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The annual epidemics of seasonal influenza is partly attributed to the continued virus evolution. It is challenging to evaluate the effect of influenza virus mutations on evading population immunity. In this study, we introduce a novel statistical and computational approach to measure the dynamic molecular determinants underlying epidemics using effective mutations (EMs), and account for the time of waning mutation advantage against herd immunity by measuring the effective mutation periods (EMPs). Extensive analysis is performed on the sequencing and epidemiology data of H3N2 epidemics in ten regions from season to season. We systematically identified 46 EMs in the hemagglutinin (HA) gene, in which the majority were antigenic sites. Eight EMs were located in immunosubdominant stalk domain, an important target for developing broadly reactive antibodies. The EMs might provide timely information on key substitutions for influenza vaccines antigen design. The EMP suggested that major genetic variants of H3N2 circulated in South-east Asia for an average duration of 4.5 years (SD 2.4) compared to a significantly shorter 2.0 years (SD 1.0) in temperate regions. The proposed method bridges population epidemics and molecular characteristics of infectious diseases, and would find broad applications in various pathogens mutation estimations.

Background Neutrophil is of the most abundant number in human immune system. During acute influenza virus infection, neutrophils are already active in the early phase of inflammation - a time in which clinical biopsy or autopsy material is not readily available. However, the role of neutrophil in virus infection is not well understood. Here, we studied the role of neutrophil in host defense during influenza A virus infection, specifically assessing if it contributes to the differential pathogenesis in H5N1 disease. Methods Neutrophils were freshly isolated from healthy volunteers and subjected to direct influenza H1N1 and H5N1 virus infection in vitro. The ability of the naïve neutrophils to infiltrate from the basolateral to the apical phase of the influenza virus infected alveolar epithelium was assessed. The viral replication, innate immune responses and Neutrophil extracellular trap (NET) formation of neutrophils upon influenza virus infection were evaluated. Results Our results demonstrated that influenza virus infected alveolar epithelium allowed neutrophil transmigration. Significantly more neutrophils migrated across the H5N1 influenza virus infected the epithelium than the counterpart infected by the seasonal influenza H1N1 virus infected. Neutrophils were equally susceptible to H5N1 and H1N1 virus infection with similar viral gene transcription. Productive replication was observed in H5N1 infected neutrophils. H5N1 induced higher cytokine and chemokine gene transcription than H1N1 infected neutrophils, including TNF-α, IFN-β, CXCL10, MIP-1α and IL-8. This inferred a more intense inflammatory response posed by H5N1 than H1N1 virus. Strikingly, NADPH oxidase-independent NET formation was only observed in H1N1 infected neutrophils at 6 hpi while no NET formation was observed upon H5N1 infection. Conclusion Our data is the first to demonstrate that NET formation is abrogated in H5N1 influenza virus infection and might contribute to the severity of H5N1 disease.

BackgroundAlthough the serological antibody responses induced by SARS-CoV-2 vaccines are well characterized, little is known about their ability to elicit mucosal immunity.ObjectivesThis study aims to examine and compare the mucosal and systemic responses of recipients of two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac).MethodsSerial blood and nasal epithelial lining fluid (NELF) samples were collected from the recipients of either Comirnaty or CoronaVac. The plasma and NELF immunoglobulins A and G (IgA and IgG) specific to SARS-CoV-2 S1 protein (S1) and their neutralization effects were quantified.ResultsComirnaty induced nasal S1-specific immunoglobulin responses, which were evident as early as 14 ± 2 days after the first dose. In 64% of the subjects, the neutralizing effects of NELF persisted for at least 50 days. Moreover, 85% of Comirnaty recipients exhibited S1-specific IgA and IgG responses in plasma by 14 ± 2 days after the first dose. By 7 ± 2 days after the booster, all plasma samples possessed S1-specific IgA and IgG responses and were neutralizing. The induction of S1-specific plasma antibodies by CoronaVac was IgG dominant, and 83% of the subjects possessed S1-specific IgG by 7 ± 2 days after the booster, with neutralizing effects.ConclusionComirnaty induces S1-specific IgA and IgG responses with neutralizing activity in the nasal mucosa; a similar response is not seen with CoronaVac.Clinical ImplicationThe presence of a nasal response with mRNA vaccine may provide additional protection compared with inactivated virus vaccine. However, whether such widespread immunological response may produce inadvertent adverse effects in other tissues warrants further investigation.

Vaccines that elicit mucosal immune responses against SARS-CoV-2 could potentially be of exceptional importance in providing first line defense at the site of viral entry. The serological antibody response induced by SARS-CoV-2 vaccines have already been well characterized. In order to understand the mucosal immune response profiles of SARS-CoV-2 vaccines, we examined both the mucosal and systemic responses of subjects vaccinated by two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac). Serial nasal epithelial lining fluid (NELF) and peripheral blood samples were collected in ten subjects who had received CoronaVac and thirty-two subjects who had received Comirnaty. We quantified IgA and IgG specific to SARS-CoV-2 S1 protein by ELISA in NELF and plasma samples. The neutralization effect of these two sample types were evaluated by surrogate ACE-SARS-CoV-2 Spike protein ELISA. Only Comirnaty induced nasal SARS-CoV-2 S1 protein-specific (S1-specific) IgA and IgG responses, which were evident as early as on 14±2 days after the first dose. The NELF samples of 72% of subjects became IgA+IgG+, while in 62.5% of subjects the samples were neutralizing by 7±2 days after the second dose. In 45% of the subjects their NELF remained neutralizing 50 days after the booster of Comirnaty. In plasma, 91% and 100% Comirnaty subjects possessed S1-specific IgA+IgG+ on 14±2 days after the first dose and 7±2 days after booster, respectively. The plasma collected on 7±2 days after booster was 100% neutralizing. The induction of S1-specific antibody by CoronaVac was IgG dominant, and 70% of the subjects possessed S1-specific IgG by 7±2 days after booster and were all neutralizing. This study reveals that Comirnaty is able to induce S1-specific IgA and IgG response with neutralizing activity in the nasal mucosa in addition to a consistent systemic response. The clinical implications and the biological mechanism of an additional nasal immune response induced by vaccines such as Comirnaty warrant further investigation.One Sentence SummarymRNA vaccine (CoronaVac) elicits mucosal IgA and IgG in the nasal epithelial lining fluid together with ELISA-detected anti-wild-type spike neutralizing antibodies as early as day 14 post vaccination.

Virus evolution drives the annual influenza epidemics in human population worldwide. However, it has been challenging to evaluate the mutation effect of the influenza virus on evading the population immunity. In this study, we introduce a novel statistical and computational approach to measure the dynamic molecular determinants underlying epidemics by the effective mutations (EMs), and account for the time of waning mutation advantage against herd immunity by the effective mutation periods (EMPs). Extensive analysis is performed on the genome and epidemiology data of 13-year worldwide H3N2 epidemics involving nine regions in four continents. We showed that the identified EM processed similar profile in geographically adjacent regions, while only 40% are common to Europe, North America, Asia and Oceania, indicating that the regional specific mutations also contributed significantly to the global H3N2 epidemics. The mutation dynamics calibrated that around 90% of the common EMs underlying global epidemics were originated from South East Asia, led by Thailand and India, and the rest were originated from North America. New Zealand was found to be the dominate sink region of H3N2 circulation, followed by UK. All regions might act as the intersection in the H3N2 transmission network. The proposed methodology provided a way to characterize key amino acids from the genetic epidemiology point of view. This approach is not restricted by the genomic region or type of the virus, and will find broad applications in identifying therapeutic targets for combating infectious diseases.

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.

Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

The initial stage of host cell infection by influenza A viruses (IAV) is mediated through interaction of the viral haemagglutinin (HA) with cell surface glycans. The binding requirement of IAVs for Galβ(1,4)Glc/ GlcNAc (lactose/lactosamine) glycans with a terminal α(2,6)-linked (human receptors) or α(2,3)-linked (avian receptors) N-acetylneuraminic residue commonly found on N-glycans, is well-established. However the role and significance of sialylated Galβ(1,3)GalNAc (core 1) epitopes that are typical O-glycoforms in influenza virus pathogenesis remains poorly detailed. Here we report a multidisciplinary study using NMR spectroscopy, virus neutralization assays and molecular modelling, into the potential for IAV to engage sialyl-Galβ(1,3)GalNAc O-glycoforms for cell attachment. H5 containing virus like particles (VLPs) derived from an H5N1 avian IAV strain show a significant involvement of the O-glycan-specific GalNAc residue, coordinated by a EQTKLY motif conserved in highly pathogenic avian influenza (HPAI) strains. Notably, human pandemic H1N1 influenza viruses shift the preference from ‘human-like’ α(2,6)-linkages in sialylated Galβ(1,4)Glc/GlcNAc fragments to ‘avian-like’ α(2,3)-linkages in sialylated Galβ(1,3)GalNAc without involvement of the GalNAc residue. Overall, our study suggests that sialylated Galβ(1,3)GalNAc as O-glycan core 1 glycoforms are involved in the influenza A virus life cycle and play a particularly crucial role during infection of HPAI strains.

