Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720

Cytocidal Effect of Irradiation on Gastric Cancer Cells Infected with a Recombinant Mammalian Orthoreovirus Expressing a Membrane-Targeted KillerRed

“…In contrast, we generated recombinant MRV with the KRmem expression cassette inserted into the 3 ′ end of the M1 segment, T3D-L(M1/3 ′ KRmem), in the present study; however, it has not been evaluated further because it clearly showed lower growth activity than T3D-L(S2/3 ′ KRmem). Unlike iRFP720 gene (951 bp) insertion [32], T3D-L(S2/3 ′ KRmem) was genetically stable in the present study. This stability was likely attributed to the insertion of a smaller gene (774 bp), which had a smaller effect on viral replication activity.…”

“…T3D-L(S2/3 ′ KRmem) could infect and replicate in GC cell lines but not in normal gastrointestinal epithelial cells. However, the infectivity in GC cell lines was not high, as shown previously [32]. Junctional adhesion molecule A (JAM-A) has been identified as an entry receptor for MRVs [46,47] and is downregulated in several cancer cells [48].…”

“…Previously, we evaluated the possible insertion sites of the iRFP720 expression cassette for bicistronic expression and found that the 3 ′ end of the M1 segment was suitable for insertion [32]. Furthermore, recombinant MRV with the iRFP720 expression cassette inserted into the 3 ′ end of the S2 segment could be recovered; however, the insertion was genetically unstable [32]. In contrast, we generated recombinant MRV with the KRmem expression cassette inserted into the 3 ′ end of the M1 segment, T3D-L(M1/3 ′ KRmem), in the present study; however, it has not been evaluated further because it clearly showed lower growth activity than T3D-L(S2/3 ′ KRmem).…”

“…Multi-step viral growth in BHK/T7-9 cells was examined as described previously [32]. In brief, each virus was inoculated into monolayered cells at a multiplicity of infection (MOI) of 0.01, and the inoculated cells were incubated at 37 • C. The culture medium was harvested after 24, 48, 72, and 96 h of incubation, and viral titers were determined as described above.…”

“…This system has enabled the generation of recombinant MRVs that express foreign genes [ 30 , 31 ]. Recently, we also generated a recombinant MRV expressing iRFP720 (a near-infrared fluorescent protein) for use in fluorescence-guided surgery and identified the genomic site that tolerates the functional expression of a foreign gene [ 32 ].…”