Replication and pathogenesis of avian influenza A (H5N1) virus infection in polarised human bronchial and alveolar epithelium

1. In vitro models of polarised human respiratory epithelial cells were established to investigate the tropism and innate host responses of influenza A (H5N1 and H1N1) viruses. 2. Both viruses efficiently infected alveolar epithelial cells of both the apical and basolateral surfaces of the epithelium, whereas release of newly formed virus was mainly from the apical surface of the epithelium. 3. H5N1 virus was a more potent inducer of cytokines and chemokines in alveolar epithelial cells than H1N1 virus. Such chemokines were secreted onto both the apical and basolateral surfaces of the polarised alveolar epithelium. 4. In bronchial epithelium, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells than did H1N1 virus. In contrast, in well-differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response than did H1N1 virus. 5. Recombinant virus with vRNPs of a mammalian PB2 and an avian PB1 had the strongest polymerase activities, and replicated better in human cell cultures, especially at a high incubation temperature. These viruses were potent inducers of cytokines and chemokines in primary human alveolar epithelial cells.published_or_final_versio

Pathogenesis of the novel avian-origin influenza A (H7N9) virus Influenza H7N9 virus in human lower respiratory tract

Poster Session: News and Views from H7N9 OutbreakBackground: As of May 2013, 131 laboratory-confirmed human infections with a novel influenza H7N9 virus had been reported from China. The source of human infection appears to be poultry. There is so far no evidence of sustained human-to-human transmission. Genetic analysis revealed that all eight gene segments of H7N9 were of avian origin; six internal gene segments from avian influenza H7N9 viruses, while hemagglutinin and neuraminidase genes were derived from influenza viruses circulating in ducks and wild ducks, respectively. The emergence of the H7N9 influenza virus catches global attention about whether the new virus could spark another pandemic. The majority of the infected patients were hospitalized and suffered from ARDS, with a fatality rate of about 37%. Our study aimed to determine the mechanism contributing to the pathogenesis of the H7N9 virus. A panel of proinflammatory cytokines and chemokines will be examined upon influenza H7N9 virus infection in alveolar epithelial cells in order to examine if these mediators were induced differentially when compared with the highly pathogenic avian influenza (HPAI) H5N1 and the 2009 pandemic H1N1 virus. Moreover, because cleaved caspase 3 is commonly employed as a marker for the indication of apoptosis, we further examined the extensiveness of cleaved caspase 3 in influenza virus infection in human lung ex vivo cultures. Materials and Methods: Fresh biopsies of human lung tissue were obtained from patients undergoing surgical resection of lung tissues. Lung tissue fragments were cultured with F12K medium incubated at 37°C. For viral infection experiments, influenza viruses A/Shanghai/1/2013 (SH1, H7N9), A/Shanghai/2/2013 (SH2, H7N9), A/Hong Kong/483/97 (H5N1), and A/California/07 (Ca07, H1N1pdm) at a viral titer of 106 TCID50/mL were used for ex vivo lung culture infection. Infected lung tissues were collected in 10% formalin at 24, 48, and 72 hpi for immunohistochemical staining. Costaining of cleaved caspase 3 and influenza virus nucleoprotein was carried out for the detection of apoptosis. Furthermore, primary culture of human alveolar epithelial cells was isolated from human lungs by mincing the lung, followed by filtration and centrifugation. Human alveolar epithelial cells were infected with the novel influenza H7N9, the HPAI H5N1, and the pandemic H1N1 virus. Virus replication was monitored by measuring infectious viral particles using TCID50. mRNA and protein expression of proinflammatory cytokines and chemokines were quantified by real time qPCR and ELISA. Results: We found extensive apoptosis in influenza H7N9 (both SH1 and SH2) and H5N1, but not H1N1pdm infected ex vivo lung tissues, suggesting that both avian influenza viruses can induce apoptosis and cause severe cell death in human lung tissue. Furthermore, unlike HPAI H5N1 which induces dysregulated proinflammatory cytokine responses, the novel influenza H7N9 virus elicited poor proinflammatory cytokine responses, inducing type I and III interferon in ex vivo human lung explant cultures. The novel influenza H7N9 virus is an intrinsically more potent inducer of proinflammatory cytokine than the H1N1pdm virus but less than the H5N1 virus. Conclusions: The proinflammatory cytokine and chemokine responses may contribute modestly to the severity of human H7N9 disease, but it is likely that direct viral cytopathology is probably playing a more important role in pathogenesis of human H7N9 diseases. The recognition of the role of cleaved caspase 3 in severe human infection of avian influenza virus can provide insights on the development of novel therapeutic approaches for the preparedness of the future outbreak of pandemics.published_or_final_versio

