Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

Torovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen.