Fig 1.
Genome structure diagram of recombinant viruses.
Various oncolytic viruses were obtained by genetically modifying the viral genome of the vaccine strain LC16m8 as a starting material. The gene nomenclature used in this paper is that used for the vaccinia virus Copenhagen strain. Genes associated with viral attenuation and EEV productivity are indicated by dashed lines and black arrows, respectively. Black arrows are genes from the IHD-J (except for F12L) and IHD-W (F12L) strains. Env means the genes of extracellular enveloped virus-associated proteins.
Table 1.
Primers used to confirm modified BACmids and MD-RVV-ΔRR-EEV6.
Fig 2.
Growth of F4L-deficient (ΔRR) virus in HeLa cells.
HeLa cells were inoculated with LC16m8-BACgfp, LC16m8-B5RmO-BACgfp, MD-RVV-BACgfp, and MD-RVV-ΔRR-BACgfp at MOI 1 PFU/cell. After 24 and 48 hours, the growth of each virus was observed under a fluorescence microscope.
Fig 3.
Effects of F4L-deficient (ΔRR) modified viruses on the cytotoxicity of cancer cells and normal cells.
Cancer cells (A; HeLa) and normal cells (B; NHDF) were inoculated with 8.0 × 104 (white) and 4.0 × 105 (black) PFU/well of the indicated modified viruses. After culturing for 72 hours, the cell viability of each virus-inoculated group was quantified using a cell counting kit, with the absorbance of the control group without the virus inoculation being set to 100%. Data are shown the mean ± SEM (n = 3). Statistical significance for between LC16m8-B5RmO, MD-RVV, and MD-RVV-ΔRR in experiments using NHDF is indicated by two-tailed paired Student’s t-test p-values (B).
Fig 4.
Effects of the modification of EEV-related genes on viral productivity in HeLa cells.
HeLa cells were inoculated with the modified virus without (white) and with (black) the deletion of F4L (ΔRR) at MOI 1 PFU/cell. After 16, 24, 32, and 48 hours, the infectivity titer of the viruses was evaluated using RK13 cells. Data are shown the mean ± SEM (n = 2).
Fig 5.
Increased production of extracellular and intracellular viruses by modifying EEV-related genes.
HeLa cells were adherently cultured in a serum-containing medium and then inoculated with the modified virus in a chemically defined medium at MOI 0.01 PFU/cell. After 48 hours of culture, the extracellular virus in the culture supernatant (A) and intracellular (B) viruses were collected. The infectivity titers of the viruses were evaluated using RK13 cells. Data are shown the mean ± SEM (n = 2).
Fig 6.
Survival rates of immunodeficient mice intravenously administered modified viruses.
The modified viruses LC16m8-B5RmO (blue), MD-RVV (green), and MD-RVV-ΔRR-EEV6 (red) were intravenously administered once to five immunodeficient mice in each group at doses of 2.5 × 104 (Low), 5.0 × 105 (Medium), and 1.0 × 107 (High) PFU/mouse. The log-rank test was used to compare survival distributions between the indicated groups to determine p-values.
Fig 7.
Body weight changes in immunodeficient mice intravenously administered modified viruses.
The modified viruses LC16m8-B5RmO (blue), MD-RVV (green), and MD-RVV-ΔRR-EEV6 (red) were intravenously administered once to five immunodeficient mice in each group at doses of 2.5 × 104 (triangle marks), 5.0 × 105 (square marks), and 1.0 × 107 (round marks) PFU/mouse. As a control group (black; cross marks), the same volume of phosphate-buffered saline was administered. Body weights are expressed as the mean values of surviving mice.
Fig 8.
Virus symptom score changes in immunodeficient mice intravenously administered modified viruses.
The modified viruses LC16m8-B5RmO (blue), MD-RVV (green), and MD-RVV-ΔRR-EEV6 (red) were intravenously administered once to five immunodeficient mice in each group at doses of 2.5 × 104 (triangle marks), 5.0 × 105 (square marks), and 1.0 × 107 (round marks) PFU/mouse. As a control group (black; cross marks), the same volume of phosphate-buffered saline was administered. Scores represent the sum of virus symptom scores in each treatment group. The definition of an individual’s viral symptom score is as follows: 0: no symptoms; 1: 1 or 2 tail lesion(s); 2: 3 or 4 tail lesions; 3: more than 4 tail lesions, a rough coat, piloerection; 4: dyspnea, moribund; 5: death.
Fig 9.
Typical symptoms in mice treated with modified viruses.
Photographs of viral symptoms in representative mice from each group 15 days after the administration of modified viruses are shown. The modified viruses LC16m8-B5RmO, MD-RVV, and MD-RVV-ΔRR-EEV6 were intravenously administered once to five immunodeficient mice in each group at doses of 5.0 × 105 PFU/mouse. As a control group, the same volume of phosphate-buffered saline (PBS) was administered.