tetano
Editor, Senior Moderator
J Gen Virol
. 2020 Jun 22.
doi: 10.1099/jgv.0.001453. Online ahead of print.
SARS-coronavirus-2 Replication in Vero E6 Cells: Replication Kinetics, Rapid Adaptation and Cytopathology
Natacha S Ogando[SUP] 1 [/SUP], Tim J Dalebout[SUP] 1 [/SUP], Jessika C Zevenhoven-Dobbe[SUP] 1 [/SUP], Ronald W A L Limpens[SUP] 2 [/SUP], Yvonne van der Meer[SUP] 1 [/SUP], Leon Caly[SUP] 3 [/SUP], Julian Druce[SUP] 3 [/SUP], Jutte J C de Vries[SUP] 4 [/SUP], Marjolein Kikkert[SUP] 1 [/SUP], Montserrat B?rcena[SUP] 2 [/SUP], Igor Sidorov[SUP] 1 [/SUP], Eric J Snijder[SUP] 1 [/SUP]
Affiliations
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.
. 2020 Jun 22.
doi: 10.1099/jgv.0.001453. Online ahead of print.
SARS-coronavirus-2 Replication in Vero E6 Cells: Replication Kinetics, Rapid Adaptation and Cytopathology
Natacha S Ogando[SUP] 1 [/SUP], Tim J Dalebout[SUP] 1 [/SUP], Jessika C Zevenhoven-Dobbe[SUP] 1 [/SUP], Ronald W A L Limpens[SUP] 2 [/SUP], Yvonne van der Meer[SUP] 1 [/SUP], Leon Caly[SUP] 3 [/SUP], Julian Druce[SUP] 3 [/SUP], Jutte J C de Vries[SUP] 4 [/SUP], Marjolein Kikkert[SUP] 1 [/SUP], Montserrat B?rcena[SUP] 2 [/SUP], Igor Sidorov[SUP] 1 [/SUP], Eric J Snijder[SUP] 1 [/SUP]
Affiliations
- PMID: 32568027
- DOI: 10.1099/jgv.0.001453
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.