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1.
Hum Genomics ; 15(1): 18, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726831

RESUMO

BACKGROUND: In the novel coronavirus pandemic, the high infection rate and high mortality have seriously affected people's health and social order. To better explore the infection mechanism and treatment, the three-dimensional structure of human bronchus has been employed in a better in-depth study on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We downloaded a separate microarray from the Integrated Gene Expression System (GEO) on a human bronchial organoids sample to identify differentially expressed genes (DEGS) and analyzed it with R software. After processing with R software, Gene Ontology (GO) and Kyoto PBMCs of Genes and Genomes (KEGG) were analyzed, while a protein-protein interaction (PPI) network was constructed to show the interactions and influence relationships between these differential genes. Finally, the selected highly connected genes, which are called hub genes, were verified in CytoHubba plug-in. RESULTS: In this study, a total of 966 differentially expressed genes, including 490 upregulated genes and 476 downregulated genes were used. Analysis of GO and KEGG revealed that these differentially expressed genes were significantly enriched in pathways related to immune response and cytokines. We construct protein-protein interaction network and identify 10 hub genes, including IL6, MMP9, IL1B, CXCL8, ICAM1, FGF2, EGF, CXCL10, CCL2, CCL5, CXCL1, and FN1. Finally, with the help of GSE150728, we verified that CXCl1, CXCL8, CXCL10, CCL5, EGF differently expressed before and after SARS-CoV-2 infection in clinical patients. CONCLUSIONS: In this study, we used mRNA expression data from GSE150819 to preliminarily confirm the feasibility of hBO as an in vitro model to further study the pathogenesis and potential treatment of COVID-19. Moreover, based on the mRNA differentiated expression of this model, we found that CXCL8, CXCL10, and EGF are hub genes in the process of SARS-COV-2 infection, and we emphasized their key roles in SARS-CoV-2 infection. And we also suggested that further study of these hub genes may be beneficial to treatment, prognostic prediction of COVID-19.


Assuntos
Brônquios/virologia , Regulação da Expressão Gênica , Brônquios/fisiologia , Quimiocina CXCL10/genética , Fator de Crescimento Epidérmico/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Interleucina-8/genética , Organoides , Mapas de Interação de Proteínas/genética , Software
2.
Nature ; 592(7852): 122-127, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636719

RESUMO

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.


Assuntos
/transmissão , Mutação , /fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , /genética , Animais , Brônquios/citologia , Brônquios/virologia , Linhagem Celular , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Aptidão Genética , Humanos , Masculino , Mesocricetus , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Ligação Proteica , RNA Viral/análise , /metabolismo , /patogenicidade
3.
PLoS Comput Biol ; 17(2): e1008685, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33534793

RESUMO

The SARS-CoV-2 pandemic of 2020 has mobilised scientists around the globe to research all aspects of the coronavirus virus and its infection. For fruitful and rapid investigation of viral pathomechanisms, a collaborative and interdisciplinary approach is required. Therefore, we have developed ViralLink: a systems biology workflow which reconstructs and analyses networks representing the effect of viruses on intracellular signalling. These networks trace the flow of signal from intracellular viral proteins through their human binding proteins and downstream signalling pathways, ending with transcription factors regulating genes differentially expressed upon viral exposure. In this way, the workflow provides a mechanistic insight from previously identified knowledge of virally infected cells. By default, the workflow is set up to analyse the intracellular effects of SARS-CoV-2, requiring only transcriptomics counts data as input from the user: thus, encouraging and enabling rapid multidisciplinary research. However, the wide-ranging applicability and modularity of the workflow facilitates customisation of viral context, a priori interactions and analysis methods. Through a case study of SARS-CoV-2 infected bronchial/tracheal epithelial cells, we evidence the functionality of the workflow and its ability to identify key pathways and proteins in the cellular response to infection. The application of ViralLink to different viral infections in a context specific manner using different available transcriptomics datasets will uncover key mechanisms in viral pathogenesis.


