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

Abstract

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.

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

“…Replication-competent, reporter-expressing, recombinant viruses have been previously shown to represent an excellent approach to study, among others, viral infection, replication, pathogenesis, and transmission (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). We and others have described the feasibility of generating rSARS-CoV-2 expressing reporter genes encoding either fluorescent or luciferase proteins (36,43,44,46,47).…”

“…The copyright holder for this preprint this version posted June 24, 2022. ; https://doi.org/10.1101/2022.06. 23.497376 doi: bioRxiv preprint Reverse genetics systems have permitted the generation of recombinant RNA viruses entirely from cloned cDNA, facilitating studies to better understand multiple aspects of the biology of viruses, including, among others, mechanisms of viral infection, pathogenesis, transmission, and disease (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Another application of reverse genetics is the generation of recombinant viruses containing gene mutations and/or deletions that result in viral attenuation for their implementation as safe, immunogenic, and protective liveattenuated vaccines (LAV) (20,(30)(31)(32)(33)(34)(35).…”