Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions

Abstract

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

Figure 1

Native infectious SARS-CoV-2 virions imaged by AFM (a) Topographic image and 3D projection of native infectious SARS-CoV-2 virions adsorbed on a poly-l-lysine (PLL)-coated mica surface using quantitative imaging (QI) mode AFM in buffer. (b,c) Zoom-in view of SARS-CoV-2 virions adsorbed on poly-l-lysine-coated mica (b) or glass coverslips coated with an alkyl silane (c) (scale bars: 50 nm). In (c) height and stiffness images acquired in QI mode on individual virions are shown. (d) Example of a TEM image of fixed infected VeroE6 cells producing SARS-CoV-2 that can be seen as spherical particles studded with S trimers (see arrows). (e) Examples of topographical profile plots measured along the horizontal diameter of viral particles. (f) Virion diameter distribution of infectious SARS-CoV2 samples imaged by AFM in liquid (height profile, n = 190 objects) and compared to fixed samples imaged by TEM (n = 319 objects).

Figure 2

SARS-CoV-2 inactivation monitored by topographic imaging using QI mode AFM in buffer. (a) Native infectious, control SARS-CoV-2 sample kept for 30 min at room temperature. (b) Virus particles inactivated with 1% and 2% FA for 30 min at 20 °C, or incubated at 58 °C for 30 min. Scale bars are 50 nm. The color gradient for Z scale is the same for all AFM panels. (c) Topographical profile plots measured along the dotted lines in the lower panel in (a). (d) 3D projection of the particles in the lower panel (a). (e) Distribution of the topographical maximal height of treated and untreated SARS-CoV-2 particles (control n = 39; 1% FA n = 40; 2% FA n = 43; 58 °C n = 39). P values are indicated and were computed by using two-sided independent t tests and comparing the results to those for the untreated sample.