Assuntos
/metabolismo , Biologia Computacional/métodos , Regulação Viral da Expressão Gênica , Transdução de Sinais , Algoritmos , Brônquios/virologia , Análise por Conglomerados , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Pesquisa Interdisciplinar , Pulmão/virologia , Modelos Estatísticos , Biologia de Sistemas , Transcriptoma , Fluxo de Trabalho
4.
Antiviral Res ; 187: 105015, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33444702

RESUMO

The newly emerged severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) coronavirus initiated a pneumonia outbreak (COVID-19) that rapidly spread worldwide and quickly became a public health emergency of international concern; However to date, except Remdesivir, there are no clinically approved specific or effective medicines to prevent or treat COVID-19. Therefore, the development of novel treatments against coronavirus infections caused by the current SARS-CoV-2 virus, as well as other highly pathogenic human coronaviruses, represents an urgent unmet need. Stimulator of interferon genes (STING) plays a central role in host defense mechanisms against microbial infections. STING activation leads to the induction of both type I interferon and autophagy responses, which elicit strong inhibitory effect against the infections caused by a broad range of microbial pathogens. However, whether STING activation can impact infections from SARS-CoV-2 or other coronaviruses remains largely unknown. In this study, we investigated the anti-coronavirus activity triggered by STING activation. We discovered that dimeric amidobenzimidazole (diABZI), a synthetic small molecule STING receptor agonist, showed potent anti-coronavirus activity against both the common cold human coronavirus 229E (HCoV-229E) and SARS-CoV-2 in cell culture systems. In addition, we demonstrated that the antiviral activity of diABZI was dependent on the interferon pathway in HCoV-229E infected normal human fibroblast lung cells (MRC-5) and reconstituted primary human airway air-liquid interface (ALI) cultures. Furthermore, low-dose of diABZI treatment at 0.1 µM effectively reduced the SARS-CoV-2 viral load at the epithelial apical surface and prevented epithelial damage in the reconstituted primary human bronchial airway epithelial ALI system. Our findings have thus revealed the therapeutic potential of STING agonists, such as diABZI, as treatments for SARS-CoV-2 and other human coronavirus infections.


Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Proteínas de Membrana/agonistas , /efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Antivirais/química , Autofagia/efeitos dos fármacos , Brônquios/virologia , Linhagem Celular , Infecções por Coronavirus/virologia , Células Epiteliais/virologia , Humanos , Interferon Tipo I/farmacologia , Pulmão/virologia , Replicação Viral
5.
Antiviral Res ; 186: 105012, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33422611

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a severe respiratory disease with varying clinical presentations and outcomes, and responsible for a major pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against multiple RNA viruses including coronaviruses. Specifically, rocaglates inhibit eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. Here, we assessed the antiviral activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In primary human airway epithelial cells, CR-31-B (-) reduced viral titers to undetectable levels at a concentration of 100 nM. Reduced virus reproduction was accompanied by substantially reduced viral protein accumulation and replication/transcription complex formation. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.


Assuntos
Antivirais/farmacologia , Benzofuranos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Benzofuranos/química , Brônquios/virologia , Células Cultivadas , Chlorocebus aethiops , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Humanos , Ácidos Hidroxâmicos/química , Mucosa Respiratória/virologia , /fisiologia , Células Vero , Carga Viral/efeitos dos fármacos , /efeitos dos fármacos
6.
Viruses ; 12(11)2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33114247

RESUMO

Porcine respiratory coronavirus (PRCoV) infects the epithelial cells in the respiratory tract of pigs, causing a mild respiratory disease. We applied air-liquid interface (ALI) cultures of well-differentiated porcine airway cells to mimic the respiratory tract epithelium in vitro and use it for analyzing the infection by PRCoV. As reported for most coronaviruses, virus entry and virus release occurred mainly via the apical membrane domain. A novel finding was that PRCoV preferentially targets non-ciliated and among them the non-mucus-producing cells. Aminopeptidase N (APN), the cellular receptor for PRCoV was also more abundantly expressed on this type of cell suggesting that APN is a determinant of the cell tropism. Interestingly, differentiation-dependent differences were found both in the expression of pAPN and the susceptibility to PRCoV infection. Cells in an early differentiation stage express higher levels of pAPN and are more susceptible to infection by PRCoV than are well-differentiated cells. A difference in the susceptibility to infection was also detected when tracheal and bronchial cells were compared. The increased susceptibility to infection of bronchial epithelial cells was, however, not due to an increased abundance of APN on the cell surface. Our data reveal a complex pattern of infection in porcine differentiated airway epithelial cells that could not be elucidated with immortalized cell lines. The results are expected to have relevance also for the analysis of other respiratory viruses.


Assuntos
Antígenos CD13/metabolismo , Células Epiteliais/metabolismo , Coronavirus Respiratório Porcino/fisiologia , Receptores Virais/metabolismo , Mucosa Respiratória/virologia , Tropismo Viral , Animais , Brônquios/metabolismo , Brônquios/virologia , Diferenciação Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/virologia , Suínos , Traqueia/metabolismo , Traqueia/virologia , Internalização do Vírus , Liberação de Vírus , Replicação Viral
7.
Am J Physiol Lung Cell Mol Physiol ; 319(3): L481-L496, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32640839

RESUMO

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide. While most develop a mild, self-limiting illness, some develop severe acute lower respiratory infection and persistent airway disease. Exposure to ambient particulate matter has been linked to asthma, bronchitis, and viral infection in multiple epidemiological studies. We hypothesized that coexposure to nanoparticles worsens RSV-induced airway epithelial barrier dysfunction. Bronchial epithelial cells were incubated with titanium dioxide nanoparticles (TiO2-NP) or a combination of TiO2-NP and RSV. Structure and function of epithelial cell barrier were analyzed. Viral titer and the role of reactive oxygen species (ROS) generation were evaluated. In vivo, mice were intranasally incubated with TiO2-NP, RSV, or a combination. Lungs and bronchoalveolar lavage (BAL) fluid were harvested for analysis of airway inflammation and apical junctional complex (AJC) disruption. RSV-induced AJC disruption was amplified by TiO2-NP. Nanoparticle exposure increased viral infection in epithelial cells. TiO2-NP induced generation of ROS, and pretreatment with antioxidant, N-acetylcysteine, reversed said barrier dysfunction. In vivo, RSV-induced injury and AJC disruption were augmented in the lungs of mice given TiO2-NP. Airway inflammation was exacerbated, as evidenced by increased white blood cell infiltration into the BAL, along with exaggeration of peribronchial inflammation and AJC disruption. These data demonstrate that TiO2-NP exposure exacerbates RSV-induced AJC dysfunction and increases inflammation by mechanisms involving generation of ROS. Further studies are required to determine whether NP exposure plays a role in the health disparities of asthma and other lung diseases, and why some children experience more severe airway disease with RSV infection.


Assuntos
Células Epiteliais/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/patogenicidade , Infecções Respiratórias/tratamento farmacológico , Titânio/farmacologia , Animais , Asma/tratamento farmacológico , Asma/etiologia , Brônquios/efeitos dos fármacos , Brônquios/virologia , Líquido da Lavagem Broncoalveolar/citologia , Células Epiteliais/virologia , Inflamação/complicações , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Vírus Sinciciais Respiratórios/efeitos dos fármacos
8.
Viruses ; 12(6)2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32599823

RESUMO

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host-pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/epidemiologia , Junções Intercelulares , Células Madin Darby de Rim Canino , Modelos Biológicos , Mucina-5AC/metabolismo , Pandemias , Cultura de Vírus
9.
BMC Infect Dis ; 20(1): 536, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703188

RESUMO

BACKGROUND: As the COVID-19 pandemic continues to spread, early, ideally real-time, identification of SARS-CoV-2 infected individuals is pivotal in interrupting infection chains. Volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. METHODS: Eight detection dogs were trained for 1 week to detect saliva or tracheobronchial secretions of SARS-CoV-2 infected patients in a randomised, double-blinded and controlled study. RESULTS: The dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of 82.63% (95% confidence interval [CI]: 82.02-83.24%) and specificity of 96.35% (95% CI: 96.31-96.39%). During the presentation of 1012 randomised samples, the dogs achieved an overall average detection rate of 94% (±3.4%) with 157 correct indications of positive, 792 correct rejections of negative, 33 incorrect indications of negative or incorrect rejections of 30 positive sample presentations. CONCLUSIONS: These preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased SARS-CoV-2 infected individuals by discriminating between samples from SARS-CoV-2 infected patients and negative controls. This data may form the basis for the reliable screening method of SARS-CoV-2 infected people.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Programas de Rastreamento/métodos , Odorantes/análise , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Animais , Brônquios/química , Brônquios/virologia , Estudos de Casos e Controles , Cães , Método Duplo-Cego , Humanos , Pandemias/prevenção & controle , Projetos Piloto , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
10.
Viruses ; 12(7)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32698440

