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.
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.
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<0.0001 to 0.0046) and A/Shanghai/1/2013 replicated to higher titres than did H7N7 (p=0.0002-0.01). Both human A H7N9 viruses predominantly infected type II alveolar epithelial cells and alveolar macrophages in the human lung and replicated to higher titres than did H5N1 (p<0.0001 to 0.0078); A/Shanghai/1/2013 replicated to higher titres than did H1N1 (p=0.0052-0.05) and H7N7 (p=0.0031-0.0151). Human H7N9 viruses were less potent inducers of proinflammatory cytokines compared with H5N1 virus.
Collectively, the results suggest that the novel H7N9 viruses are better adapted to infect and replicate in the human conducting and lower airways than are other avian influenza viruses, including H5N1, and pose an important pandemic threat.
Area of Excellence Scheme of the University Grants Committee (AoE/M-12/96), Hong Kong Special Administrative Region.
Abstract
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The authors have declared that no competing interests exist In a global pandemic involving respiratory pathogens such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), intensified ...scientific research is required to delineate pathways involved in infectivity, transmissibility, and pathogenicity of the causative pathogen. ...these cell lines have been used to evaluate drugs and vaccines response 9. Infected hamsters showed mild–moderate symptoms and elevated inflammatory responses that mirror clinical pathologies observed in humans, and severe symptoms manifest when the animals were infected with high viral dosage 20–22. ...hamsters are useful as preclinical models to screen for therapeutic agents; however, the models may be limited by the availability of hamster-specific reagents for assays 23. Nonhuman primates are attractive models due to their close phylogenetic and physiological similarities to humans; however, differences in the clinical course of disease have been reported.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Since their first isolation in 2013, influenza A/H5N6 viruses have spread amongst poultry across multiple provinces in China and to Laos, Vietnam and Myanmar. So far, there have been 14 human H5N6 ...infections with 10 fatalities.We investigated the tropism, replication competence and cytokine induction of one human and two avian H5N6 isolates in
and
cultures derived from the human respiratory tract. Virus tropism and replication were studied in
cultures of human nasopharynx, bronchus and lung. Induction of cytokines and chemokines was measured
in virus-infected primary human alveolar epithelial cells.Human H5N6 virus replicated more efficiently than highly pathogenic avian influenza (HPAI) H5N1 virus and as efficiently as H1N1pdm in
human bronchus and lung and was also able to replicate in
cultures of human nasopharynx. Avian H5N6 viruses replicated less efficiently than H1N1pdm in human bronchial tissues and to similar titres as HPAI H5N1 in the lung. While the human H5N6 virus had affinity for avian-like receptors, the two avian isolates had binding affinity for both avian- and human-like receptors. All three H5N6 viruses were less potent inducers of pro-inflammatory cytokines compared with H5N1 virus.Human H5N6 virus appears better adapted to infect the human airways than H5N1 virus and may pose a significant public health threat.
Despite causing regular seasonal epidemics with substantial morbidity, mortality and socioeconomic burden, there is still a lack of research into influenza B viruses (IBVs). In this study, we provide ...for the first time a systematic investigation on the tropism, replication kinetics and pathogenesis of IBVs in the human respiratory tract.Physiologically relevant
explant cultures of human bronchus and lung, human airway organoids, and
cultures of differentiated primary human bronchial epithelial cells and type-I-like alveolar epithelial cells were used to study the cellular and tissue tropism, replication competence and induced innate immune response of 16 IBV strains isolated from 1940 to 2012 in comparison with human seasonal influenza A viruses (IAVs), H1N1 and H3N2. IBVs from the diverged Yamagata- and Victoria-like lineages and the earlier undiverged period were included.The majority of IBVs replicated productively in human bronchus and lung with similar competence to seasonal IAVs. IBVs infected a variety of cell types, including ciliated cells, club cells, goblet cells and basal cells, in human airway organoids. Like seasonal IAVs, IBVs are low inducers of pro-inflammatory cytokines and chemokines. Most results suggested a higher preference for the conducting airway than the lower lung and strain-specific rather than lineage-specific pathogenicity of IBVs.Our results highlighted the non-negligible virulence of IBVs which require more attention and further investigation to alleviate the disease burden, especially when treatment options are limited.
Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the ...individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.
Allergic asthma, driven by T helper 2 cell-mediated immune responses to common environmental antigens, remains the most common respiratory disease in children. Perfluorinated chemicals (PFCs) are ...environmental contaminants of great concern, because of their wide application, persistence in the environment, and bioaccumulation. PFCs associate with immunological disorders including asthma and attenuate immune responses to vaccines. The influence of PFCs on the immunological response to allergens during childhood is unknown. We report here that a major PFC, perfluorooctane sulfonate (PFOS), inactivates house dust mite (HDM) to dampen 5-wk-old, early weaned mice from developing HDM-induced allergic asthma. PFOS further attenuates the asthma protective effect of the microbial product lipopolysaccharide (LPS). We demonstrate that PFOS prevents desensitization of lung epithelia by LPS, thus abolishing the latter's protective effect. A close mechanistic study reveals that PFOS specifically binds the major HDM allergen Der p1 with high affinity as well as the lipid A moiety of LPS, leading to the inactivation of both antigens. Moreover, PFOS at physiological human (nanomolar) concentrations inactivates Der p1 from HDM and LPS in vitro, although higher doses did not cause further inactivation because of possible formation of PFOS aggregates. This PFOS-induced neutralization of LPS has been further validated in primary human cell models and extended to an in vivo bacterial infection mouse model. This study demonstrates that early life exposure of mice to a PFC blunts airway antigen bioactivity to modulate pulmonary inflammatory responses, which may adversely affect early pulmonary health.
Prior studies illustrate the presence and clinical importance of detecting
species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of ...PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of
in specimens from the human airway. Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway
when compared to standard qPCR.
The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in
= 20 sputum specimens obtained from non-diseased (
= 4), chronic obstructive pulmonary disease (COPD;
= 8) and non-cystic fibrosis bronchiectasis (
= 8) patients where
status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed.
Standard qPCR and ddPCR were both able to detect, even at low abundance,
species (
and
) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of
particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR.
ddPCR has greater sensitivity for
detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of
species in chronic respiratory disease states such as bronchiectasis.
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