Background: The ferret is a key animal model to study the transmission characteristics of influenza viruses. Results: Characterization of ferret respiratory tract tissues identified influenza virus glycan receptors. Conclusion: Species-specific influenza virus glycan receptors were identified. Significance: Our findings provide new insights into the usefulness of ferrets in the study of influenza virus infection., The initial recognition between influenza virus and the host cell is mediated by interactions between the viral surface protein hemagglutinin and sialic acid-terminated glycoconjugates on the host cell surface. The sialic acid residues can be linked to the adjacent monosaccharide by α2–3- or α2–6-type glycosidic bonds. It is this linkage difference that primarily defines the species barrier of the influenza virus infection with α2–3 binding being associated with avian influenza viruses and α2–6 binding being associated with human strains. The ferret has been extensively used as an animal model to study the transmission of influenza. To better understand the validity of this model system, we undertook glycomic characterization of respiratory tissues of ferret, which allows a comparison of potential viral receptors to be made between humans and ferrets. To complement the structural analysis, lectin staining experiments were performed to characterize the regional distributions of glycans along the respiratory tract of ferrets. Finally, the binding between the glycans identified and the hemagglutinins of different strains of influenza viruses was assessed by glycan array experiments. Our data indicated that the respiratory tissues of ferret heterogeneously express both α2–3- and α2–6-linked sialic acids. However, the respiratory tissues of ferret also expressed the Sda epitope (NeuAcα2-3(GalNAcβ1–4)Galβ1–4GlcNAc) and sialylated N,N′-diacetyllactosamine (NeuAcα2–6GalNAcβ1–4GlcNAc), which have not been observed in the human respiratory tract surface epithelium. The presence of the Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in the study of influenza virus infection.

Toll-like receptors (TLRs) play an important role in innate immunity to virus infections. We investigated the role of TLR3 in the pathogenesis of H5N1 and pandemic H1N1 (pH1N1) influenza virus infections in mice. Wild-type mice and those defective in TLR3 were infected with influenza A/HK/486/97 (H5N1) or A/HK/415742/09 (pH1N1) virus. For comparison, mice defective in the gene for myeloid differential factor 88 (MyD88) were also infected with the viruses, because MyD88 signals through a TLR pathway different from TLR3. Survival and body weight loss were monitored for 14 days, and lung pathology, the lung immune-cell profile, viral load and cytokine responses were studied. H5N1-infected TLR3−/− mice had better survival than H5N1-infected WT mice, evident by significantly faster regain of body weight, lower viral titre in the lung and fewer pathological changes in the lung. However, this improved survival was not seen upon pH1N1 infection of TLR3−/− mice. In contrast, MyD88−/− mice had an increased viral titre and decreased leukocyte infiltration in the lungs after infection with H5N1 virus and poorer survival after pH1N1 infection. In conclusion, TLR3 worsens the pathogenesis of H5N1 infection but not of pH1N1 infection, highlighting the differences in the pathogenesis of these two viruses and the different roles of TLR3 in their pathogenesis.

Human disease caused by highly pathogenic avian influenza A (HPAI) (H5N1) is associated with fulminant viral pneumonia and mortality rates in excess of 60%. Acute respiratory syndrome (ARDS) has been found to be the most severe form of acute lung injury caused by H5N1 virus infection while cytokine dysregulation and viral replication are thought to contribute to its pathogenesis. In this study, the antiviral and anti-inflammatory effects of two indirubin derivatives: indirubin-3′-oxime (IM) and E804 on primary human peripherial blood-derived macrophages and type-I like pneumocytes (human alveolar epithelial cells) during influenza A (H5N1) virus infection were investigated. We found that both of the indirubin derivatives strongly suppress the pro-inflammatory cytokines including IP-10 (CXCL10), one of the key factors which contribute to the lung inflammation during H5N1 virus infection. In addition, we also demonstrated that the indirubin derivative delays the virus replication in the primary cell culture models. Our results showed that indirubin derivatives have a potential to be used as an adjunct to antiviral therapy for the treatment of severe human H5N1 disease.

