PMC:7091888 / 28818-30190 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7091888","sourcedb":"PMC","sourceid":"7091888","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7091888","text":"SARS-CoV-2 pseudovirus was generated, as previously described, with some modifications.25,27,29 Briefly, 293T cells were cotransfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding SARS-CoV-2 S protein using the calcium phosphate method. SARS-CoV and MERS-CoV pseudoviruses were packaged as controls. The transfected medium was changed into fresh Dulbecco’s Modified Eagle’s Medium (DMEM) 8 h later, and pseudovirus-containing supernatants were collected 72 h later for single-cycle infection in target cells. Pseudovirus neutralization assay was then performed by incubation of SARS-CoV-2, SARS-CoV, or MERS-CoV pseudovirus with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera for 1 h at 37 °C, followed by addition of the mixture into hACE2/293T (for SARS-CoV-2 pseudovirus and SARS-CoV pseudovirus) or hDPP4/293T (for MERS-CoV pseudovirus) target cells. Fresh medium was added 24 h later, and the cells were lysed 72 h later in cell lysis buffer (Promega, Madison, WI). The lysed cell supernatants were incubated with luciferase substrate (Promega) and detected for relative luciferase activity using the Infinite 200 PRO Luminator (Tecan). The 50% MERS pseudovirus neutralizing antibody titer (NT50) was calculated using the CalcuSyn computer program, as previously 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