Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

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. Importance ToVs are diarrhea-causing pathogens that have been detected in many species, including humans. BToV has spread worldwide, leading to economic losses. We developed the first reverse genetics system for Tobaniviridae using a BAC-based BToV. Using this system, we showed that recombinant BToVs with HEf and HEs showed no significant differences in cell growth. In contrast, clinical BToVs generally lose the HE gene after a few passages but some recombinant viruses retained the HE gene for up to 20 passages, suggesting some benefits of HE retention. The EGFP gene of the recombinant viruses was unstable and was rapidly deleted, likely via negative selection. Interestingly, one virus variant with mutations in NSPs was more stable, resulting in improved EGFP-expression and viral growth, suggesting that the mutations contributed to some acceptance of the exogenous EGFP gene without clear positive selection. The recombinant BToVs and reverse genetics developed here are powerful tools for understanding fundamental viral processes and their pathogenesis and for developing BToV vaccines. ### Competing Interest Statement The authors have declared no competing interest.