Summary Background Since March, 2013, an avian-origin influenza A H7N9 virus has caused severe pneumonia in China. The aim of this study was to investigate the pathogenesis of this new virus in human beings. Methods We obtained ex-vivo cultures of the human bronchus, lung, nasopharynx, and tonsil and in-vitro cultures of primary human alveolar epithelial cells and peripheral blood monocyte-derived macrophages. We compared virus tropism and induction of proinflammatory cytokine responses of two human influenza A H7N9 virus isolates, A/Shanghai/1/2013 and A/Shanghai/2/2013; a highly pathogenic avian influenza H5N1 virus; the highly pathogenic avian influenza H7N7 virus that infected human beings in the Netherlands in 2003; the 2009 pandemic influenza H1N1 virus, and a low pathogenic duck H7N9 virus that was genetically different to the human disease causing A H7N9 viruses. Findings Both human H7N9 viruses replicated efficiently in human bronchus and lung ex-vivo cultures, whereas duck/H7N9 virus failed to replicate in either. Both human A H7N9 viruses infected both ciliated and non-ciliated human bronchial epithelial cells and replicated to higher titres than did H5N1 (p

Objectives Swine have been regarded as intermediate hosts in the spread of influenza from birds to humans but studies of the sialylated glycans that comprise their respiratory tract have not been extensively studied in the past. This study analyzed the sialylated N-glycan and O-glycan profile of swine trachea and lung and correlated this with ex-vivo infection of swine explants with avian influenza viruses. Sample Lungs and tracheal samples were obtained from normal farm and laboratory raised swine and used for ex vivo infection as well as mass spectrometric analysis. Infection of the ex vivo tissues used high pathogenic and low pathogenic avian viruses including the novel H7N9 virus that emerged in China in early 2013. Main outcome measures Assessment of successful replication was determined by TCID50 as well as virus immunohistochemistry. The N-glycan and O-glycan profiles were measured by MALDI-TOF and sialylated linkages were determined by sialidase treatment. Lectin binding histochemistry was also performed on formalin fixed tissue samples with positive binding detected by chromogen staining. Results The swine respiratory tract glycans differed from the human respiratory tact glycans in two main areas. There was a greater abundance of Gal-α-Gal linkages resulting in a relative decrease in sialylated glycans. The swine respiratory tract also had a greater proportion of glycans containing Neu5Gc and Siaα2-6 glycans than the human respiratory tract. Infection with avian viruses was confined primarily to lung bronchioles rather than trachea and parenchyma. Conclusions In contrast to previous studies we found that there was not as much expression of Siaα2-3 glycans on the surface of the trachea. Infection of Siaα2-3 binding avian viruses was restricted to the lower respiratory tract bronchioles. This finding may diminish the ability of the swine to act as an intermediary in the transmission of avian viruses to humans.

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.

International audience; Tissues infected with highly pathogenic avian influenza viruses such as H5N1 and H7N7 are normally required to be fixed in formalin or paraformaldehyde before examination in order to inactivate the virus. In this study commercially available monoclonal antibodies to the influenza nucleoprotein (NP) were evaluated in order to determine which antibodies would identify positive cells in tissues fixed in formalin or paraformaldehyde. An assessment of which antigen retrieval process would unmask antigens blocked by formalin fixation was also made. Of six commercially available monoclonal antibodies tested, only one (HB65, European Veterinary Laboratories) was able to identify all formalin fixed avian, swine and human influenza virus infected tissues, and this was after pronase induced epitope retrieval. This monoclonal antibody is recommended for routine diagnostic use for the detection of influenza A infected tissues that have been fixed in formalin or paraformaldehyde.

Objectives The influenza virus (IFV) infection models commonly used to evaluate antiviral agents (e.g. MDCK cell line and mice) are limited by physiological differences from the human respiratory tract in vivo. Here we report the pharmacodynamics of DAS181, a sialidase fusion protein that inhibits influenza infection, in the model systems of well-defined human airway epithelium (HAE) culture and ex vivo culture of fresh human bronchial tissue, both of which are close mimics of the human respiratory tract in vivo. Methods HAE culture and ex vivo human bronchi were used to evaluate the sialic acid removal and regeneration efficiency and IFV inhibition after various DAS181 treatment levels and regimens. Results DAS181 effectively desialylates HAE cultures and ex vivo bronchi tissues and therefore potently inhibits replication of different IFV strains. The treatment effect of DAS181 occurs immediately upon application to the epithelial surface and is unaffected by the respiratory mucus. In both HAE and human bronchial tissue, the inhibitory effect of DAS181 treatment lasts for at least 2 days. Approximately 80% epithelial surface desialylation and significant anti-IFV efficacy can be achieved at topical concentrations of DAS181 in the range of 5-10 microg/cm(2) when applied once daily. An additional treatment or a higher loading dose of DAS181 on the first day provides significant additional treatment benefit. Comparing the effect of DAS181 versus its two analogues, DAS180 and DAS185, has confirmed that sialidase function is critical for DAS181, and the cell-binding domain (amphiregulin tag) prolongs DAS181 retention and potentiates its function. Conclusions These results provide valuable insights into DAS181 treatment dose and potential regimens in the clinical setting.

Sialic acids (Sias) are regarded as receptors for influenza viruses and are usually bound to galactose (Gal) in an alpha2-3 or alpha2-6 configuration. The detection of these Sia configurations in tissues has commonly been through the use of plant lectins that are able to identify which cells contain Siaalpha2-3- and Siaalpha2-6-linked glycans, although other techniques for receptor distribution have been used. Initial experiments indicated that avian versus human influenza virus binding was determined by either Siaalpha2-6 or Siaalpha2-3 expression. In this review, we suggest that the distribution and detection of these terminal Siaalpha2-3- and Siaalpha2-6-linked receptors within the respiratory tract might not be as clear cut as has been reported. We will also review how other viral and receptor components might act as determinants for successful viral replication and transmission. Understanding these additional components is important in comprehending the infection and the transmission of both existing human influenza viruses and newly emerging avian influenza viruses.

Limited antiviral compounds are available for the control of influenza, and the emergence of resistant variants would further narrow the options for defense. The H275Y neuraminidase (NA) mutation, which confers resistance to oseltamivir carboxylate, has been identified among the seasonal H1N1 and 2009 pandemic influenza viruses; however, those H275Y resistant variants demonstrated distinct epidemiological outcomes in humans. Specifically, dominance of the H275Y variant over the oseltamivir-sensitive viruses was only reported for a seasonal H1N1 variant during 2008-2009. Here, we systematically analyze the effect of the H275Y NA mutation on viral fitness and transmissibility of A(H1N1)pdm09 and seasonal H1N1 influenza viruses. The NA genes from A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal), and A/Brisbane/59/2007 (Brisbane) were individually introduced into the genetic background of CA04. The H275Y mutation led to reduced NA enzyme activity, an increased K(m) for 3'-sialylactose or 6'-sialylactose, and decreased infectivity in mucin-secreting human airway epithelial cells compared to the oseltamivir-sensitive wild-type counterparts. Attenuated pathogenicity in both RG-CA04(NA-H275Y) and RG-CA04 x Brisbane(NA-H275Y) viruses was observed in ferrets compared to RG-CA04 virus, although the transmissibility was minimally affected. In parallel experiments using recombinant Brisbane viruses differing by hemagglutinin and NA, comparable direct contact and respiratory droplet transmissibilities were observed among RG-NewCal(HA,NA), RG-NewCal(HA,NA-H275Y), RG-Brisbane(HA,NA-H275Y), and RG-NewCal(HA) x Brisbane(NA-H275Y) viruses. Our results demonstrate that, despite the H275Y mutation leading to a minor reduction in viral fitness, the transmission potentials of three different antigenic strains carrying this mutation were comparable in the naive ferret model., published_or_final_version