Influenza B viruses in swine: virus tropism in swine respiratory organ explant cultures

Poster Session: Virology and Viral ReceptorsBackground: Swine has been considered an animal reservoir of pandemic influenza A virus (IAV), for example, the 2009 H1N1 pandemic virus, swine is acting as a “mixing vessel” for the reassortment of swine, human and avian IAVs. Certain influenza B virus (IBV) strains were also found to be readily infecting piglets as early as in 1969. However, tissue tropism of IBV in swine is understudied, at least in 2000s, mainly due to the misconception that IBV causes milder disease than IAV. IBV has in fact circulated in many parts of the world causing regular seasonal epidemics in humans with mortality rates sometimes higher than that in IAV seasons. Here, our research group hypothesizes that swine could be a neglected host of IBV, apart from human and seal, due to the previous infectivity of IBV in this animal, as well as the fact that swine has close contact with human and possesses a similar sialic acid (influenza virus receptor) distribution profile as the human respiratory tract. We aim to examine the characteristics of IBV tissue tropism using swine tracheal and lung explant models, and risk assess swine susceptibility to a panel of IBV strains from both Yamagata and Victoria lineages of different years. Materials and Methods: The tracheal and lung explants were prepared from fresh swine respiratory organs from approximately 6-month-old pigs, and cultured with maximal similarity to the in vivo conditions. A panel of IBV strains, from both Yamagata and Victoria lineages and from different years, were used to infect the tissue explants at 37oC or 39oC according to the original physiological temperature of the tissue. The virus replication efficiencies were evaluated through viral titration and immunohistochemistry of the collected supernatant and formalin-fixed tissue explants respectively at 1, 24, 48 and 72 h postinfection. Seasonal IAVs (H1N1 - A/OK/447/08 and H3N2 - A/OK/370/05) were used as controls. Results: Most of the tested IBVs showed productive replication in the swine lung explants. Swine tracheal explants, on the other hand, supported the replication of limited IBV strains. Most of these IBVs belong to the Victoria lineage, which spread across the years from 2005 to 2011. IBVs that could replicate in swine lung explants reached their maxima at 48 hpi or sometimes later. This is comparatively slower than the replication rates of seasonal IAVs (H1N1 & H3N2) used in the study, which usually showed significant increase at 24 hpi with still increasing virus yields at 48 hpi in some cases. However, the overall increase in titres between the IBVs and seasonal IAVs were similar. In swine tracheal explants, both IBVs and seasonal IAVs showed limited replications with similar trends of having maxima being reached at 24 hpi. Conclusions: The successful replication of IBVs in swine explants cultures indicates the possible susceptibility of swine to IBV and provides the essential basis for further investigation on the likelihood for swine to be an animal reservoir of the virus, as well as the threat it may pose to humans. Continuous studies on the replication kinetics of a greater number of IBVs in swine explant cultures across a wider range of years, countries and lineages will probably be our future target.published_or_final_versio

Proinflammatory cytokine response and viral replication in mouse bone marrow derived macrophages infected with influenza H1N1 and H5N1 viruses

The pathogenesis of human influenza H5N1 virus infection remains poorly understood and controversial. Cytokine dysregulation in human infection has been hypothesized to contribute to disease severity. We developed in vitro cultures of mouse bone marrow derived macrophages (BMDMΦ) from C57BL/6N mouse to compare influenza A (H5N1 and H1N1) virus replication and pro-inflammatory cytokine and chemokine responses. While both H1N1 and H5N1 viruses infected the mouse bone marrow derived macrophages, only the H1N1 virus had showed evidence of productive viral replication from the infected cells. In comparison with human seasonal influenza H1N1 (A/HK/54/98) and mouse adapted influenza H1N1 (A/WSN/33) viruses, the highly pathogenic influenza H5N1 virus (A/HK/483/97) was a more potent inducer of the chemokine, CXCL 10 (IP-10), while there was not a clear differential TNF-α protein expression pattern. Although human influenza viruses rarely cause infection in mice without prior adaption, the use of in vitro cell cultures of primary mouse cells is of interest, especially given the availability of gene-defective (knock-out) mice for specific genes.published_or_final_versio

