Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2020 Sep;101(9):925-940.
doi: 10.1099/jgv.0.001453.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Natacha S Ogando  1 , Tim J Dalebout  1 , Jessika C Zevenhoven-Dobbe  1 , Ronald W A L Limpens  2 , Yvonne van der Meer  1 , Leon Caly  3 , Julian Druce  3 , Jutte J C de Vries  4 , Marjolein Kikkert  1 , Montserrat Bárcena  2 , Igor Sidorov  1 , Eric J Snijder  1
Affiliations
Comparative Study

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Natacha S Ogando et al. J Gen Virol. 2020 Sep.

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.

Keywords: RNA synthesis; antisera; antiviral drugs; evolution; furin-like cleavage site; plaque phenotype.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Rapid evolution of SARS-CoV-2 during passaging in Vero E6 cells. (a) Outline of a plaque-picking experiment that was initiated when the p2 stock of SARS-CoV-2 Australia/VIC01/2020 showed remarkable plaque heterogeneity on Vero E6 cells (leftmost well). Following a plaque assay of the p1 virus stock, small and large plaques were picked and these virus clones were passaged three times in Vero E6 cells, while their plaque phenotype was monitored. In contrast to the large plaque viruses (example L8; bottom row), the plaque phenotype of the small plaque viruses (example S5; top row) rapidly evolved within these three passages. (b) Evolution/adaptation of the S protein gene during Vero E6 passaging. Overview of NGS data obtained for the p2 stock, S5p1/p2/p3 and S8p1 in the S1/S2 region of the SARS-CoV-2 S protein gene that encodes the so-called furin-like cleavage site. The analysis was based on NGS reads spanning nt 23 576 to 23 665 of the SARS-CoV genome (see Methods for details) and their translation in the S protein ORF. Deletions are indicated with Δ followed by the affected amino acid residues.
Fig. 2.
Fig. 2.
Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells. (a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n=4; mean±sd is presented). (b) Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.
Fig. 3.
Fig. 3.
Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells. (a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV-infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome [110, 111]
Fig. 4.
Fig. 4.
Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins. Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double) labelled with (a) a rabbit antiserum recognizing nsp4 and a mouse mAb recognizing dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c–e) rabbit antisera recognizing against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33 258. Bar is 25 µm for (a) and (b); 100 µm for (c), (d) and (e).
Fig. 5.
Fig. 5.
Visualization of SARS-CoV-2 and SARS-CoV infection by electron microscopy. Electron micrographs of Vero E6 cells infected with either SARS-CoV-2 or SARS-CoV at the indicated time points (c–j). Images from a mock-infected cell are included for comparison (a–b). (c–j) Regions containing viral replication organelles. These virus-induced structures accumulated in large clusters in the perinuclear region by 8 h p.i. [(c), (g), boxed regions enlarged in (d) and (h), respectively]. These regions primarily contained DMVs [(d–e), (h–i), black asterisks]. Additionally, virus-induced convoluted membranes [(e), white arrowhead] were observed in SARS-CoV infection, whereas zippered ER [(i), white arrowheads] appeared to be more common in SARS-CoV-2-infected cells. At 10 h p.i., vesicle packets [(f), (j), white asterisks], which seem to arise by fusion of two or more DMVs through their outer membrane, became abundant in the RO regions. (k–r) Examples of virion assembly and release in infected cells. Virus particles budding into membranes of the ERGIC [(k–l), (o–p), arrowheads]. The black arrowheads in the boxed areas highlight captured budding events, enlarged in (l) and (p). Subsequently, virus particles are transported to the plasma membrane which, at 10 h p.i., is surrounded by a large number of released virions [(m), (q), boxed areas enlarged in (n) and (r), respectively]. N, nucleus; m, mitochondria; G, Golgi apparatus. Scale bars: 1 µm (a, c, g); 500 nm (b, d–f, h–j, k, m, o, q); 100 nm (l, n, p, r).
Fig. 6.
Fig. 6.
Assay to screen for compounds that inhibit SARS-CoV-2 replication. Inhibition of SARS-CoV-2 replication (coloured symbols and curves) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey symbols and curves). For each compound a twofold serial dilution series in the low-micromolar range was tested; (a) remdesivir, (b) chloroquine, (c) alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96 Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of three independent experiments, each performed using quadruplicate samples (mean±sd are shown). A non-linear regression analysis was applied.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020;5:536–544. doi: 10.1038/s41564-020-0695-z. - DOI - PMC - PubMed
    1. Li X, Song Y, Wong G, Cui J. Bat origin of a new human coronavirus: there and back again. Sci China Life Sci. 2020;63:461–462. doi: 10.1007/s11427-020-1645-7. - DOI - PMC - PubMed
    1. McIntosh K, Chao RK, Krause HE, Wasil R, Mocega HE, et al. Coronavirus infection in acute lower respiratory tract disease of infants. J Infect Dis. 1974;130:502–507. doi: 10.1093/infdis/130.5.502. - DOI - PMC - PubMed
    1. Pyrc K, Berkhout B, van der Hoek L. Identification of new human coronaviruses. Expert Rev Anti Infect Ther. 2007;5:245–253. doi: 10.1586/14787210.5.2.245. - DOI - PubMed
    1. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med. 2003;348:1953–1966. doi: 10.1056/NEJMoa030781. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources