SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19.

Importance: The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. Objective: To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. Design, Setting, and Participants: The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Main Outcomes and Measures: Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Results: Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. Conclusions and Relevance: The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

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.

SARS-CoV-2: basic concepts, origin and treatment advances.

The first cases of COVID-19, caused by the virus called SARS-CoV-2, were recorded in Wuhan, China, in December 2019; however, its transmission ability caused for the infection to be practically present throughout the world six months later. The origin of the virus appears to be zoonotic; it has been proposed that it comes from a bat and that it may have had an intermediate host that led to its introduction in the human population. SARS-CoV-2 is an enveloped virus, with a positive single-stranded RNA genome, and it binds to the angiotensin-converting enzyme, present in susceptible cells, to infect the human respiratory system. Although other coronaviruses have been previously known, they have not had the same impact, and, therefore, research on pharmacological treatments is not sufficiently developed to face the current challenge. Almost since the beginning of the epidemic, several molecules have been proposed for the treatment of infection; however, there is not yet a drug available with sufficient effectiveness for treatment. This review describes SARS-CoV-2 main characteristics, its replicative cycle, its possible origin and some advances in the development of antiviral treatments.

Los primeros casos de COVID-19, causada por el virus denominado SARS-CoV-2, se registraron en Wuhan, China, en diciembre de 2019; sin embargo, su capacidad de transmisión ocasionó que seis meses después la infección prácticamente estuviera presente en todo el mundo. El origen del virus parece ser zoonótico; se propone que proviene del murciélago y podría haber tenido un hospedero intermediario que llevó a su introducción en la población humana. SARS-CoV-2 es un virus envuelto, con genoma de ARN de cadena sencilla en sentido positivo y se ancla a la enzima convertidora de angiotensina, presente en las células susceptibles para infectar el sistema respiratorio de los humanos. Aunque previamente se han conocido otros coronavirus, no han tenido el mismo impacto, por lo que la investigación en tratamientos farmacológicos no tiene el desarrollo suficiente para afrontar el reto actual. Casi desde el comienzo de la epidemia se han propuesto moléculas para el tratamiento de la infección, sin embargo, aún no se cuenta con un fármaco con suficiente efectividad terapéutica. En esta revisión se describen las características principales de SARS-CoV-2, su ciclo replicativo, su posible origen y algunos avances en el desarrollo de tratamientos antivirales.

Intracellular host cell membrane remodelling induced by SARS-CoV-2 infection in vitro.

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.

SARS-CoV-2: generalidades, origen y avances en el tratamiento / SARS-CoV-2: basic concepts, origin and treatment advances

Resumen Los primeros casos de COVID-19, causada por el virus denominado SARS-CoV-2, se registraron en Wuhan, China, en diciembre de 2019; sin embargo, su capacidad de transmisión ocasionó que seis meses después la infección prácticamente estuviera presente en todo el mundo. El origen del virus parece ser zoonótico; se propone que proviene del murciélago y podría haber tenido un hospedero intermediario que llevó a su introducción en la población humana. SARS-CoV-2 es un virus envuelto, con genoma de ARN de cadena sencilla en sentido positivo y se ancla a la enzima convertidora de angiotensina, presente en las células susceptibles para infectar el sistema respiratorio de los humanos. Aunque previamente se han conocido otros coronavirus, no han tenido el mismo impacto, por lo que la investigación en tratamientos farmacológicos no tiene el desarrollo suficiente para afrontar el reto actual. Casi desde el comienzo de la epidemia se han propuesto moléculas para el tratamiento de la infección, sin embargo, aún no se cuenta con un fármaco con suficiente efectividad terapéutica. En esta revisión se describen las características principales de SARS-CoV-2, su ciclo replicativo, su posible origen y algunos avances en el desarrollo de tratamientos antivirales.

