Part of the challenge in controlling COVID-19 is the innovative features of this coronavirus. New knowledge on virus genetics and morphology needs to be analyzed concurrently with viral “behavior” within the host cell as well as the dynamics that determine the fate of the particle. To approach SARS-CoV-2/cell interactions, we investigate several steps of virus infection in Vero cells at 2 and 48 hpi by SEM. Vero cells are a widely used model used in viral infection studies and is an adequately supports coronavirus replication12,14,15,19. This microscopic approach detailed virus-induced changes in the cell.
Our assays were performed using three MOIs (0.01; 0.1 and 1), and we could discern the MOI of 0.1 as the more adequate for this type of study. This MOI allowed the best cell conditions and distribution and also allowed visualization of virions through the cell surface into the cell interior.
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The absence of virions adhered to the cells surface at 2 hpi corroborates recent studies performed by Belhaouari et al.19 in which SARS-CoV-2 particles were only observed at these loci after 12 hpi. In contrast, SARS-CoV-2 particles were found lying on the cellular surface at 48 hpi between surface projections and adhered to them. We also observed probable viral particles inside vacuoles suggesting a secretion route. These aggregates of cell organelles and components (Fig. 2F) may reflect the polarized release of virus previously described for SARS-CoV20.
All viruses measured by SEM display a spiky round shape with a size of around 70-85 nm in diameter considering a platinum coating of 5 nm. This agrees with the dimensions described in recent studies1,21,22.
Viral particles adhered to smooth surface and microvilli-like surface projetions
The effects on the surface morphology of infected cells varies among viruses. Infection by several viruses including HTLV-IIIB leads to a loss of cell SP that are then replaced by blebs23. Microvilli induction or increases were reported in several cases of DNA or RNA viral infection24,25. For RNA viruses that egress by budding, e.g., influenza, the increase in SP of infected cells coincides with higher budding rates26.
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Similar to prior studies on SARS-CoV infection of Vero cells27, we also observed a ruffled host cell and thickened edges displaying a layered shape. These sites were appropriate to register the attachment of SARS-CoV-2 particles (Fig. 2A) similar to transmission electron microscopy images of the same early step of SARS-CoV infection of Vero cells28.
Likewise, the proliferation of SP on the infected cells, especially at the apical region of these cells, is similar to SARS-CoV and SARS-CoV-2. In addition, the abundance of SARS-CoV-2 particles held on SP, was recently showed29 and may facilitate the speed of viral propagation in the epithelium of conducting airways from the lumen of the respiratory superior tract because this environment is colonized by ciliated cells.
Vacuoles containing viral particles
Cell scraping is a very useful expedient that is occasionally used in studies of host cell/parasite interactions30,31. Infected cells are artificially devoid of plasma membranes and exposed to a myriad of vacuoles (Fig. 3A). Drastic vacuolization due to viral infection was previously described for other RNA viruses including SARS-CoV20,32. Similar sites were recently reported as virus morphogenesis matrix vesicae (VMMV)19. The particles observed in the interior of these VMMVs (Fig. 3C-E) were previously described as doughnut-like particles when observed by electron microscopy19,33. SARS-CoV immature particles are presumed to bud into vesicles as part of the assembly process, and thus the observed particles were probably immature viruses devoid of the representative (corona) spikes of this virion. Bordering vesicles were found in close association with the vacuoles (Fig. 3D), and thus we speculate that their role in viral pre-components leads to discharge into the compartments.
Studies with other coronaviruses identified large virion-containing vacuoles (LVCVs) where the complete particle would bud. There is correlation between these structures as observed by transmission electron microscopy and our data suggesting the occurrence of both phenomena.
Translocation of vacuoles towards the plasma membrane
Coronaviruses infection leads to massive remodeling of cell membranes34,35; the more condensed area depicted in the cytoplasm at 48 hpi (Fig. 2F) may correspond to the main locus of viral morphogenesis. The proposed mechanism for the export of viruses to the extracellular space is via fusion of the transport compartment membrane with the cell plasma membrane20.
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The size of the vacuoles we observed in the cell periphery was not compatible with the identified clathrin-coated pits because the vacuoles measure approximately 1 µm; clathrin-coated pits measure near 200 nm in diameter. The presence of these endocytosis-associated players was recently reported in SARS-CoV-2-infected cells. They are likely receptacles to the nucleocapsid after the incoming virus is uncoated19.
Thus, our observations suggest that a boost in vacuoles is restricted only to a specific and more condensed part of the cytoplasm. This suggests translocation to the plasma membrane is required for release the viral particles by a fusion mechanism.
Cellular bridges containing viral particles
Viral particles adhered to cell surface protrusions that were shown to connect two cells. This observation suggests viral “cell surfing” previously described by other enveloped viruses such as HIV and human metapneumovirus36,37. This mechanism is presumed to allow the in vivo penetration of virus in mucosal surfaces that display microvilli-rich cells.
Actin filaments play a fundamental role in viral extrusion by the cell for both RNA and DNA viruses. Actin offers the strength to discharge the progeny virus particles to the extracellular medium, as occurs to some viruses that leave the cell by budding, including Fowlpox and West Nile viruses38,39. Other examples include actin comets—these are an efficient form of poxvirus dissemination and cell-to-cell HIV spreading, which involves the direct engagement of GAG proteins and F-actin40,41.
Previous studies have shown that the cytoskeleton network plays an important role in the maturation and, possibly, in the replication process of SARS-CoV27. Communication between the two cells in Fig. 4C-D suggests the occurrence of a thin (< 0.7 µm) strand of F-actin containing tunneling nanotube (TNT). These intercellular membranous connections may provide the transference of molecular information especially viruses42. Similarly, virus cell surfing was shown on SARS-CoV-2 infection, which offers new insights into cell-to-cell propagation and virus transmission.
