RR:C19 Evidence Scale rating by reviewer:
Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.
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Review: This paper titled- SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system aims to answer a timely question of neural cell tropism for various strains of SARS-CoV2. Although the authors ask an important question, that can be leveraged to better therapeutics for neurotropic SARS-CoV2 infection, the manuscript lacks major information. This manuscript has a potential to be published in journals, but needs some major and minor revisions that may help authors do better justification for their data. Overall, the claims made by the authors, are not fully supported by the results mentioned in the manuscript.
Major: In the result section 3.1; the authors claim that "ACE-2 expression was predominantly observed in astrocytes, with expression also observed in neurons. In contrast, TMPRSS2 expression was predominant in neurons, with expression also observed in astrocytes". However, it should be noted that this claim cannot be made without quantification data of the expression level or signal intensity. Moreover, histological analysis of the tissue does not always guarantee that correct cell types are being looked at. Thus it would better if the authors used cell specific markers like Tuj1 (neurons) and GFAP (astrocytes) to conclude their claims.
Major: In result section3.2; the authors state that "Astrocytes supported the highest levels of infection, with 235 approximately 25-fold higher levels of infected cells than that of pericytes". It should be noted, although the graphs indicate that astrocytes are higher, this comparison cannot be done with proper statistical analysis. Based on the figure legends and the figure itself, it does not seem that the authors has applied any statistical test. It would be good to see if the authors can compare the results statically by one-way ANOVA and then add this result in this section. This would make the publication stronger. As mentioned, it does not support their claim.
Major: In the result section 3.3; the authors write "Neurons and choroid plexus epithelial cells displayed similar levels of intracellular infectious virus and virus released into the extracellular medium. In contrast, astrocytes released more infectious virus into the extracellular medium, with a titre of 3.675 x 105/mL compared to 4.39 x 103/mL for cell lysate (Figure 2C)". Again, as mentioned earlier, if such statements are made in a publication. it needs to be supported by strong statistical analysis. The authors show no indication that any statistical tests were run on this set of data. If it has been statistically compared, then it would be great if the authors showed this in this figure. Overall this result section is not supported with the data shown.
Major: In the result section 3.5; The authors write "Neurons supported the highest levels of infection by all variants, and astrocytes and choroid plexus epithelial cells were infected at similar levels (Figure 4A)". The way the data is presented in the manuscript, the only conclusions that can be made is that neurons are shown to be infected by the variants of SARS-CoV2. Similarly astrocytes and choroid plexus are also shown to be infected by the variants. However, this graph is not correct to make conclusions that neurons support higher levels of infection and that astrocytes and choroid plexus support similar levels. If the authors wish to make statements on the level of infection, the neurons, astrocytes, and choroid plexus must be compared to one another and must not be graphed separately as done currently. Overall the results in this section are not fully supported by the data shown.
Minor: Methods section needs to be more detailed about various techniques, controls used, antibodies used, and dilutions of the antibodies used for various experiments in the manuscript.
Minor: It would be informative if the authors could label the images shown in the manuscript to better inform the reader.
Minor: The introduction section needs to include more details about ACE2 and TMPRSS2 and to inform the reader as to what the rationale was for selecting anti-ACE2 neutralizing technique.
Discussion: Overall, the manuscript can be informative to the field to better understand neurological disease after CoV2 infection albeit not in the current form as the authors claims are not fully supported by their data.
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