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1.
J Pathol ; 253(1): 17-30, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991738

RESUMO

Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a target for disease prevention. However, the relationship between ACE2 expression and its clinical implications in SARS-CoV-2 pathogenesis remains unknown. Here, we explored the location and expression of ACE2, and its correlation with gender, age, and cigarette smoke (CS), in a CS-exposed mouse model and 224 non-malignant lung tissues (125 non-smokers, 81 current smokers, and 18 ex-smokers) by immunohistochemistry. Moreover, the correlations of ACE2 with CS-induced oxidative stress-related markers, hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and 4-hydroxynonenal (4-HNE) were investigated. Chromatin immunoprecipitation and luciferase reporter assays identified the cause of ACE2 overexpression in human primary lung epithelial cells. We demonstrated that ACE2 was predominantly overexpressed on the apical surface of bronchial epithelium, while reduced in alveolar epithelium, owing to the dramatically decreased abundance of alveolar type II pneumocytes in CS-exposed mouse lungs. Consistent with this, ACE2 was primarily significantly overexpressed in human bronchial and alveolar epithelial cells in smokers regardless of age or gender. Decreased ACE2 expression was observed in bronchial epithelial cells from ex-smokers compared with current smokers, especially in those who had ceased smoking for more than 10 years. Moreover, ACE2 expression was positively correlated with the levels of HIF-1α, iNOS, and 4-HNE in both mouse and human bronchioles. The results were further validated using a publicly available dataset from The Cancer Genome Atlas (TCGA) and our previous integrated data from Affymetrix U133 Plus 2.0 microarray (AE-meta). Finally, our results showed that HIF-1α transcriptionally upregulates ACE2 expression. Our results indicate that smoking-induced ACE2 overexpression in the apical surface of bronchial epithelial cells provides a route by which SARS-CoV-2 enters host cells, which supports clinical relevance in attenuating the potential transmission risk of COVID-19 in smoking populations by smoking cessation. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Células Epiteliais Alveolares/enzimologia , Brônquios/enzimologia , Células Epiteliais/virologia , Fumar/efeitos adversos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/virologia , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Humanos , Lactente , Pulmão/metabolismo , Pulmão/virologia , Pessoa de Meia-Idade , Adulto Jovem
2.
Nat Commun ; 11(1): 6319, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33298930

RESUMO

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.


Assuntos
Interações entre Hospedeiro e Microrganismos , Interferons/metabolismo , Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Aspártico Endopeptidases/metabolismo , Autopsia , /metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Humanos , Imunidade , Imuno-Histoquímica , Hibridização In Situ , Interferons/genética , Pulmão/patologia , Pulmão/virologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Mucinas/genética , Mucinas/metabolismo , Tensoativos/metabolismo , Transcriptoma , Carga Viral
3.
PLoS Pathog ; 16(12): e1009130, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33284849

RESUMO

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.


Assuntos
/virologia , Mucosa Nasal/virologia , /patogenicidade , Proteínas Virais/genética , Quimiocina CXCL10/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Cinética , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Transcriptoma , Proteínas Virais/imunologia , Replicação Viral/fisiologia
4.
PLoS Pathog ; 16(12): e1009128, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33284859

RESUMO

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.


Assuntos
/imunologia , Interleucina-6/imunologia , Receptores de Angiotensina/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , /metabolismo , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/virologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Interleucina-6/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Transdução de Sinais/fisiologia , Ativação Transcricional
5.
Cell Death Dis ; 11(12): 1042, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293527

RESUMO

COVID-19, caused by SARS-CoV-2, is an acute and rapidly developing pandemic, which leads to a global health crisis. SARS-CoV-2 primarily attacks human alveoli and causes severe lung infection and damage. To better understand the molecular basis of this disease, we sought to characterize the responses of alveolar epithelium and its adjacent microvascular endothelium to viral infection under a co-culture system. SARS-CoV-2 infection caused massive virus replication and dramatic organelles remodeling in alveolar epithelial cells, alone. While, viral infection affected endothelial cells in an indirect manner, which was mediated by infected alveolar epithelium. Proteomics analysis and TEM examinations showed viral infection caused global proteomic modulations and marked ultrastructural changes in both epithelial cells and endothelial cells under the co-culture system. In particular, viral infection elicited global protein changes and structural reorganizations across many sub-cellular compartments in epithelial cells. Among the affected organelles, mitochondrion seems to be a primary target organelle. Besides, according to EM and proteomic results, we identified Daurisoline, a potent autophagy inhibitor, could inhibit virus replication effectively in host cells. Collectively, our study revealed an unrecognized cross-talk between epithelium and endothelium, which contributed to alveolar-capillary injury during SARS-CoV-2 infection. These new findings will expand our understanding of COVID-19 and may also be helpful for targeted drug development.


Assuntos
/patologia , Comunicação Celular/fisiologia , /fisiologia , /metabolismo , Linhagem Celular , Técnicas de Cocultura , Regulação para Baixo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/patologia , Mitocôndrias/virologia , Proteoma/metabolismo , Proteômica/métodos , Alvéolos Pulmonares/citologia , /isolamento & purificação , Serina Endopeptidases/metabolismo , Regulação para Cima
7.
Cell Res ; 30(12): 1078-1087, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33159154

RESUMO

Silent hypoxia has emerged as a unique feature of coronavirus disease 2019 (COVID-19). In this study, we show that mucins are accumulated in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients and are upregulated in the lungs of severe respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected mice and macaques. We find that induction of either interferon (IFN)-ß or IFN-γ upon SARS-CoV-2 infection results in activation of aryl hydrocarbon receptor (AhR) signaling through an IDO-Kyn-dependent pathway, leading to transcriptional upregulation of the expression of mucins, both the secreted and membrane-bound, in alveolar epithelial cells. Consequently, accumulated alveolar mucus affects the blood-gas barrier, thus inducing hypoxia and diminishing lung capacity, which can be reversed by blocking AhR activity. These findings potentially explain the silent hypoxia formation in COVID-19 patients, and suggest a possible intervention strategy by targeting the AhR pathway.


Assuntos
Interferons/metabolismo , Muco/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , /virologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Hipóxia , Interferon beta/farmacologia , Interferon gama/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Macaca , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Mucinas/metabolismo , /patogenicidade , Transdução de Sinais , Regulação para Cima/efeitos dos fármacos
8.
mBio ; 11(6)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33158999

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.


Assuntos
Betacoronavirus/fisiologia , Modelos Biológicos , Mucosa Respiratória/virologia , Brônquios/citologia , Células Cultivadas , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Cinética , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Carga Viral , Tropismo Viral , Liberação de Vírus , Replicação Viral
9.
BMC Infect Dis ; 20(1): 832, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176697

RESUMO

BACKGROUND: Carvacrol, as the major components of aromatic plants used for treating human skin diseases including origanum, Satureja, thymus, and coridothymus species, presented a kind of antiviral activity. To explore the mechanisms of carvacrol against herpes simplex virus (HSV) in vitro. METHOD: The BSC-1 cells model of HSV infection was established, and from the two aspects of viral replication level and cell death pathway, the antiviral effects of carvacrol on HSV infected cells were also evaluated by plaque assay under the three modes including prevention, treatment, and direct inactivation. RESULTS: In the three ways, the half-maximal effective concentration (EC50) of 2% true carvacrol solution on HSV-2 infected cells were severally 0.43, 0.19 and 0.51 mmol/L, and the corresponding therapeutic index (TI) were 4.02, 9.11 and 3.39, respectively. It's the opposite of the increased levels caused by HSV-2 infection, that both the expressions at the transcription genes and protein levels of virus own replication key factors (including ICP4, ICP27, VP16, gB, and UL30) and cytokines (including RIP3, TNF-α, and MLKL) of infected cells treated with carvacrol were dose-dependently inhibited. Besides, HSV-2 infection can cause the decrease of intracellular protein ubiquitination level, and carvacrol can reverse the ubiquitination decrease level caused by HSV-2 infection. CONCLUSION: Carvacrol exhibits significant antiviral activity by inhibiting the HSV-2 proliferation process and HSV-2-induced TNF-α increasing levels, decreasing RIP3 and MLKL protein expressions through the intracellular RIP3-mediated programmed cell necrosis pathway. In addition, carvacrol also may exhibit anti-HSV-2 activity by reversing the ubiquitination decrease level caused by HSV-2 infection on the ubiquitin-proteasome system, which provides insights into the molecular mechanism.


