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

Abstract

35 36 The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 37 at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread 38 highlight the importance of understanding the full molecular details of coronavirus infection 39 and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and 40 SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the 41 commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher 42 levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was 43 recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-44 infected cells established extensive cross-reactivity of antisera previously raised against a 45 variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron 46 microscopy revealed that the ultrastructural changes induced by the two SARS viruses are 47

The coding capacity of SARS-CoV-2

“…We noticed three of these junctions represent short in-frame deletions in the spike protein (5aa, 7aa and 10aa long) that overlap deletions that were recently described by other groups 15,20,21 , in which the furin-like cleavage site is deleted ( Figure S11). The re-occurrence of the same genomic deletion strongly supports the conclusion that this deletion is being selected for during passage in Vero cells.…”

“…When we compared the frequency of junctions between 5h and 24h time points, the leader dependent junctions (both canonical and non-canonical) and the genomic deletions correlated well but the leader independent junctions were specifically increased at 24 hpi ( Figure 2C). Recent kinetic measurements show viral particles already bud out of infected Vero cells at 8 hpi 21 . Therefore, this time-dependent increase in non-canonical RNA junctions indicates that the leader independent RNA junctions are likely associated with genomic replication.…”

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

“…However, Vero-E6 cells are a model cell line for isolating viruses and were selected based on their susceptibility to SARS-CoV-2-induced death. Further, African green monkeys are susceptible to SARS-CoV-2 infection (19)(20)(21). Interestingly, we did not detect several genes previously implicated in SARS-CoV-2 infection including TMPRSS2, PIKFyve, and TPC2 suggesting that these genes are either not essential in a Vero cell-intrinsic manner or that functional redundancies exist with other genes (15,45).…”

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers

“…Each viral sample (2 mL) was incubated in duplicate in a 25 cm 2 cell culture flask, together with 5 mL of DMEM medium, at 37°C and at 5% CO 2 atmospheric pressure for 72 h. At the end of the 72 h waiting period, 5 mL of new DMEM medium was added to the culture flask, which was again incubated at 37°C with a CO 2 level of 5% for 48 h. Finally, infectivity was assessed daily by screening cells for cytopathic effects (CPE) under reverse-phase light microscope. Limit of Detection (LOD) of infectivity for SARS-CoV-2 on VERO E6 cells is not available, although replication kinetics, adaptation and CPE on these cells has been verified and described (Ogando et al, 2020). Estimates of LOD based on CPE observation on VERO cells were found to be in the range of 1 × 10 1 -1 × 10 −2 TCID 50 for a wide group of viral families (Gombold et al, 2014).…”