Immunomodulatory and anti-viral effects of statins in influenza H5N1 virus infection of human alveolar epithelial cells and peripheral blood–derived macrophages

Poster Session: Novel TherapeuticsBackground: Highly pathogenic avian influenza (HPAI) H5N1 virus panzootic in poultry continues to spread. It causes zoonotic human disease with a high (> 60%) fatality rate and continues to pose a pandemic threat. Based on clinical, animal, and in vitro cell studies, we and others have suggested that differences in viral replication competence, tissue tropism, and cytokine dysregulation between H5N1 and low pathogenic viruses may contribute to disease pathogenesis. Statins as HMG-CoA inhibitors act to reduce cholesterol and have been demonstrated to have anti-inflammatory and immune-modulatory activities. However, there is controversy about the benefits of statin use on influenza infection in mice and humans. In this study, we aimed to evaluate the effects of statin treatment in influenza infection using physiologically relevant in vitro models—human alveolar epithelial cells (AECs) and peripheral blood–derived macrophages (PBDMs). Materials and Methods: Primary human AECs and PBDMs were infected with HPAI H5N1 (A/HK/483/97) and seasonal H1N1 (A/HK/54/98) viruses in the presence or absence of statin (simvastatin and sevastatin) treatment. Virus replication was monitored by measuring infectious viral particles in cell culture supernatants using TCID50. Immuno-modulatory effects of statins were examined by measuring the mRNA and protein expression of cytokines and chemokines using qPCR and ELISA. In order to understand the intervention of statins and influenza infection, the gene expression profile of selected members of the sterol-biosynthesis pathway in influenza virus–infected AECs and PBDMs were also monitored. The responses of a variety of cytokine treatments on the genes of the sterol-biosynthesis pathway were investigated in AECs. Furthermore, the intracellular free cholesterol level was also examined by enzymatic assay in AECs infected with influenza virus. Results: We demonstrated that both simvastatin and mevastatin exhibited a dose-dependent inhibition of influenza virus replication for both HPAI H5N1 and seasonal H1N1 viruses in human AECs and PBDMs. The observed inhibitory effect of simvastatin and mevastatin occurred below the non-specific toxic effects to cells, which were measured by MTT assay. Treatment of simvastatin and mevastatin significantly suppressed H5N1 virus–induced pro-inflammatory cytokines such as TNF-α in PBDMs and chemokines, including IP-10 and MCP-1 secretion in both AECs and PBDMs at 24 hours post-infection. We further showed that human AECs and PBDMs infected with both HPAI H5N1 and seasonal H1N1 viruses had significant down-regulation of sterol pathway gene expression at 24 hours post-infection. AECs and PBDMs treated with IFN-γ or IFN-β but not IL-1β, TNF, or IL-6, showed down-regulation of sterol pathway gene expression. In addition, we found that the free cholesterol level was significantly reduced at 24 and 48 h post-H5N1 virus infection in AECs and in IFN-β–treated AECs. These results further support a specific modulation of the sterol metabolic pathway upon influenza virus infection. Conclusions: Taken together, the controversy about the beneficial effects of statin use in influenza infection and our data suggest that statins possess both the antiviral and immune-regulatory effects in H5N1-infected in vitro cell models. We also demonstrated a highly specific response of AECs and PBDMs through a coordinated negative regulation of multiple sterol pathway members upon influenza virus infection or treatment of interferon. Identification of a reduction in sterol pathway gene expression and cholesterol levels with IFN treatment in human AECs offers new insights on the host-mediated antiviral responses through the sterol metabolism pathway and opens new therapeutic options for human influenza disease.published_or_final_versio

Polarity of influenza H5N1 virus infection in respiratory epithelial cells and the impact of basolateral release of cytokines in disease pathogenesis

