RESUMO
IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.
Assuntos
Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteínas não Estruturais Virais , Animais , Humanos , Camundongos , Substituição de Aminoácidos , Camelus , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , Proteínas não Estruturais Virais/genéticaRESUMO
Middle East respiratory syndrome coronavirus (MERS-CoV) causes zoonotic disease. Dromedary camels are the source of zoonotic infection. We identified a mutation of amino acid leucine to phenylalanine in the codon 232 position of the non-structural protein 6 (nsp6) (nsp6 L232F) that is repeatedly associated with zoonotic transmission. We generated a pair of isogenic recombinant MERS-CoV with nsp6 232L and 232F residues, respectively, and showed that the nsp6 L232F mutation confers higher replication competence in ex-vivo culture of human nasal and bronchial tissues and in lungs of mice experimentally infected in-vivo. Mechanistically, the nsp6 L232F mutation appeared to modulate autophagy and was associated with higher exocytic virus egress, while innate immune responses and zippering activity of the endoplasmic reticulum remained unaffected. Our study suggests that MERS-CoV nsp6 may contribute to viral adaptation to humans. This highlights the importance of continued surveillance of MERS-CoV in both camels and humans.
RESUMO
The emergence of SARS-CoV-2 variants of concern with progressively increased transmissibility between humans is a threat to global public health. The Omicron variant of SARS-CoV-2 also evades immunity from natural infection or vaccines1, but it is unclear whether its exceptional transmissibility is due to immune evasion or intrinsic virological properties. Here we compared the replication competence and cellular tropism of the wild-type virus and the D614G, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) variants in ex vivo explant cultures of human bronchi and lungs. We also evaluated the dependence on TMPRSS2 and cathepsins for infection. We show that Omicron replicates faster than all other SARS-CoV-2 variants studied in the bronchi but less efficiently in the lung parenchyma. All variants of concern have similar cellular tropism compared to the wild type. Omicron is more dependent on cathepsins than the other variants of concern tested, suggesting that the Omicron variant enters cells through a different route compared with the other variants. The lower replication competence of Omicron in the human lungs may explain the reduced severity of Omicron that is now being reported in epidemiological studies, although determinants of severity are multifactorial. These findings provide important biological correlates to previous epidemiological observations.
Assuntos
Brônquios/virologia , Pulmão/virologia , SARS-CoV-2/crescimento & desenvolvimento , Tropismo Viral , Replicação Viral , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/epidemiologia , COVID-19/transmissão , COVID-19/virologia , Catepsinas/metabolismo , Chlorocebus aethiops , Endocitose , Humanos , Técnicas In Vitro , SARS-CoV-2/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Técnicas de Cultura de Tecidos , Células VeroRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health globally. Patients with severe COVID-19 disease progress to acute respiratory distress syndrome, with respiratory and multiple organ failure. It is believed that dysregulated production of proinflammatory cytokines and endothelial dysfunction contribute to the pathogenesis of severe diseases. However, the mechanisms of SARS-CoV-2 pathogenesis and the role of endothelial cells are poorly understood. METHODS: Well-differentiated human airway epithelial cells were used to explore cytokine and chemokine production after SARS-CoV-2 infection. We measured the susceptibility to infection, immune response, and expression of adhesion molecules in human pulmonary microvascular endothelial cells (HPMVECs) exposed to conditioned medium from infected epithelial cells. The effect of imatinib on HPMVECs exposed to conditioned medium was evaluated. RESULTS: We demonstrated the production of interleukin-6, interferon gamma-induced protein-10, and monocyte chemoattractant protein-1 from the infected human airway cells after infection with SARS-CoV-2. Although HPMVECs did not support productive replication of SARS-CoV-2, treatment of HPMVECs with conditioned medium collected from infected airway cells induced an upregulation of proinflammatory cytokines, chemokines, and vascular adhesion molecules. Imatinib inhibited the upregulation of these cytokines, chemokines, and adhesion molecules in HPMVECs treated with conditioned medium. CONCLUSIONS: We evaluated the role of endothelial cells in the development of clinical disease caused by SARS-CoV-2 and the importance of endothelial cell-epithelial cell interaction in the pathogenesis of human COVID-19 diseases.
Assuntos
COVID-19 , SARS-CoV-2 , Comunicação Celular , Células Endoteliais , Células Epiteliais , HumanosRESUMO
We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells.