Abstract The first cases of COVID-19, caused by the virus called SARS-CoV-2, were recorded in Wuhan, China, in December 2019; however, its transmission ability caused for the infection to be practically present throughout the world six months later. The origin of the virus appears to be zoonotic; it has been proposed that it comes from a bat and that it may have had an intermediate host that led to its introduction in the human population. SARS-CoV-2 is an enveloped virus, with a positive single-stranded RNA genome, and it binds to the angiotensin-converting enzyme, present in susceptible cells, to infect the human respiratory system. Although other coronaviruses have been previously known, they have not had the same impact, and, therefore, research on pharmacological treatments is not sufficiently developed to face the current challenge. Almost since the beginning of the epidemic, several molecules have been proposed for the treatment of infection; however, there is not yet a drug available with sufficient effectiveness for treatment. This review describes SARS-CoV-2 main characteristics, its replicative cycle, its possible origin and some advances in the development of antiviral treatments.

Emergencia de sars-cov-2. aspectos básicos sobre su origen, epidemiología, estructura y patogenia para clínicos / Emerging sars-cov-2. basic information about epidemiology, origin source, structure and pathogenicity of sars-cov-2 for clinicians

SARS-CoV-2 es el tercer coronavirus que emerge en las últimas dos décadas y produce la enfermedad denominada COVID-19 (enfermedad infecciosa por coronavirus 2019). Ha demostrado ser fácilmente transmisible entre humanos con una rápida diseminación mundial y declarada como pandemia el 11 de marzo 2020. A la fecha ha causado millones de casos y muertes, disrupción de servicios sanitarios y severas consecuencias sociales, económicas y políticas en todos los países. Los estudios filogenéticos lo relacionan con SARS-CoV presentes en murciélagos. Comparte características de patogenicidad con sus parientes más cercanos, SARS-CoV y MERS-CoV. 15 a 20% de los afectados presentan cuadros graves. A la fecha no se cuenta con antivirales efectivos ni vacunas. Para un adecuado control se hace imprescindible dilucidar aspectos epidemiológicos, moleculares y de patogenicidad. En esta revisión se presenta información básica sobre epidemiología, origen, estructura y patogenia de SARS-CoV-2. i:es

SARS-CoV-2 is the third coronavirus emerging in the last two decades producing a disease denominated COVID-19 (infectious disease by coronavirus 2019). It has demostrated to be easily transmisible between humans with a fast world wide dissemination and declared a pandemia in March 11, 2020. Up to date it has caused millions of cases, deaths, disruptions in medical services and severe social, political and economic consecuences, all around the globe. Phylogenetic studies relate it to SARS-CoV present in bats. It shares pathogenic characteristics with its close relatives, SARS-CoV and MERS-CoV. 15 to 20% of the patients develop a severe clinical course. Up to date there are no effective antiviral drugs or vaccines. For an adecuate control it is essential to learn about epidemiological, pathogenic and molecular aspects. In this review some basic information about epidemiology, origin source, structure and pathogenicity of SARS-CoV-2 is presented. i:en

Cathepsin L in COVID-19: From Pharmacological Evidences to Genetics.

The coronavirus disease 2019 (COVID-19) pandemics is a challenge without precedent for the modern science. Acute Respiratory Discomfort Syndrome (ARDS) is the most common immunopathological event in SARS-CoV-2, SARS-CoV, and MERS-CoV infections. Fast lung deterioration results of cytokine storm determined by a robust immunological response leading to ARDS and multiple organ failure. Here, we show cysteine protease Cathepsin L (CatL) involvement with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 from different points of view. CatL is a lysosomal enzyme that participates in numerous physiological processes, including apoptosis, antigen processing, and extracellular matrix remodeling. CatL is implicated in pathological conditions like invasion and metastasis of tumors, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, viral infection, and other diseases. CatL expression is up-regulated during chronic inflammation and is involved in degrading extracellular matrix, an important process for SARS-CoV-2 to enter host cells. In addition, CatL is probably involved in processing SARS-CoV-2 spike protein. As its inhibition is detrimental to SARS-CoV-2 infection and possibly exit from cells during late stages of infection, CatL could have been considered a valuable therapeutic target. Therefore, we describe here some drugs already in the market with potential CatL inhibiting capacity that could be used to treat COVID-19 patients. In addition, we discuss the possible role of host genetics in the etiology and spreading of the disease.