Extracellular vesicles from primary human macrophages stimulated with VIP or PACAP mediate anti-SARS-CoV-2 activities in monocytes through NF-κB signaling pathway.

Infection by SARS-CoV-2 is associated with uncontrolled inflammatory response during COVID-19 severe disease, in which monocytes are as one of the main sources of pro-inflammatory mediators leading to acute respiratory distress syndrome. Extracellular vesicles (EVs) from different cells play important roles during SARS-CoV-2 infection, but investigations describing the involvement of EVs from primary human monocyte-derived macrophages (MDM) on the regulation of this infection are not available. Here, we describe the effects of EVs released by MDM stimulated with the neuropeptides VIP and PACAP on SARS-CoV-2-infected monocytes. MDM-derived EVs were isolated by differential centrifugation of medium collected from cells cultured for 24 h in serum-reduced conditions. Based on morphological properties, we distinguished two subpopulations of MDM-EVs, namely large (LEV) and small EVs (SEV). We found that MDM-derived EVs stimulated with the neuropeptides inhibited SARS-CoV-2 RNA synthesis/replication in monocytes, protected these cells from virus-induced cytopathic effects and reduced the production of pro-inflammatory mediators. In addition, VIP- and PACAP-treated MDM-derived EVs prevented the SARS-CoV-2-induced NF-κB activation. Overall, our findings suggest that MDM-EVs are endowed with immunoregulatory properties that might contribute to the antiviral and anti-inflammatory responses in SARS-CoV-2-infected monocytes and expand our knowledge of EV effects during COVID-19 pathogenesis.

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.