Review 1: "SARS-CoV-2 infects brain choroid plexus and disrupts the blood-CSF-barrier"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

The paper by Pellegrini et al. investigates SARS-CoV-2 tropism in brain in organoid systems. The authors have provided convincing evidence that choroid-plexus epithelial cells exhibit a significant degree of susceptibility to SARS-CoV-2 infection, whereas neurons and glia cells appear to be highly refractory of SARS-CoV-2. The authors identified an apoliprotein and ACE2 expressing subset of epithelial barrier cells within the choroid plexus as the main target of SARS-CoV-2. The authors have also provided evidence that SARS-CoV-2 can infect the choroid plexus epithelium from the basal side and that infection was associate with altered cell-cell junctions suggesting that SARS-CoV-2 infection can lead to a breakdown of the blood-CSF-barrier.

The paper is well-written, and the experimental section has been well executed. The results have been clearly presented and nicely illustrated and support the central message of the paper, namely that a subpopulation of choroid plexus epithelial cells, rather than neurons or glia, is a main target of SARS-CoV-2 under the experimental conditions of the study.

As with other studies using brain organoid systems to study the effect of infectious agent on the brain, there is the general limitation of whether organoids accurate reflect the environment of infections, like SARS-CoV-2, resulting in neurological symptoms observed in adults. Nevertheless, regardless of this limitation, this study is interesting and can provide insights into cell type specific susceptibility to infection.

The authors have convincingly shown that SARS-CoV-2 infection causes alterations in cell-cell junctions contributing to the blood-CSF-barrier. This finding would be strengthened by the incorporation of RNAseq studies to examine whether the observed physical injury to the blood-CSF-barrier is associate with changes in gene expression that have been linked to neurological symptoms. The observation by the authors of an apoliprotein and ACE2 expressing subset of choroid plexus cells as the main target of SARS-CoV-2 would be further enhanced by confirming that blocking of the ACE2 receptor prevents SARS-CoV-2 infection of these cells.

Based on their finding of abundant infection from the basal side of the choroid plexus epithelium, the authors suggest that the vascular compartment of the choroid plexus could be the SARS-CoV-2 entry point to the central nervous system. Although this a plausible scenario, there is very limited evidence of viremia in COVID-19 patients. The discussion section could incorporate comments and comparison with other known neurotropic viruses that target the choroid plexus.