Extended Data Fig. 2: Exclusion of the possible effect of anti-HBV agents on VG161 replication in vitro and in vivo.

a, b, Comparison of the blood VG161 viral DNA concentration between patients who received (n = 6 biologically independent patients) or did not receive (n = 5 biologically independent patients) oral administration of anti-HBV agents within 24 h. c, Female NCG mice were implanted subcutaneously with Hep3B cells and gavaged with entecavir (0.1 mg/kg/day) or PBS (n = 3 biologically independent animals). Intratumoral injection of 1 × 10⁷ PFU VG161 was performed 3 days after gavage. Hep3B tumors were excised at indicated time (0, 0.5, 1, 2, 3, and 7 days). The virus titers of tumor lysate were measured by plaque assay on Vero cells. d, The in vitro cytotoxicity of different anti-HBV agents on the tumor cells. Cells were treated with anti-HBV agents, and cell viability was measured using the CCK-8 assay at 65 h (6.7 × 10⁴ cells examined over 4 independent experiments). e, Hep3B cells were treated with or without anti-HBV agents for 12 h, followed by replacement with fresh medium and infection with VG161. The cell viability upon different MOI and pretreatments was measured using the CCK-8 assay at 65 h (n = 6.7 × 10⁴ cells examined over 4 independent experiments). f, The effect of anti-HBV agent treatment on VG161 replication ability in Hep3B cells. Hep3B cells were treated with or without anti-HBV agents for 12 h, followed by replacement with fresh medium and infection with VG161 for 24, 48, 72 h. The culture supernatant were collected, mixed, and lysed, then titrated by plaque assay (n = 0.8 × 10⁶ cells examined over 3 independent experiments). For b, Fisher’s exact test was used for analysis. For d and e, No statistical tests were performed to compare differences. For a, c and f, Data are presented as the mean ± standard error and were analyzed using the unpaired two-sided Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and NS, not significant).

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