Live Imaging of Oncolytic Mammalian Orthoreovirus Expressing NanoLuc Luciferase in Tumor Xenograft Mice.

Wild-type mammalian reoviruses (MRVs) have been evaluated as oncolytic agents against various cancers; however, genetic modification methods for improving MRV agents have not been exploited fully. In the present study, using MRV strain T1L, we generated a reporter MRV that expresses a NanoLuc luciferase (NLuc) gene and used it for noninvasive imaging of MRV infection in tumor xenograft mice. NLuc and a P2A self-cleaving peptide gene cassette were placed upstream of the L1 gene open reading frame to enable bicistronic expression of NLuc and the L1 gene product. BALB/c nude mice intranasally infected with MRV expressing NLuc (rsT1L-NLuc) displayed bioluminescent signals in the chest area at 4 days postinfection (dpi), which is consistent with natural MRV infection in the lung. Furthermore, to monitor tumor-selective infection by MRV, nude mice bearing human cancer xenografts were infected intravenously with rsT1L-NLuc. Bioluminescent signals were detected in tumors as early as 3 dpi and persisted for 2 months. The results demonstrate the utility of an autonomous replicating reporter MRV for noninvasive live imaging of replicating oncolytic MRV agents. Engineering of recombinant MRV for improved oncolytic activity has not yet been achieved due to difficulty in generating autonomous replicating MRV harboring transgenes. Here, we constructed a reporter MRV that can be used to monitor cancer-selective infection by oncolytic MRV in a mouse model. Among the numerous oncolytic viruses, MRV has an advantage in that the wild-type virus shows marked oncolytic activity in patients without any notable adverse effects. The reporter MRV developed herein will open avenues to the development of recombinant MRV vectors armed with anticancer transgenes.