RESUMO

The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects almost everyone in the world in many ways. We previously predicted antivirals (atazanavir, remdesivir and lopinavir/ritonavir) and non-antiviral drugs (tiotropium and rapamycin) that may inhibit the replication complex of SARS-CoV-2 using our molecular transformer-drug target interaction (MT-DTI) deep-learning-based drug-target affinity prediction model. In this study, we dissected molecular pathways upregulated in SARS-CoV-2-infected normal human bronchial epithelial (NHBE) cells by analyzing an RNA-seq data set with various bioinformatics approaches, such as gene ontology, protein-protein interaction-based network and gene set enrichment analyses. The results indicated that the SARS-CoV-2 infection strongly activates TNF and NFκB-signaling pathways through significant upregulation of the TNF, IL1B, IL6, IL8, NFKB1, NFKB2 and RELB genes. In addition to these pathways, lung fibrosis, keratinization/cornification, rheumatoid arthritis, and negative regulation of interferon-gamma production pathways were also significantly upregulated. We observed that these pathologic features of SARS-CoV-2 are similar to those observed in patients with chronic obstructive pulmonary disease (COPD). Intriguingly, tiotropium, as predicted by MT-DTI, is currently used as a therapeutic intervention in COPD patients. Treatment with tiotropium has been shown to improve pulmonary function by alleviating airway inflammation. Accordingly, a literature search summarized that tiotropium reduced expressions of IL1B, IL6, IL8, RELA, NFKB1 and TNF in vitro or in vivo, and many of them have been known to be deregulated in COPD patients. These results suggest that COVID-19 is similar to an acute mode of COPD caused by the SARS-CoV-2 infection, and therefore tiotropium may be effective for COVID-19 patients.


Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Brometo de Tiotrópio/uso terapêutico , Transcriptoma , Brônquios/virologia , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/patologia , Células Epiteliais/virologia , Humanos , Pandemias , Pneumonia Viral/etiologia , Pneumonia Viral/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Transdução de Sinais/fisiologia
11.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32699094

RESUMO

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Células Epiteliais/imunologia , Interferon Tipo I/imunologia , Interferons/imunologia , Pneumonia Viral/imunologia , Animais , Betacoronavirus/fisiologia , Brônquios/citologia , Brônquios/imunologia , Brônquios/virologia , Linhagem Celular , Células Cultivadas , Quimiocinas/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Citocinas/imunologia , Cães , Células Epiteliais/virologia , Humanos , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Pandemias , Pneumonia Viral/virologia , Células Vero , Replicação Viral
12.
Antiviral Res ; 180: 104860, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32565134

RESUMO

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory in human with high mortality and it has been a challenge to determine optimum treatment for MERS-CoV-induced respiratory infection. Here, we observed the distribution of MERS-CoV receptors using human respiratory mucosa and also evaluated the contribution of interferon-lambdas (IFN-λs) in response to MERS-CoV infection using in vitro normal human nasal epithelial (NHNE) and bronchial epithelial (NHBE) cells. We found that the gene and protein expression of DPPIV, MERS-CoV receptor, were more dominantly located in nasal and bronchial epithelium although human nasal mucosa exhibited relatively lower DPPIV expression than lung parenchymal tissues. The quantitative mRNA level of the MERS-CoV envelope (upE) gene was significantly induced in MERS-CoV-infected cultured NHNE and NHBE cells until 3 days after infection. The induction of IFNs was identified in NHNE and NHBE cells after MERS-CoV infection and IFN-λs were predominantly increased in MERS-CoV-infected respiratory epithelial cells. Inoculation of IFN-λs to NHNE and NHBE cells suppressed MERS-CoV replication and in particular, IFN-λ4 showed a strong therapeutic effect in reducing MERS-CoV infection with higher induction of IFN-stimulated genes. Thus, IFN-λ has a decisive function in the respiratory epithelium that greatly limits MERS-CoV replication, and may be a key cytokine for better therapeutic outcomes against MERS-CoV infection in respiratory tract.


