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

Debora Ferreira Barreto-Vieira,Maria Eduarda Monteiro,Wendell Girard-Dias,Fernando Couto Motta,Marcos Alexandre Nunes Da Silva,Ortrud Monika Barth,Patricia T Bozza,Milene Dias Miranda,Thiago Moreno L Souza,Suelen Silva Gomes Dias,Vinicius Cardoso Soares,Arthur Da Costa Rasinhas,Bráulio Soares Archanjo,Marilda M Siqueira,Ana Luisa Teixeira De Almeida

doi:10.3390/v14020201

Debora Ferreira Barreto-Vieira, Maria Eduarda Monteiro + Show 13 more

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https://doi.org/10.3390/v14020201

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Abstract

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.

Highlights

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic [1], belongs to the Coronaviridae family (Coronavirinae subfamily) which includes four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus
Analysis of Vero-E6 cells infected with SARS-CoV-2 under inverted light microscopy demonstrated a cytopathic effect (CPE), that was mostly evident from 48 h pi
Showed changes associated to SARS-CoV-2 infection, including: (I) Cell activation—Cell activation was evidenced by a marked plasmatic membrane projection (Figure 1B–F and Figure 2B–F), connection between adjacent cells mediated by these filopodia (Figure 1B–C, Figure 2B–D, and Figure 3B), viral particles associated with filopodia and microvilli (Figure 1B–F, Figure 2B–F, and Figure 3A) and to the cell membrane (Figure 1B–F and Figure 2B–F)

Summary

Introduction

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic [1], belongs to the Coronaviridae family (Coronavirinae subfamily) which includes four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. While Alphacoronavirus and Betacoronavirus exclusively infect mammalian species, Gammacoronavirus and Deltacoronavirus have a wider host range that includes avian species [1,2]. The SARS-CoV-2 is grouped within the Betacoronavirus genus together with Middle East Respiratory Syndrome Coronavirus (MERSCoV) and SARS-CoV, which are both highly pathogenic, and HCoV-HKU1, HCoV-OC43, which causes seasonal and usually mild respiratory tract infections. Coronaviruses are enveloped with positive-sense single-stranded RNA genome of. Phylogenetic analysis of SARS-CoV-2 revealed that is closely related (88–89% similarity) to SARS-like coronaviruses from bats—such as bat-SL-CoVZC45

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