The transcription of HAdV-3 hexon gene of rAd3H was confirmed by RT-PCR analysis as follows: Total RNA was extracted by RNAiso (TAKARA; Tokyo, Japan) and reverse transcribed into cDNA using the primer HVRR (5′-TTTCTGAAGTTCCACTCGTAGGTGTA-3′). This resultant cDNA was used as the template in a PCR amplification with the primer pairs, HVRF1 (5′-CAGGATGCTTCGGAGTACCTGAG-3′) and HVRR (Zhang et al. 2020 (link)). The PCR product was identified using agarose gel electrophoresis and DL10000 markers. Subsequent Western blot analysis confirmed expression of the hexon protein. A culture of rAd3H was frozen and thawed three times, centrifuged at 1000 ×g for 10 min, and the supernatant was collected. The samples were added to loading buffer, boiled for 10 min, electrophoresed in 5% concentrated gel, and 10% Tris-Tricine SDS-PAGE. After PAGE, the proteins were transferred onto nitrocellulose membrane by electrophoresis at 25 V for 40 min. The membrane was probed first by binding with mouse anti-HAdV-3-hexon antibody (Guangzhou HuYanSuo Medical Technology; Guangzhou, China) as the epitope-specific antibody (1:1000 dilution), and followed with goat anti-mouse IgG-HRP (Bioworld; Georgia, USA) as the secondary amplifying antibody. Luminol and hydrogen peroxide were used as chromogenic substrates to score the fluorescence signal.