Assuntos
Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Cimenos/farmacologia , Células Epiteliais/virologia , Herpes Simples/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina/metabolismo , Animais , Chlorocebus aethiops , Citocinas/metabolismo , Herpes Simples/virologia , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
10.
Front Cell Infect Microbiol ; 10: 586592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33194826

RESUMO

The present study focuses on the role of human miRNAs in SARS-CoV-2 infection. An extensive analysis of human miRNA binding sites on the viral genome led to the identification of miR-1207-5p as potential regulator of the viral Spike protein. It is known that exogenous RNA can compete for miRNA targets of endogenous mRNAs leading to their overexpression. Our results suggest that SARS-CoV-2 virus can act as an exogenous competing RNA, facilitating the over-expression of its endogenous targets. Transcriptomic analysis of human alveolar and bronchial epithelial cells confirmed that the CSF1 gene, a known target of miR-1207-5p, is over-expressed following SARS-CoV-2 infection. CSF1 enhances macrophage recruitment and activation and its overexpression may contribute to the acute inflammatory response observed in severe COVID-19. In summary, our results indicate that dysregulation of miR-1207-5p-target genes during SARS-CoV-2 infection may contribute to uncontrolled inflammation in most severe COVID-19 cases.


Assuntos
/imunologia , MicroRNAs/genética , RNA Viral/genética , /genética , /genética , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/imunologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/virologia , RNA Viral/metabolismo , /fisiologia
11.
Sci Signal ; 13(659)2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33234691

RESUMO

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus , Células Epiteliais , Calicreínas/metabolismo , Mucosa Respiratória , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/genética , Linhagem Celular Tumoral , Infecções por Coronavirus/enzimologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Calicreínas/genética , Mucosa Respiratória/enzimologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Glicoproteína da Espícula de Coronavírus/genética
12.
PLoS One ; 15(10): e0241212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095800

RESUMO

Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.


Assuntos
Coinfecção/veterinária , Síndrome de Imunodeficiência Adquirida Felina/virologia , Infecções por Hepadnaviridae/veterinária , Hepadnaviridae/genética , Animais de Estimação/virologia , Animais , Brônquios/citologia , Brônquios/virologia , Gatos , Coinfecção/virologia , Citoplasma/virologia , Células Epiteliais/citologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Síndrome de Imunodeficiência Adquirida Felina/sangue , Feminino , Fibroblastos/citologia , Fibroblastos/virologia , Variação Genética , Genoma Viral/genética , Hepadnaviridae/isolamento & purificação , Infecções por Hepadnaviridae/virologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Hepatócitos/virologia , Vírus da Imunodeficiência Felina/isolamento & purificação , Masculino , Microscopia Eletrônica de Transmissão , Filogenia , Recombinação Genética , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Tailândia , Replicação Viral , Eliminação de Partículas Virais
13.
Signal Transduct Target Ther ; 5(1): 235, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037188

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to respiratory illness and multi-organ failure in critically ill patients. Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019 (COVID-19) clinical manifestations, the underlying mechanisms of virus-triggered inflammatory responses are currently unknown. Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells. The processed inflammatory cytokines are released through the virus-induced necroptosis pathway. Virus-induced apoptosis, necroptosis, and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model, a valid model for studying SARS-CoV-2 pathogenesis. Furthermore, analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration, necrotic cell debris, and pulmonary interstitial fibrosis, typical of immune pathogenesis in the lung. The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might assist the development of therapeutic strategies to treat COVID-19.


