SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology
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Abstract
The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that – upon passaging in Vero E6 cells – SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.
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SciScore for 10.1101/2020.04.20.049924: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antisera and immunofluorescence microscopy: The SARS-CoV-specific rabbit or mouse antisera/antibodies used in this study are listed in Table 1. Antiserasuggested: NoneSecondary antibodies used were an Alexa488-conjugated goat anti-rabbit IgG antibody (Invitrogen), a Cy3-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) and an Alexa488-conjugated goat anti-mouse IgG antibody (Invitrogen). anti-rabbit IgGsuggested: Noneanti-mouse…SciScore for 10.1101/2020.04.20.049924: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antisera and immunofluorescence microscopy: The SARS-CoV-specific rabbit or mouse antisera/antibodies used in this study are listed in Table 1. Antiserasuggested: NoneSecondary antibodies used were an Alexa488-conjugated goat anti-rabbit IgG antibody (Invitrogen), a Cy3-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories) and an Alexa488-conjugated goat anti-mouse IgG antibody (Invitrogen). anti-rabbit IgGsuggested: Noneanti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell and virus culture: Vero E6 cells and HuH7 cells were grown as described previously (35). HuH7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)In Leiden, the virus was passaged two more times at low multiplicity of infection (m.o.i.) in Vero E6 cells to obtain a working stock (p2 stock). Vero E6suggested: RRID:CVCL_XD71)Experimental Models: Organisms/Strains Sentences Resources For plaque picking, plaque assays were performed using our p1 stock, while using an overlay containing 1% of agarose instead of Avicel (RC-581; FMC Biopolymer). p1suggested: NoneSoftware and Algorithms Sentences Resources After hybridization, RNA bands were visualised and quantified by phosphorimaging using a Typhoon-9410 variable mode scanner (GE Healthcare) and ImageQuant TL software (GE Healthcare). ImageQuantsuggested: (ImageQuant, RRID:SCR_014246)Subsequently, sequencing reads were screened for the presence of human (GRCh37.75), mouse (GRCm38.p4), E. coli MG1655 (EMBL U00096.2), phiX (RefSeq NC_001422.1) and common vector sequences (UniVec and ChlSab1.1). EMBLsuggested: (ChEMBL, RRID:SCR_014042)RefSeqsuggested: (RefSeq, RRID:SCR_003496)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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