The 2009 pandemic influenza H1N1 (H1N1pdm) virus was generated by reassortment of swine influenza viruses of different lineages. This was the first influenza pandemic to emerge in over 4 decades and the first to occur after the realization that influenza pandemics arise from influenza viruses of animals. In order to understand the biological determinants of pandemic emergence, it is relevant to compare the tropism of different lineages of swine influenza viruses and reassortants derived from them with that of 2009 pandemic H1N1 (H1N1pdm) and seasonal influenza H1N1 viruses in ex vivo cultures of the human nasopharynx, bronchus, alveoli, and conjunctiva. We hypothesized that virus which can transmit efficiently between humans replicated well in the human upper airways. As previously reported, H1N1pdm and seasonal H1N1 viruses replicated efficiently in the nasopharyngeal, bronchial, and alveolar epithelium. In contrast, representative viruses from the classical swine (CS) (H1N1) lineage could not infect human respiratory epithelium; Eurasian avian-like swine (EA) (H1N1) viruses only infected alveolar epithelium and North American triple-reassortant (TRIG) viruses only infected the bronchial epithelium albeit inefficiently. Interestingly, a naturally occurring triple-reassortant swine virus, A/SW/HK/915/04 (H1N2), with a matrix gene segment of EA swine derivation (i.e., differing from H1N1pdm only in lacking a neuraminidase [NA] gene of EA derivation) readily infected and replicated in human nasopharyngeal and bronchial epithelia but not in the lung. A recombinant sw915 with the NA from H1N1pdm retained its tropism for the bronchus and acquired additional replication competence for alveolar epithelium. In contrast to H1N1pdm, none of the swine viruses tested nor seasonal H1N1 had tropism in human conjunctiva. Recombinant viruses generated by swapping the surface proteins (hemagglutinin and NA) of H1N1pdm and seasonal H1N1 virus demonstrated that these two gene segments together are key determinants of conjunctival tropism. Overall, these findings suggest that ex vivo cultures of the human respiratory tract provide a useful biological model for assessing the human health risk of swine influenza viruses. © 2011, American Society for Microbiology., link_to_OA_fulltext

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility., link_to_OA_fulltext

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1 virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocytederived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease. © 2011, American Society for Microbiology., link_to_OA_fulltext

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections. © 2010 Jia et al., published_or_final_version

The novel pandemic influenza H1N1 (H1N1pdm) virus of swine origin causes mild disease but occasionally leads to acute respiratory distress syndrome and death. It is important to understand the pathogenesis of this new disease in humans. We compared the virus tropism and host-responses elicited by pandemic H1N1pdm and seasonal H1N1 influenza viruses in ex vivo cultures of human conjunctiva, nasopharynx, bronchus, and lung, as well as in vitro cultures of human nasopharyngeal, bronchial, and alveolar epithelial cells. We found comparable replication and host-responses in seasonal and pandemic H1N1 viruses. However, pandemic H1N1pdm virus differs from seasonal H1N1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in its receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection with H1N1pdm. A greater viral replication competence in bronchial epithelium at 33°C may also contribute to the slight increase in virulence of the pandemic influenza virus. In contrast with highly pathogenic influenza H5N1 virus, pandemic H1N1pdm does not differ from seasonal influenza virus in its intrinsic capacity for cytokine dysregulation. Collectively, these results suggest that pandemic H1N1pdm virus differs in modest but subtle ways from seasonal H1N1 virus in its intrinsic virulence for humans, which is in accord with the epidemiology of the pandemic to date. These findings are therefore relevant for understanding transmission and therapy. Copyright © American Society for Investigative Pathology., link_to_OA_fulltext

Background: The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase)™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses. Methods and Findings: The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus. Conclusions: The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV., published_or_final_version

DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both α2-6-linked and α2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1. Copyright © 2009, American Society for Microbiology., link_to_OA_fulltext

Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern. © 2009 Li et al., published_or_final_version

The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses., Author Summary This study was performed to determine what possible glycan receptors for influenza were present in the human respiratory tract. We compared the glycans present on existing published glycan arrays with the actual glycans identified in the human respiratory tract by mass spectrometric analysis to determine how representative these arrays would be for potential binding. The most comprehensive array to date only contained approximately half the range of the actual glycans present. Over the past 5 years we have performed ex-vivo infection of 113 bronchial and 185 lung samples with seasonal, avian and swine influenza viruses, and have demonstrated that the lung is able to be infected by all types of influenza viruses but that the bronchus can also be infected by a limited range of avian, swine and seasonal viruses. The key findings are that there is wide spectrum of glycans present in the respiratory tract which can be used by influenza viruses for infection, and the currently available arrays are not predictive of successful infection. Our findings will be of use for researchers in developing more comprehensive and focused arrays for the screening of emerging influenza viruses and bacteria in order to determine their potential threat to humans.

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection., IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Background: Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-α) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells. Methods: We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro. Results: We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus. Conclusion: The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease. © 2005 Chan et al., licensee BioMed Central Ltd., published_or_final_version