Supplementary file 1_Biological characterization of a clinical human adenovirus type 3 isolate with oncolytic potential.docx

Viral genome amplification kinetics were evaluated in human respiratory-related and tumor-derived cell lines and a murine cell line by quantitative PCR. Comparative apoptosis induction by HAdV-3-TZ01 and HAdV-5 was assessed in 293F cells infected at a low multiplicity of infection using Annexin V/PI flow cytometry. Antitumor activity was evaluated following repeated intratumoral administration of HAdV-3-TZ01 in an A549-luciferase xenograft model in BALB/c-nu nude mice, with tumor growth and bioluminescence monitored longitudinally.</p>Results<p>Genomic and phylogenetic analyses confirmed that HAdV-3-TZ01 belonged to HAdV-3 within species B and exhibited a genomic backbone highly conserved with contemporary circulating HAdV-3 strains.

A short putative HAdV-7-derived recombinant fragment was identified. HAdV-3-TZ01 showed efficient viral genome amplification in multiple human respiratory-related cell lines, including A549, H3122, BEAS-2B, MRC-5, and HEK293FT cells, whereas genome amplification was markedly restricted in murine L929 cells. HAdV-3-TZ01 induced a significantly higher proportion of late apoptotic cells than HAdV-5 in 293F cells under low-multiplicity infection conditions, although the absolute increase in total apoptosis was modest.

In the A549-luciferase xenograft model, intratumoral administration of HAdV-3-TZ01 significantly suppressed tumor progression. By day 43, mean tumor volume was reduced by approximately 54% and tumor-associated bioluminescence by approximately 50% compared with mock-treated controls. No significant body-weight loss or overt signs of distress were observed under the experimental conditions.</p>Conclusion<p>HAdV-3-TZ01 retains the characteristic human epithelial tropism of HAdV-3 and exhibits efficient viral genome amplification in human epithelial-derived cells, apoptosis induction, and measurable antitumor activity in vivo.

The apoptosis difference between HAdV-3-TZ01 and HAdV-5 should be interpreted cautiously because the analysis was limited to 293F cells and did not establish a tumor-specific pro-apoptotic mechanism. As an unmodified respiratory pathogen, HAdV-3-TZ01 should not be considered a directly translatable therapeutic agent. Rather, these findings support HAdV-3-TZ01 as a potential starting backbone for future genetic engineering aimed at improving tumor selectivity and safety.</p>