Assuntos
Apoptose/imunologia , Betacoronavirus/patogenicidade , Caspase 8/imunologia , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/imunologia , Necroptose/imunologia , Pneumonia Viral/imunologia , Fibrose Pulmonar/imunologia , Animais , Caspase 8/genética , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocina CCL5/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/genética , Síndrome da Liberação de Citocina/patologia , Síndrome da Liberação de Citocina/virologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-7/genética , Interleucina-7/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Transgênicos , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Fibrose Pulmonar/virologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
14.
Immun Inflamm Dis ; 8(4): 753-762, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124193

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the global pandemic of the coronavirus disease-2019 (COVID-19). To date, there are still no effective approaches for the prevention and treatment of COVID-19. OBJECTIVE: The present study aims to explore the possible mechanisms of SARS-CoV-2 infection in human lung cells. METHODS: Data interpretation was conducted by recruiting bioinformatics analysis, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways analysis using downloaded data from the NCBI Gene Expression Omnibus database. RESULTS: The present study demonstrated that SARS-CoV-2 infection induces the upregulation of 14 interferon-stimulated genes, indicative of immune, and interferon responses to the virus. Notably, genes for pyrimidine metabolism and steroid hormone biosynthesis are selectively enriched in human lung cells after SARS-CoV-2 infection, suggesting that altered pyrimidine metabolism and steroid biosynthesis are remarkable, and perhaps druggable features after SARS-CoV-2 infection. Besides, there is a strong positive correlation between viral ORF1ab, ORF6, and angiotensin-converting enzyme 2 (ACE2) expression in human lung cells, implying that ACE2 facilitates SARS-CoV-2 infection and replication in host cells probably through the induction of ORF1ab and ORF6.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/etiologia , Interferons/metabolismo , Pulmão/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etiologia , Betacoronavirus/metabolismo , Biologia Computacional , Infecções por Coronavirus/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Pandemias , Pneumonia Viral/patologia , Poliproteínas , Pirimidinas/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Transdução de Sinais/imunologia , Esteroides/biossíntese , Regulação para Cima/imunologia , Proteínas Virais/metabolismo
15.
Signal Transduct Target Ther ; 5(1): 221, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024073
16.
Artigo em Inglês | MEDLINE | ID: mdl-33031994

RESUMO

As the infected cases of COVID-19 reach more than 20 million with more than 778,000 deaths globally, an increase in psychiatric disorders including anxiety and depression has been reported. Scientists globally have been searching for novel therapies and vaccines to fight against COVID-19. Improving innate immunity has been suggested to block progression of COVID-19 at early stages, while omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to have immunomodulation effects. Moreover, n-3 PUFAs have also been shown to improve mood disorders, thus, future research is warranted to test if n-3 PUFAs may have the potential to improve our immunity to counteract both physical and mental impact of COVID-19.


Assuntos
Ansiedade/prevenção & controle , Infecções por Coronavirus/prevenção & controle , Depressão/prevenção & controle , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Fatores Imunológicos/administração & dosagem , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Ansiedade/imunologia , Ansiedade/metabolismo , Ansiedade/virologia , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Citocinas/biossíntese , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Depressão/imunologia , Depressão/metabolismo , Depressão/virologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Ácidos Graxos Ômega-3/imunologia , Ácidos Graxos Ômega-3/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/imunologia , Fatores Imunológicos/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/virologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia
17.
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
18.
J Proteome Res ; 19(11): 4470-4485, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33045833

