key: cord-0045151-0toeuh72
authors: Kniss, Douglas A.
title: Letter-to-the-Editor: Alternative Interpretation to the Findings Reported in Visualization of SARS-CoV-2 Virus Invading the Human Placenta Using Electron Microscopy
date: 2020-06-10
journal: Am J Obstet Gynecol
DOI: 10.1016/j.ajog.2020.06.016
sha: 5ac0696da8b46cb8bc5a83fceb4913401dddcf3d
doc_id: 45151
cord_uid: 0toeuh72

nan

I am writing in response to the In Press article by Algarroba et al. describing a woman infected with SARS-CoV-2 diagnosed by RT-PCR. 1 The authors examined the placenta by transmission electron microscopy (TEM) to identify SARS-CoV-2 virus particles. They identified circular inclusions in the cytoplasm of several syncytiotrophoblasts 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/vesicles. Clathrin-coated vesicles are spherical structures employed by trophoblasts and other cell types to internalize cargos from the extracellular space. 2 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 TEM 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 S 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 Pre-proof paper are clathrin-coated vesicles and not SARS-CoV-2 virus particles. This conclusion is based upon 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 3 ; (2) there is no evidence of virions bound to the apical surface of syncytiotrophoblast (ACE2/SARS-CoV-2 receptor) 4 

(3) U-shaped, corona-studded structures are apparent at the surface of syncytiotrophoblast that represent newly forming coated vesicles that have not yet pinched off (i.e., endocytosed their cargo) (see Figure 2) ; and (4) the neonate was demonstrated to be virus-negative by RT-PCR.

To provide more convincing evidence of the presence of SARS-CoV-2 virons 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 diagnose the mother as COVID-19-positive. In addition, the authors could have examined placentas from patients who were negative for presence of the virus as a control. If they failed to visualize any coated vesicular structures in placentas from uninfected (control) patients, that would have provided evidence that the observed structures in the trophoblasts of the infected woman may have been SARS-CoV-2 virus particles, albeit 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 highresolution 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 our understanding the nature of vertical transmission necessitates that extensive

Visualization of SARS-CoV-2 virus invading the human placenta using electron microscopy

Sorting of major cargo glycoproteins into clathrin-coated vesicles

Molecular mechanism and physiological functions of clathrinmediated endocytosis

The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single-cell transcriptome study