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

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

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 nonstructural 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.

Authors 11

1. Natacha S. Ogando Aachen Molecular Virology Laboratory Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

2. Tim J. Dalebout Aachen Molecular Virology Laboratory Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

3. Jessika C. Zevenhoven-Dobbe Aachen Molecular Virology Laboratory Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

4. Ronald W.A.L. Limpens Aachen Section Electron Microscopy Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

5. Leon Caly

   Victorian Infectious Diseases Reference Laboratory · Peter Doherty Institute

   Affiliation as printed

   Virus Identification Laboratory, Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia

6. Julian D Druce

   Victorian Infectious Diseases Reference Laboratory · Peter Doherty Institute

   Affiliation as printed

   Virus Identification Laboratory, Victorian Infectious Diseases Reference Laboratory, Doherty Institute, Melbourne, Australia

7. Jutte J.C. de Vries Aachen Clinical Microbiology Laboratory Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Clinical Microbiology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

8. Marjolein Kikkert Aachen Molecular Virology Laboratory Department of Medical Microbiology

   Leiden University Medical Center

   Affiliation as printed

   Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

9. Montserrat Bárcena Aachen Section Electron Microscopy Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

10. Igor A. Sidorov Aachen Molecular Virology Laboratory Department of Medical Microbiology

    Leiden University Medical Center

    Affiliation as printed

    Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands

11. Eric J. Snijder corresponding Aachen Molecular Virology Laboratory Department of Medical Microbiology

    Leiden University Medical Center

    Affiliation as printed

    Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands