ABSTRACT
BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.
Subject(s)
COVID-19/pathology , Nasopharynx/ultrastructure , SARS-CoV-2/ultrastructure , Antigens, Viral/metabolism , COVID-19/diagnosis , COVID-19/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Humans , Image Enhancement , Microscopy , Microvilli/ultrastructure , Nasal Mucosa/ultrastructure , Nasal Mucosa/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Virion/ultrastructureSubject(s)
Betacoronavirus/ultrastructure , Coronavirus Infections/pathology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Pneumonia, Viral/pathology , Bronchi/cytology , COVID-19 , Cells, Cultured , Humans , Microscopy, Electron, Scanning , Pandemics , SARS-CoV-2 , Virus CultivationSubject(s)
Cilia/metabolism , Eye Diseases/metabolism , Flagella/metabolism , Heart Diseases/metabolism , Lung Diseases/metabolism , Polycystic Kidney Diseases/metabolism , Centrosome/metabolism , Centrosome/ultrastructure , Cilia/ultrastructure , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Eye Diseases/genetics , Eye Diseases/pathology , Flagella/ultrastructure , Gene Expression Regulation , Heart Diseases/genetics , Heart Diseases/pathology , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Lung Diseases/genetics , Lung Diseases/pathology , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/pathology , Wnt Signaling PathwayABSTRACT
In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.