural state in the environment. Immunofluorescence confocal microscopy and scanning electron microscopy revealed that EV71 particles persisted for up to 10 days when incubated with the H. pylori biofilm. Furthermore, the presence of the H. pylori biofilm significantly augmented viral viability, as verified through virus plaque assays. Interestingly, the viability of EV71 was dependent on the quantity of H. pylori biofilm formation. Thus, two H. pylori strains able to generate large amounts of biofilm could facilitate EV71 viability for up to 17 days, whereas two other H. pylori strains that produced moderate or low quantities of biofilm could not prolong virus viability. It is interesting that biofilm contains N-acetyl-glucosamine and glycosaminoglycan, and that EV71 has binding affinity to cell-surface heparan sulfate glycosaminoglycan, which acts as an EV71 attachment receptor. The synergistic ability of H. pylori biofilm to promote EV71 viability for extended periods implies that H. pylori biofilm may serve as an additional pathway of EV71 transmission.

Viral Load of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Respiratory Aerosols Emitted by Patients With Coronavirus Disease 2019 (COVID-19) While Breathing, Talking, and Singing.

BACKGROUND: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 µm) and fine (≤5 µm) respiratory aerosols produced when breathing, talking, and singing. METHODS: Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing. RESULTS: Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63-5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative. CONCLUSIONS: Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies.

Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist, SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection.

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

Transcriptomics of rhinovirus persistence reveals sustained expression of RIG-I and interferon-stimulated genes in nasal epithelial cells in vitro.

BACKGROUND: Human rhinoviruses (HRVs) are frequently associated with asthma exacerbations, and have been found in the airways of asthmatic patients. While HRV-induced acute infection is well-documented, it is less clear whether the nasal epithelium sustains prolonged HRV infections along with the associated activation of host immune responses. OBJECTIVE: To investigate sustainably regulated host responses of human nasal epithelial cells (hNECs) during HRV persistence. METHODS: Using a time-course study, HRV16 persistence and viral replication dynamics were established using an in vitro infection model of hNECs. RNA sequencing was performed on hNECs in the early and late stages of infection at 3 and 14 days post-infection (dpi), respectively. The functional enrichment of differentially expressed genes (DEGs) was evaluated using gene ontology (GO) and Ingenuity pathway analysis. RESULTS: HRV RNA and protein expression persisted throughout prolonged infections, even after decreased production of infectious virus progeny. GO analysis of unique DEGs indicated altered regulation of pathways related to ciliary function and airway remodeling at 3 dpi and serine-type endopeptidase activity at 14 dpi. The functional enrichment of shared DEGs between the two time-points was related to interferon (IFN) and cytoplasmic pattern recognition receptor (PRR) signaling pathways. Validation of the sustained regulation of candidate genes confirmed the persistent expression of RIG-I and revealed its close co-regulation with interferon-stimulated genes (ISGs) during HRV persistence. CONCLUSIONS: The persistence of HRV RNA does not necessarily indicate an active infection during prolonged infection. The sustained expression of RIG-I and ISGs in response to viral RNA persistence highlights the importance of assessing how immune-activating host factors can change during active HRV infection and the immune regulation that persists thereafter.

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus.

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Serial Passaging of Seasonal H3N2 Influenza A/Singapore/G2-31.1/2014 Virus in MDCK-SIAT1 Cells and Primary Chick Embryo Cells Generates HA D457G Mutation and Other Variants in HA, NA, PB1, PB1-F2, and NS1.

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

Combination Therapy Targeting Platelet Activation and Virus Replication Protects Mice against Lethal Influenza Pneumonia.

Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P2Y12 platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia.

Upregulation of cell-surface mucin MUC15 in human nasal epithelial cells upon influenza A virus infection.

BACKGROUND: Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections. METHODS: To further identify mucins significant in influenza infection, we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. RESULTS: We found that the expression of MUC15 was significantly upregulated upon infection, and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly, positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines, chemokines, EGFR and phosphorylated ERK) started to peak and plateau, MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. CONCLUSIONS: Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus, we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection.

CD151, a novel host factor of nuclear export signaling in influenza virus infection.