RESUMO

Porcine deltacoronavirus (PDCoV) is an emergent enteropathogenic coronavirus associated with swine diarrhea. Porcine small intestinal epithelial cells (IPEC) are the primary target cells of PDCoV infection in vivo. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively identify differentially expressed proteins (DEPs) in PDCoV-infected IPEC-J2 cells. A total of 78 DEPs, including 23 upregulated and 55 downregulated proteins, were identified at 24 h postinfection. The data are available via ProteomeXchange with identifier PXD019975. To ensure reliability of the proteomics data, two randomly selected DEPs, the downregulated anaphase-promoting complex subunit 7 (ANAPC7) and upregulated interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), were verified by real-time PCR and Western blot, and the results of which indicate that the proteomics data were reliable and valid. Bioinformatics analyses, including GO, COG, KEGG, and STRING, further demonstrated that a majority of the DEPs are involved in numerous crucial biological processes and signaling pathways, such as immune system, digestive system, signal transduction, RIG-I-like receptor, mTOR, PI3K-AKT, autophagy, and cell cycle signaling pathways. Altogether, this is the first study on proteomes of PDCoV-infected host cells, which shall provide valuable clues for further investigation of PDCoV pathogenesis.


Assuntos
Cromatografia Líquida/métodos , Infecções por Coronavirus/metabolismo , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular , Coronavirus , Células Epiteliais/química , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Proteoma/química , Proteoma/metabolismo , Proteômica , Suínos
19.
Sci Rep ; 10(1): 16631, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024252

RESUMO

The aim of this study was to test in vitro the ability of a mixture of citrus extract, maltodextrin, sodium chloride, lactic acid and citric acid (AuraShield L) to inhibit the virulence of infectious bronchitis, Newcastle disease, avian influenza, porcine reproductive and respiratory syndrome (PRRS) and bovine coronavirus viruses. Secondly, in vivo, we have investigated its efficacy against infectious bronchitis using a broiler infection model. In vitro, these antimicrobials had expressed antiviral activity against all five viruses through all phases of the infection process of the host cells. In vivo, the antimicrobial mixture reduced the virus load in the tracheal and lung tissue and significantly reduced the clinical signs of infection and the mortality rate in the experimental group E2 receiving AuraShield L. All these effects were accompanied by a significant reduction in the levels of pro-inflammatory cytokines and an increase in IgA levels and short chain fatty acids (SCFAs) in both trachea and lungs. Our study demonstrated that mixtures of natural antimicrobials, such AuraShield L, can prevent in vitro viral infection of cell cultures. Secondly, in vivo, the efficiency of vaccination was improved by preventing secondary viral infections through a mechanism involving significant increases in SCFA production and increased IgA levels. As a consequence the clinical signs of secondary infections were significantly reduced resulting in recovered production performance and lower mortality rates in the experimental group E2.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Coronavirus Bovino/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Vírus da Doença de Newcastle/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Células Epiteliais/virologia , Humanos , Influenza Aviária/metabolismo , Influenza Aviária/virologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Doença de Newcastle/metabolismo , Doença de Newcastle/virologia , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Doenças das Aves Domésticas/virologia , Suínos
20.
Sci Rep ; 10(1): 15878, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985508

RESUMO

Alcohol-based disinfectants are widely used for the sanitization of microorganisms, especially those that cause infectious diseases, including viruses. However, since the germicidal mechanism of alcohol is lipolysis, alcohol-based disinfectants appear to have a minimal effect on non-enveloped viruses, such as noroviruses. Because there is no cultivation method for human norovirus (HuNoV) in vitro, murine norovirus and feline calicivirus have been used as surrogates for HuNoV to analyze the efficacy of disinfectant regents. Therefore, whether these disinfectants and their conditions are effective against HuNoVs remain unknown. In this study, we report that ethanol or isopropanol alone can sufficiently suppress GII.4 genotype HuNoV replication in human iPSC-derived intestinal epithelial cells. Additionally, pH adjustments and salting-out may contribute toward the virucidal effect of alcohol against other HuNoV genotypes and cancel the impediment of organic substance contamination, respectively. Therefore, similar to sodium hypochlorite, alcohol-based disinfectants containing electrolytes can be used for HuNoV inactivation.


Assuntos
2-Propanol/farmacologia , Desinfetantes/farmacologia , Etanol/farmacologia , Norovirus/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Infecções por Caliciviridae/virologia , Células Epiteliais/virologia , Humanos , Concentração de Íons de Hidrogênio
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