Poster Presentations: Virus Host Interaction/PathogenesisINTRODUCTION: Highly pathogenic avian influenza virus (H5N1) is the first avian influenza virus that documented to cause respiratory disease and death in human. The biological basis for the severe human disease and high fatality rate remains unclear. We have previously demonstrated that when compared to human H1N1 and H3N2 influenza viruses, infection of influenza H5N1 virus led to the hyper-induction of pro-inflammatory cytokines in human primary macrophages and ...postprin

Evaluation of the human adaptation of influenza A/H7N9 virus in PB2 protein using human and swine respiratory tract explant cultures

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.published_or_final_versio

Replication of H9 influenza viruses in the human ex vivo respiratory tract, and the influence of neuraminidase on virus release

H9N2 viruses are the most widespread influenza viruses in poultry in Asia. We evaluated the infection and tropism of human and avian H9 influenza virus in the human respiratory tract using ex vivo respiratory organ culture. H9 viruses infected the upper and lower respiratory tract and the majority of H9 viruses had a decreased ability to release virus from the bronchus rather than the lung. This may be attributed to a weak neuraminidase (NA) cleavage of carbon-6-linked sialic acid (Sia) rather than carbon-3-linked Sia. The modified cleavage of N-acetlylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) by NA in H9 virus replication was observed by reverse genetics, and recombinant H9N2 viruses with amino acids (38KQ) deleted in the NA stalk, and changing the amino acid at position 431 from Proline-to-Lysine. Using recombinant H9 viruses previously evaluated in the ferret, we found that viruses which replicated well in the ferret did not replicate to the same extent in the human ex vivo cultures. The existing risk assessment models for H9N2 viruses in ferrets may not always have a strong correlation with the replication in the human upper respiratory tract. The inclusion of the human ex vivo cultures would further strengthen the future risk-assessment strategies.published_or_final_versio

Angiopoietin-1 and keratinocyte growth factor restore the impaired alveolar fluid clearance induced by influenza H5N1 virus infection

Poster Session: Novel TherapeuticsBackground: Acute respiratory distress syndrome (ARDS) caused by high pathogenic avian influenza (HPAI) H5N1 virus infection has resulted in severe illness and high mortality rates among patients. Patients with ARDS are often characterized by impaired alveolar fluid clearance and alveolar edema. An understanding of the mechanism responsible for human alveolar edema will lead to the development of novel therapeutic treatments for ARDS patients. We hypothesized that the paracrine soluble factors angiopoietin-1 (Ang-1) and keratinocyte growth factor (KGF) can resolve alveolar fluid clearance by up-regulating the expression of major sodium and chloride transporters impaired by HPAI H5N1 virus infection. Materials and Methods: Human alveolar epithelial cells grown on transwell inserts were infected with HPAI H5N1 (A/HK/483/97) and low pathogenic avian influenza (LPAI) H1N1 (A/HK/54/98) viruses at MOI 0.1 or incubated with conditioned culture medium containing Ang-1 and/or KGF. At 24 and 48 h post-infection, the rate of alveolar fluid transport and protein permeability across the alveolar epithelium was measured. Protein expression of sodium and chloride transporters (Na-K-ATPase, CFTR, and epithelial sodium channel alpha subunit) was measured by qPCR, ELISA, and Western blot. Results: HPAI H5N1 (A/HK/483/97) virus infection significantly reduced net alveolar fluid transport and protein permeability when compared with H1N1 (A/HK/54/98) virus infection at 24 h post-infection and further reduced it at 48 h post-infection. This reduction in alveolar fluid clearance was associated with a substantial reduction in protein expression of Na-K-ATPase, CFTR, and epithelial sodium channel alpha subunit. The influenza virus–infected cells treated with Ang-1 and KGF restored the impaired alveolar edema fluid clearance and protein permeability after HPAI H5N1 virus infection. Furthermore, the paracrine soluble factors Ang-1 and KGF up-regulated the protein expression of the major sodium and chloride transporters resulting from the HPAI influenza virus infection. Conclusions: The paracrine soluble factors Ang-1 and KGF play an important role in maintaining human alveolar fluid clearance by up-regulating the sodium and chloride transporting systems in human alveolar epithelium. This study enriches the understanding of the development of ARDS in human H5N1 disease and may aid in the development of possible therapeutic applications.published_or_final_versio

Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.published_or_final_versio