Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells
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Abstract
Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments.
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SciScore for 10.1101/2021.06.24.449252: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical approval was granted by the institutional review board of Amsterdam UMC (METC 2020.167).
Consent: As described by Schurink et al., 2020 COVID-19 was confirmed by quantitative real-time RT-PCR, and informed consent was obtained from the decedents’ next of kin.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Then, for LM, semi-thin sections were incubated on primary antibody for 1 hour in PBS + 0.1% bovine serum albumin (Sigma, A4503-50G) and washed with PBS + 0.02M glycine. A4503-50Gsuggested: NoneIn this case, background blocking was … SciScore for 10.1101/2021.06.24.449252: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical approval was granted by the institutional review board of Amsterdam UMC (METC 2020.167).
Consent: As described by Schurink et al., 2020 COVID-19 was confirmed by quantitative real-time RT-PCR, and informed consent was obtained from the decedents’ next of kin.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Then, for LM, semi-thin sections were incubated on primary antibody for 1 hour in PBS + 0.1% bovine serum albumin (Sigma, A4503-50G) and washed with PBS + 0.02M glycine. A4503-50Gsuggested: NoneIn this case, background blocking was done by 0.1% BSA in PBS + 0.02M glycine, followed by incubation on rabbit anti mouse antibody (Z0259, DAKO) for 20 minutes and washed with PBS + 0.02M glycine. anti mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources EM Infection and fixation of Cultured Vero Cells: Vero E6 were seeded (2.5×106 cells/T75 flask) one day before infection in MEM/25mM HEPES/2% fetal calf serum with penicillin and streptomycin. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Images were analyzed using imageJ FIJI. imageJsuggested: (ImageJ, RRID:SCR_003070)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Double membrane vesicles (DMVs), have been described in several EM studies [2,3,5,15–18] but are not so obvious in our immuno-EM images; only a few double membranes were identified surrounding e-lucent compartments (Fig S4, blue arrows), possible due to fixation limitations, as shown before by Snijder et al., 2006 [16]. As double membranes were not recognizable, DMVs were not annotated in this study, and thus, it remains unclear if the DMVs detected in other studies are lipid filled. Interestingly, our CLEM data (Figs 2 and 4) demonstrated that only part of the e-lucent compartments are lipid-filled. This could be due to the fact that only a subclass of the compartments are lipid filled, or could have a technical explanation. Lipids are notoriously difficult to fix with glutaraldehyde and paraformaldehyde alone [61], and thus part of the compartments might have lost the lipid content. High resolution EM studies on cryo-preserved cells suggest DMVs to be filled with viral RNA with LD lying next to the DMVs [18]. For other viruses, lipid accumulation has been shown to be involved in viral replication [62–69] and some studies have demonstrated lipid accumulations in SARS-CoV-2-infected Vero cells [54] and also in infected human pulmonary epithelial Calu-3 cells [13]. Nardacci et al., 2021, demonstrated that lipid accumulation is specific for SARS-CoV-2 and not for SARS-CoV-1 in a comparative electron microscopy study and established an increase of LD in lungs from deceased COVID...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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