Helium-ion microscopy reveals an intra-cytoplasmic route for SARS-CoV-2 cell-cell transmission

The usual picture of SARS-CoV-2 transmission is extra-cytoplasmic, that is: virions (the basic virus complex) from outside enter the host cell by docking their spike glycoproteins to the Angiotensin Converting Enzyme 2 (ACE2) present on the host cell membrane. After intracellular entry, newly replicated virions are released outside the host cell to propagate the infection via the same ACE2 docking mechanism. Antibodies may attack the extra-cellular virions, thus providing immunity. We used scanning Helium-ion microscopy to study the virion propagation process in in-vitro culture of Vero E6 cells infected with mNeonGreen-SARS-CoV-2. The unprecedented resolution of HeIM and its capacity to scan a large number of samples, showed the presence of: 1)-long tunneling nanotubes that connect two or more cells over submillimeter distances; 2)-large scale multiple cell fusion events; and 3)-abundant extracellular vesicles of various sizes. Tunneling nanotubes and cell fusion events are not significanlty present in uninfected samples. Taken together, these three ultrastructural features reveal a new intra-cytoplasmic modality to connect SARS-CoV-2 infected cells; this modality may act as an alternative route of viral transmission, different from the extra-cytoplasmic ACE2 docking mechanism. Our findings may explain the ability of SARS-CoV-2 to survive the immune surveillance of the host organism and may explain clinical observations such as the breakthrough infections.