Assuntos
COVID-19 , Epidemias , China , Hong Kong/epidemiologia , Humanos , SARS-CoV-2RESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, causing a respiratory disease (coronavirus disease 2019, COVID-19) of varying severity in Wuhan, China, and subsequently leading to a pandemic. The transmissibility and pathogenesis of SARS-CoV-2 remain poorly understood. We evaluate its tissue and cellular tropism in human respiratory tract, conjunctiva, and innate immune responses in comparison with other coronavirus and influenza virus to provide insights into COVID-19 pathogenesis. METHODS: We isolated SARS-CoV-2 from a patient with confirmed COVID-19, and compared virus tropism and replication competence with SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and 2009 pandemic influenza H1N1 (H1N1pdm) in ex-vivo cultures of human bronchus (n=5) and lung (n=4). We assessed extrapulmonary infection using ex-vivo cultures of human conjunctiva (n=3) and in-vitro cultures of human colorectal adenocarcinoma cell lines. Innate immune responses and angiotensin-converting enzyme 2 expression were investigated in human alveolar epithelial cells and macrophages. In-vitro studies included the highly pathogenic avian influenza H5N1 virus (H5N1) and mock-infected cells as controls. FINDINGS: SARS-CoV-2 infected ciliated, mucus-secreting, and club cells of bronchial epithelium, type 1 pneumocytes in the lung, and the conjunctival mucosa. In the bronchus, SARS-CoV-2 replication competence was similar to MERS-CoV, and higher than SARS-CoV, but lower than H1N1pdm. In the lung, SARS-CoV-2 replication was similar to SARS-CoV and H1N1pdm, but was lower than MERS-CoV. In conjunctiva, SARS-CoV-2 replication was greater than SARS-CoV. SARS-CoV-2 was a less potent inducer of proinflammatory cytokines than H5N1, H1N1pdm, or MERS-CoV. INTERPRETATION: The conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens. FUNDING: US National Institute of Allergy and Infectious Diseases, University Grants Committee of Hong Kong Special Administrative Region, China; Health and Medical Research Fund, Food and Health Bureau, Government of Hong Kong Special Administrative Region, China.
Assuntos
Betacoronavirus/imunologia , Túnica Conjuntiva/virologia , Infecções por Coronavirus/imunologia , Imunidade Inata/imunologia , Pneumonia Viral/imunologia , Sistema Respiratório/virologia , Tropismo Viral/fisiologia , Replicação Viral/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/fisiologia , COVID-19 , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/fisiopatologia , Infecções por Coronavirus/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/fisiopatologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/fisiopatologia , Mucosa Respiratória/virologia , Sistema Respiratório/imunologia , Sistema Respiratório/fisiopatologia , SARS-CoV-2RESUMO
In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.
Assuntos
Equorina/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias Humanas/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/farmacologia , Equorina/química , Equorina/genética , Western Blotting , Cafeína/farmacologia , Cálcio/metabolismo , Linhagem Celular , Corantes Fluorescentes/química , Células HEK293 , Humanos , Imidazóis/química , Receptores de Inositol 1,4,5-Trifosfato/agonistas , Medições Luminescentes , Miócitos Cardíacos/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Pirazinas/química , Fatores de TempoRESUMO
Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.
Assuntos
Apoptose/fisiologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Aves , Brônquios/metabolismo , Brônquios/virologia , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Cães , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Inflamação/metabolismo , Inflamação/virologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae , Replicação Viral/fisiologiaRESUMO
The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB participate in diverse neuronal functions, including activity-dependent synaptic plasticity that is crucial for learning and memory. On binding to BDNF, TrkB is not only autophosphorylated at tyrosine residues but also undergoes serine phosphorylation at S478 by the serine/threonine kinase cyclin-dependent kinase 5 (Cdk5). However, the in vivo function of this serine phosphorylation remains unknown. We generated knock-in mice lacking this serine phosphorylation (Trkb(S478A/S478A) mice) and found that the TrkB phosphorylation-deficient mice displayed impaired spatial memory and compromised hippocampal long-term potentiation (LTP). S478 phosphorylation of TrkB regulates its interaction with the Rac1-specific guanine nucleotide exchange factor TIAM1, leading to activation of Rac1 and phosphorylation of S6 ribosomal protein during activity-dependent dendritic spine remodeling. These findings reveal the importance of Cdk5-mediated S478 phosphorylation of TrkB in activity-dependent structural plasticity, which is crucial for LTP and spatial memory formation.
Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Receptor trkB/metabolismo , Comportamento Espacial/fisiologia , Análise de Variância , Animais , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Quinase 5 Dependente de Ciclina/deficiência , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Ácido Glutâmico/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanilato Quinases/metabolismo , Humanos , Imunoprecipitação/métodos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação/genética , Quinoxalinas/farmacologia , Ratos , Receptor trkB/genética , Proteínas Quinases S6 Ribossômicas , Serina/metabolismo , Coloração pela Prata , Sinaptofisina/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Fatores de Tempo , Transfecção , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C(18) 5microm column (length, 250mm; inner diameter, 4.6mm) using a mixture of water-acetonitrile-trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1mL/min, with detection at 229nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1-7.5microg/mL with r(2)=0.9991. The limit of detection and quantification of this method were 0.05microg/mL and 0.1microg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5mg/kg and intraperitoneal administration of 10 and 20mg/kg in rats.
Assuntos
Inibidores da Colinesterase/sangue , Inibidores da Colinesterase/farmacocinética , Quinolonas/sangue , Quinolonas/farmacocinética , Acetonitrilas/química , Animais , Área Sob a Curva , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Congelamento , Meia-Vida , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Estrutura Molecular , Controle de Qualidade , Quinolonas/química , Quinolonas/farmacologia , Ratos , Ratos Sprague-Dawley , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta/métodos , Ácido Trifluoracético/química , Água/químicaRESUMO
The present study aims to investigate the preclinical intestinal absorption of bis(7)-tacrine (B7T) using different absorption models. In addition, potential intestinal and liver first-pass metabolism was evaluated by in vitro incubation of B7T with rat intestine and liver microsome. Results showed that the permeability of B7T across artificial membrane was pH dependent with rapid diffusion achieved at both pH 6.8 and 7.4. However, the absorptive permeability of B7T in Caco-2 cell model was substantially lower than that in the artificial membrane accompanied with over 56% of B7T being trapped within Caco-2 cells. In the rat in situ intestinal perfusion model, B7T was subject to an extensive intestinal extraction (>90%) with extremely low concentration of B7T detected in mesenteric blood, which was further found to be associated with the high tissue binding (99.9%) of B7T. In vitro incubation of B7T with rat liver and intestinal microsomes revealed that hydroxylation of B7T might mainly occur in rat liver rather than intestine. In conclusion, B7T is expected to have a low oral bioavailability in vivo, which may be due to its poor intestinal permeability, significant tissue binding and hepatic hydroxylation metabolism.
Assuntos
Inibidores da Colinesterase/farmacocinética , Absorção Intestinal/fisiologia , Nootrópicos/farmacocinética , Tacrina/análogos & derivados , Doença de Alzheimer/tratamento farmacológico , Animais , Biotransformação , Células CACO-2 , Inibidores da Colinesterase/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Epitélio/metabolismo , Glucuronídeos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , Nootrópicos/metabolismo , Permeabilidade , Ratos , Ratos Sprague-Dawley , Espectrofotometria Ultravioleta , Tacrina/metabolismo , Tacrina/farmacocinéticaRESUMO
The current study aims to develop a specific and sensitive LC-MS/MS method for determination of bis(7)-tacrine (B7T) in rat plasma. A 100 microL plasma sample was extracted with ethyl acetate. B7T and the internal standard (IS), pimozide, in the samples were then analyzed with LC-MS/MS in positive electrospray ionization condition. Chromatographic separation of B7T and IS was achieved in a C(18) reversed-phase HPLC column (150 x 2.1 mm i.d.) by isocratic elution with a mobile phase consisting of 0.05% formic acid in water and acetonitrile (1:1, v/v) at a flow rate of 0.35 mL/min. Multiple-reaction monitoring (MRM) mode was employed to measure the ion transitions: m/z 247 to 197 for B7T and m/z 462 to m/z 328 for IS, respectively. The method was linear over the studied ranges of 100-5000 and 10-100 ng/mL. The intra-day and inter-day variations of the analysis were less than 6.8% with standard errors less than 9.0%. The detection limit of B7T in rat plasma was 1 ng/mL. The developed method was successfully applied to the pharmacokinetic study of B7T after intravenous administration of 1 mg/kg B7T and further proved to be readily utilized for determination of B7T in rat plasma samples.