Examining the feasibility of replacing ORF3a with fluorescent genes to construct SARS-CoV-2 reporter viruses

The SARS-CoV-2 genome encodes at least nine accessory proteins, including innate immune antagonist and putative viroporin ORF3a. ORF3a plays a role in many stages of the viral replication cycle, including immune modulation. We constructed two recombinant (r)SARS-CoV-2 viruses in which the ORF3a gene was replaced with mScarlet (mS) or mNeonGreen (mNG), denoted rSARS-CoV-2-Δ3a-mS and rSARS-CoV-2-Δ3a-mNG, respectively. rSARS-CoV-2-Δ3a-mNG generated a fluorescent signal after infection in both A549-ACE-2-TMPRSS2 (AAT) and VeroE6-TMPRSS2 (VTN) cells, unlike rSARS-CoV-2-Δ3a-mS. rSARS-CoV-2-Δ3a-mS mS protein could be detected immunologically in VTN but not AAT cells, indicating expression of a non-fluorescent mS protein. Analysis of the viral transcriptomes in infected AAT cells by nanopore direct RNA sequencing revealed the level of mS transcript was below the limit of detection in AAT cells. rSARS-CoV-2-Δ3a-mNG virus was found to be genetically stable in AAT and VTN cells, but rSARS-CoV-2-Δ3a-mS acquired partial d