Assuntos
Antivirais/uso terapêutico , Interferons/uso terapêutico , Interleucinas/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Mucosa Respiratória/virologia , Replicação Viral/efeitos dos fármacos , Brônquios/virologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica , Humanos , Imunidade Inata/imunologia , Interferons/biossíntese , Interleucinas/biossíntese , Mucosa Laríngea/virologia , Pulmão/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Reação em Cadeia da Polimerase , Mucosa Respiratória/efeitos dos fármacos
13.
Nature ; 583(7818): 830-833, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32380511

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Pulmão/patologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Transgenes , Animais , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Brônquios/patologia , Brônquios/virologia , Infecções por Coronavirus/imunologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Humanos , Imunoglobulina G/imunologia , Pulmão/imunologia , Pulmão/virologia , Linfócitos/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Masculino , Camundongos , Camundongos Transgênicos , Pandemias , Pneumonia Viral/imunologia , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Replicação Viral , Perda de Peso
14.
Nature ; 583(7818): 834-838, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32408338

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Pulmão/patologia , Pulmão/virologia , Mesocricetus/virologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Aerossóis , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Antígenos Virais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Brônquios/patologia , Brônquios/virologia , Infecções por Coronavirus/imunologia , Duodeno/virologia , Fômites/virologia , Abrigo para Animais , Rim/virologia , Masculino , Mesocricetus/imunologia , Mucosa Nasal/virologia , Pandemias , Pneumonia Viral/imunologia , RNA Viral/análise , Carga Viral , Perda de Peso
15.
mBio ; 11(1)2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937643

RESUMO

Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection.IMPORTANCE Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species in vitro This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.


Assuntos
Vírus Defeituosos/genética , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/imunologia , Células A549 , Brônquios/citologia , Brônquios/virologia , Células Cultivadas , Vírus Defeituosos/imunologia , Genoma Viral , Humanos , Vírus da Influenza A/fisiologia , RNA Viral/genética , Análise de Sequência de RNA , Análise de Célula Única , Replicação Viral
16.
Cytokine ; 126: 154895, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31706200

RESUMO

MERS-CoV, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. Inflammatory responses have a significant impact on MERS-CoV pathogenesis and disease outcome. However, CD4+ T-cell induced immune responses during acute MERS-CoV infection are barely detectable, with potent inhibition of effector T cells and downregulation of antigen presentation. The local pulmonary immune response, particularly the Th1 and Th2-related immune response during acute severe MERS-CoV infection is not fully understood. In this study, we offer the first insights into the pulmonary gene expression profile of Th1 and Th2-related cytokines/chemokines (Th1 & Th2 responses) during acute MERS-CoV infection using RT2 Profiler PCR Arrays. We also quantified the expression level of primary inflammatory cytokines/chemokines. Our results showed a downregulation of Th2, inadequate (partial) Th1 immune response and high expression levels of inflammatory cytokines IL-1α and IL-1ß and the neutrophil chemoattractant chemokine IL-8 (CXCL8) in the lower respiratory tract of MERS-CoV infected patients. Moreover, we identified a high viral load in all included patients. We also observed a correlation between inflammatory cytokines, Th1, and Th2 downregulation and the case fatality rate. Th1 and Th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ARDS, and a higher case fatality rate. Further study of the molecular mechanisms underlying the Th1 and Th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Citocinas/sangue , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/imunologia , Brônquios/patologia , Brônquios/virologia , Infecções por Coronavirus/mortalidade , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamação/genética , Inflamação/imunologia , Masculino , Pessoa de Meia-Idade , Carga Viral , Replicação Viral/imunologia
17.
Biomed Pharmacother ; 121: 109652, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31734578

RESUMO

BACKGROUND: The aim of the present study was to investigate the synergistic effects and interactive mechanisms of Shufeng Jiedu Capsule (SFJDC) combined with oseltamivir in the treatment of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) induced by the influenza A virus (IAV). METHODS: The extraction of SFJDC was analyzed by UHPLC/ESI Q-Orbitrap Mass Spectrometry. Human bronchial epithelial cells were isolated from COPD (DHBE) bronchial tissues, co-cultured with IAV for 24 h, and were subsequently treated with SFJDC and/or oseltamivir. Cell viability was detected by MTT assay. A rat model of COPD with IAV infection was established and treated with SFJDC and/or oseltamivir. Interleukin (IL)-1ß and IL-18 in serum and bronchoalveolar lavage fluid (BALF) were measured by ELISA. Additionally, mRNA and protein levels of NLRP3 inflammasome pathway were measured by quantitative real-time PCR and Western blotting, respectively. RESULTS: SFJDC and/or oseltamivir, at their optimal concentrations, had no significant cytotoxicity against DHBEs. The levels of NLRP3-inflammasome-associated components were significantly elevated after cells were inoculated with IAV, whereas the mRNA and protein levels of these components were significantly decreased after treatment with SFJDC and/or oseltamivir in vitro. Moreover, in vivo, the combination of SFJDC and oseltamivir improved survival rates, attenuated clinical symptoms, induced weight gain, alleviated lung damage, and significantly reduced IL-1ß and IL-18 levels in serum and BALF, as well as reduced the expression levels of NLRP3-associated components and viral titers in lung homogenates. CONCLUSION: SFJDC combined with oseltamivir treatment significantly attenuated IAV-induced airway inflammation and lung viral titers. Hence, our findings may provide a novel therapeutic strategy for IAV-induced respiratory infection.


Assuntos
Antivirais/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Oseltamivir/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/virologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/virologia , Animais , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Brônquios/virologia , Líquido da Lavagem Broncoalveolar/virologia , Linhagem Celular , Técnicas de Cocultura/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/virologia , Influenza Humana/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Carga Viral/efeitos dos fármacos
18.
Sci Rep ; 9(1): 14736, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31611566

RESUMO

Bovine Respiratory Disease (BRD) is the leading cause of mortality in calves. The objective of this study was to examine the response of the host's bronchial lymph node transcriptome to Bovine Respiratory Syncytial Virus (BRSV) in a controlled viral challenge. Holstein-Friesian calves were either inoculated with virus (103.5 TCID50/ml × 15 ml) (n = 12) or mock challenged with phosphate buffered saline (n = 6). Clinical signs were scored daily and blood was collected for haematology counts, until euthanasia at day 7 post-challenge. RNA was extracted and sequenced (75 bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and differential gene expression analysis was performed using EdgeR. There was a clear separation between BRSV challenged and control calves based on gene expression changes, despite an observed mild clinical manifestation of the disease. Therefore, measuring host gene expression levels may be beneficial for the diagnosis of subclinical BRD. There were 934 differentially expressed genes (DEG) (p < 0.05, FDR <0.1, fold change >2) between the BRSV challenged and control calves. Over-represented gene ontology terms, pathways and molecular functions, among the DEG, were associated with immune responses. The top enriched pathways included interferon signaling, granzyme B signaling and pathogen pattern recognition receptors, which are responsible for the cytotoxic responses necessary to eliminate the virus.


Assuntos
Doenças dos Bovinos/genética , Bovinos/virologia , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/fisiologia , Transcriptoma , Animais , Brônquios/metabolismo , Brônquios/virologia , Bovinos/genética , Doenças dos Bovinos/virologia , Interações Hospedeiro-Patógeno , Linfonodos/metabolismo , Linfonodos/virologia , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia
19.
Am J Physiol Lung Cell Mol Physiol ; 317(6): L893-L903, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31513433

RESUMO

Patients with frequent exacerbations represent a chronic obstructive pulmonary disease (COPD) subgroup requiring better treatment options. The aim of this study was to determine the innate immune mechanisms that underlie susceptibility to frequent exacerbations in COPD. We measured sputum expression of immune mediators and bacterial loads in samples from patients with COPD at stable state and during virus-associated exacerbations. In vitro immune responses to rhinovirus infection in differentiated primary bronchial epithelial cells (BECs) sampled from patients with COPD were additionally evaluated. Patients were stratified as frequent exacerbators (≥2 exacerbations in the preceding year) or infrequent exacerbators (<2 exacerbations in the preceding year) with comparisons made between these groups. Frequent exacerbators had reduced sputum cell mRNA expression of the antiviral immune mediators type I and III interferons and reduced interferon-stimulated gene (ISG) expression when clinically stable and during virus-associated exacerbation. A role for epithelial cell-intrinsic innate immune dysregulation was identified: induction of interferons and ISGs during in vitro rhinovirus (RV) infection was also impaired in differentiated BECs from frequent exacerbators. Frequent exacerbators additionally had increased sputum bacterial loads at 2 wk following virus-associated exacerbation onset. These data implicate deficient airway innate immunity involving epithelial cells in the increased propensity to exacerbations observed in some patients with COPD. Therapeutic approaches to boost innate antimicrobial immunity in the lung could be a viable strategy for prevention and treatment of frequent exacerbations.


Assuntos
Brônquios/imunologia , Imunidade Inata/imunologia , Infecções por Picornaviridae/complicações , Doença Pulmonar Obstrutiva Crônica/imunologia , Insuficiência Respiratória/complicações , Rhinovirus/imunologia , Escarro/imunologia , Idoso , Brônquios/patologia , Brônquios/virologia , Progressão da Doença , Feminino , Volume Expiratório Forçado , Humanos , Estudos Longitudinais , Medidas de Volume Pulmonar , Masculino , Pessoa de Meia-Idade , Fenótipo , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/virologia , Escarro/virologia
20.
Clin Exp Allergy ; 49(12): 1587-1597, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31400236

RESUMO

BACKGROUND: The temporal in vivo response of epithelial cells to a viral challenge and its association with viral clearance and clinical outcomes has been largely unexplored in asthma. OBJECTIVE: To determine gene expression profiles over time in nasal epithelial cells (NECs) challenged in vivo with rhinovirus-16 (RV16) and compare to nasal symptoms and viral clearance. METHODS: Patients with stable mild to moderate asthma (n = 20) were challenged intranasally with RV16. Nasal brush samples for RNA sequencing were taken 7 days prior to infection and 3, 6 and 14 days post-infection, and blood samples 4 days prior to infection and day 6 post-infection. Viral load was measured in nasal lavage fluid at day 3, 6 and 14. RESULTS: Top differentially (>2.5-fold increase) expressed gene sets in NECs post-RV16 at days 3 and 6, compared with baseline, were interferon alpha and gamma response genes. Patients clearing the virus within 6 days (early resolvers) had a significantly increased interferon response at day 6, whereas those having cleared the virus by day 14 (late resolvers) had significantly increased responses at day 3, 6 and 14. Interestingly, patients not having cleared the virus by day 14 (non-resolvers) had no enhanced interferon responses at any of these days. The daily Cold Symptom Scores (CSS) peaked at days 3 to 5 and correlated positively with interferon response genes at day 3 (R = 0.48), but not at other time-points. Interferon response genes were also enhanced in blood at day 6 after RV16 challenge. CONCLUSION AND CLINICAL RELEVANCE: This study shows that viral load and clearance varies markedly over time in mild to moderate asthma patients exposed to a fixed RV16 dose. The host's nasal interferon response to RV16 at day 3 is associated with upper respiratory tract symptoms. The temporal interferon response in nasal epithelium associates with viral clearance in the nasal compartment.


Assuntos
Asma , Brônquios , Interferon-alfa/imunologia , Interferon gama/imunologia , Mucosa Nasal , Infecções por Picornaviridae , Rhinovirus/imunologia , Adulto , Asma/imunologia , Asma/patologia , Asma/virologia , Brônquios/imunologia , Brônquios/patologia , Brônquios/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Mucosa Nasal/patologia , Mucosa Nasal/virologia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/patologia
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