BACKGROUND: Despite advances in our understanding of the mechanisms of influenza A virus (IAV) infection, the crucial virus-host interactions during the viral replication cycle still remain incomplete. Tetraspanin CD151 is highly expressed in the human respiratory tract, but its pathological role in IAV infection is unknown. OBJECTIVES: We sought to characterize the functional role and mechanisms of action of CD151 in IAV infection of the upper and lower respiratory tracts with H1N1 and H3N2 strains. METHODS: We used CD151-null mice in an in vivo model of IAV infection and clinical donor samples of in vitro-differentiated human nasal epithelial cells cultured at air-liquid interface. RESULTS: As compared with wild-type infected mice, CD151-null infected mice exhibited a significant reduction in virus titer and improvement in survival that is associated with pronounced host antiviral response and inflammasome activation together with accelerated lung repair. Interestingly, we show that CD151 complexes newly synthesized viral proteins with host nuclear export proteins and stabilizes microtubule complexes, which are key processes necessary for the polarized trafficking of viral progeny to the host plasma membrane for assembly. CONCLUSIONS: Our results provide new mechanistic insights into our understanding of IAV infection. We show that CD151 is a critical novel host factor of nuclear export signaling whereby the IAV nuclear export uses it to complement its own nuclear export proteins (a site not targeted by current therapy), making this regulation unique, and holds promise for the development of novel alternative/complementary strategies to reduce IAV severity.

In Vitro Model of Fully Differentiated Human Nasal Epithelial Cells Infected With Rhinovirus Reveals Epithelium-Initiated Immune Responses.

Human rhinoviruses (HRVs) are the commonest cause of the common cold. While HRV is less pathogenic than other respiratory viruses, it is frequently associated with exacerbation of chronic respiratory diseases such as rhinosinusitis and asthma. Nasal epithelial cells are the first sites of viral contact, immune initiation, and airway interconnectivity, but there are limited studies on HRV infection of nasal epithelial cells. Hence, we established a model of HRV infection of in vitro-differentiated human nasal epithelial cells (hNECs) derived from multiple individuals. Through HRV infection of hNECs, we found that HRV mainly targeted ciliated cells and preferentially induced type I and III interferon antiviral pathways. Quantitative polymerase chain reaction analysis of inflammatory genes suggested predominant type 1 immunity signaling and recruitment, with secreted CXCL9, IP-10, CXCL11, and RANTES as likely initiators of airway inflammatory responses. Additionally, we further explored HRV bidirectional release from the hNECs and identified 11 associated genes. Other HRV interactions were also identified through a systematic comparison with influenza A virus infection of hNECs. Overall, this in vitro hNEC HRV infection model provides a platform for repeatable and controlled studies of different individuals, thus providing novel insights into the roles of human nasal epithelium in HRV interaction and immune initiation.

Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses.

BACKGROUND: Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches. RESULTS: The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference. CONCLUSION: The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

MicroRNA-146a induction during influenza H3N2 virus infection targets and regulates TRAF6 levels in human nasal epithelial cells (hNECs).

We have previously shown that human nasal epithelial cells (hNECs) are highly permissive cells for respiratory viruses including influenza A virus (IAV) and respiratory syncytial virus. Recent studies have indicated that microRNAs (miRNAs) are involved in virus-host relationship, and this led us to investigate its essential roles in the in vitro hNECs model derived from multiple donors. By comparing the differential expression of miRNAs upon IAV infection among animal and cell line studies, candidates were selected with focus on the initial immune response. After infection of influenza H3N2 virus, hNECs showed constant increase virus titer at 24-72h post-infection (hpi); accompanied with a significantly elevated level of miR-146a-5p at 72 hpi. The exponential elevation of progeny virus titer correlated with a key influenza sensing Toll-like receptor (TLR)7 pathway. TLR7 downstream gene transcripts, myeloid differentiation primary response gene 88 (MyD88), interferon regulator factor 7 (IRF7), and interferon-ß (IFN-ß) were significantly upregulated at 48 and 72 hpi, while interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor associated factor-6 (TRAF6) were unchanged. Interestingly, when miR-146a was overexpressed with miRNA mimics prior to H3N2 infection, further decreased transcripts of TRAF6, but not IRAK1, were detected. By using the in vitro hNEC model, we demonstrated that H3N2-induced miR-146a specifically targets and regulates TRAF6 expression; but not IRAK expression in the nasal epithelium. We also found that unlike the cell model studies that lead to our studies, when ran across a heterogeneous model of different individual, miRNA signals were highly varied and the expression of most miRNAs, including miR-146a-5p, was more subdued compared to homogenous cell line model, highlighting a need for a more thorough analysis of miRNA signals and targets in a model more mimicking a clinical influenza infection.