Rescue of SARS-CoV-2 from a single bacterial artificial chromosome

Abstract

An infectious coronavirus disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC).Recombinant (r)SARS-CoV-2 was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, the BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the SARS-CoV-2 natural isolate. Likewise, rSARS-CoV-2 showed similar levels of replication to that of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays similar features in vivo to that of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

Generation and Characterization of Recombinant SARS-CoV-2 Expressing Reporter Genes

“…In Vero E6 cells, rSARS-CoV-2 expressing reporter genes have similar growth kinetics and plaque phenotype than that of wild-type virus (rSARS-CoV-2/WT). Importantly, we have observed a correlation between reporter gene expression and viral replication (21), and infected cells can be easily detected, without the need of secondary approaches, based on reporter gene expression. Using these reporterexpressing rSARS-CoV-2, we have developed fluorescent-based microneutralization assays that can be used to identify neutralizing antibodies (NAbs) and/or antivirals.…”

“…However, these reverse genetics protocols require laborious in vitro assembly and transcription steps prior to transfecting cells, an inconvenience that should be considered due to the laborious nature and restraint of these methods. Here, we describe the generation and characterization of replication-competent rSARS-CoV-2 expressing fluorescent Venus or mCherry, or bioluminescent Nluc reporter genes using on January 17, 2021 by guest http://jvi.asm.org/ Downloaded from our recently described bacterial artificial chromosome (BAC)-based reverse genetics approach (21,22). In Vero E6 cells, rSARS-CoV-2 expressing reporter genes have similar growth kinetics and plaque phenotype than that of wild-type virus (rSARS-CoV-2/WT).…”

Selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants

“…An important consideration for selecting an appropriate cell line for the proposed studies is permissibility to SARS-CoV-2 infection. In this study, we used Vero E6 cells, a cell line that has been shown to be permissive to SARS-CoV-2 infection and widely used for the study of SARS-CoV-2 ( Chiem et al, 2020 ; Ye et al, 2020 ; Oladunni et al, 2020 ; Park et al, 2021 ). Other permissive cell types, such as Vero E6 transfected to express transmembrane protease serine 2 (TMPRSS2), a serine protease which activates SARS-CoV-2 infection ( Matsuyama et al, 2020 ); human 293 T ( Crawford et al, 2020 ) or A549 ( Chen et al, 2020 ) cells constitutively expressing the human angiotensin converting enzyme 2 (hACE2), or Calu3 cells ( Felgenhauer et al, 2020 ), could be used instead of Vero E6 cells for the proposed experiments.…”

“…For the experiments in this manuscript we used SARS-CoV-2 isolate USA-WA1/2020 obtained from BEI Resources ( Chiem et al, 2020 ; Ye et al, 2020 ; Oladunni et al, 2020 ; Park et al, 2021 ). We recommend using a multiplicity of infection (MOI) of 0.001 for the first passage in a 12-well plate.…”

“…However, deep sequencing analysis of the entire viral genome will reveal detailed genetic alterations spanning the entire viral genome which could possibly be involved in the selection for an escape mutant. Therefore, we recommend to conduct deep sequencing analysis of the SARS-CoV-2 MARM, as previously described ( Ye et al, 2020 ). Moreover, we also recommend conducting full sequence analysis of the SARS-CoV-2 passaged in the presence of the irrelevant Ig antibody to differentiate mutations related to the passage of the virus in cultured Vero E6 cells from those responsible for the lack of neutralization by the MARMs.…”