Alternative interpretation to the findings reported in visualization of severe acute respiratory syndrome coronavirus 2 invading the human placenta using electron microscopy.

To the Editors:

I am writing in response to the in-press article by Algarroba et al describing a woman with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection tested using real-time polymerase chain reaction (RT-PCR). The authors examined the placenta by transmission electron microscopy to identify SARS-CoV-2 particles. They identified circular inclusions in the cytoplasm of several syncytiotrophoblasts that they concluded were SARS-CoV-2 virions.

The report of virus-like inclusions in syncytiotrophoblast is intriguing and thought-provoking. However, I respectfully offer an alternative interpretation of the data. The structures identified as SARS-CoV-2 virions look exactly like clathrin-coated pits or vesicles. Clathrin-coated vesicles are spherical structures employed by trophoblasts and other cell types to internalize cargos from the extracellular space. Coated vesicles and coated pits derive their name from the external scaffold coat composed of clathrin triskelions that decorate the surface of the structure. In transmission electron micrographs in which tissue-thin sections are stained with uranyl acetate and lead citrate, coated vesicles have an electron-dense studded surface that appears identical to the “corona” comprising the spike protein that decorates all coronaviruses, including SARS-CoV-2 virions. It is this studded surface or corona that gives the genus Betacoronaviridae its characteristic morphology and name.

I propose that the structures identified by Algarroba et al in their journal preproof paper are clathrin-coated vesicles and not SARS-CoV-2 particles. This conclusion is based on the following evidence: (1) the circular structures in the electron micrographs in the paper, identified as virions, have the size and shape of clathrin-coated vesicles found in nearly all eukaryotic cells; (2) there is no evidence of virions bound to the apical surface of the syncytiotrophoblast (ACE2 or SARS-CoV-2 receptor) as would be predicted in virus-infected cells; (3) U-shaped, corona-studded structures are apparent at the surface of the syncytiotrophoblast representing newly forming coated vesicles that have not yet pinched off (ie, endocytosed their cargo) (Figure); and (4) the neonate was determined to be virus-negative using RT-PCR.

To provide more convincing evidence of the presence of SARS-CoV-2 virions in the syncytiotrophoblast, the authors could have assessed the placenta for the presence of viral RNA using the same RT-PCR platform they used to test the mother for SARS-CoV-2 infection. In addition, the authors could have examined placentas of patients who tested negative for the presence of the virus as a control. If they failed to visualize any coated vesicular structures in placentas from patients without infection (control), it would have provided evidence that the observed structures in the trophoblasts of the woman with infection may have been SARS-CoV-2 particles but not definitive proof. Without such controls, it is premature to state with certainty that the structures reported in the paper are virions and not coated vesicles.

More definitive evidence to support SARS-CoV-2 infection of the trophoblast and therefore potential vertical transmission would be an experimental approach in which labeled recombinant spike (S) protein could bind to placental tissue sections and be visualized by high-resolution imaging methods. We are currently utilizing this technique to determine whether human trophoblasts bind and internalize virions as a means of vertical transmission. The importance of understanding the nature of vertical transmission necessitates that extensive controls be carried out using multidimensional approaches to address this clinically crucial question in an unambiguous manner.