Abstract
Porcine adenovirus serotype 5 (PAdV-5) is an emerging viral vector platform for veterinary vaccines; however, its genomic plasticity and essential replication elements remain incompletely characterized. This study reports the isolation and reverse genetic manipulation of a novel PAdV-5 strain (GD84) from diarrheic piglets in China. PCR screening of 167 clinical samples revealed a PAdV-5 detection rate of 38.3% (64/167), with successful isolation on ST cells after three blind passages. The complete GD84 genome is 32,620 bp in length and exhibited 99.0% nucleotide identity to the contemporary strain Ino5, but only 97.0% to the prototype HNF-70. It features an atypical GC content of 51.0% and divergent structural genes-most notably the hexon gene (89% identity to HNF-70)-suggesting altered immunogenicity. Using Red/ET recombineering, we established a rapid (less than 3 weeks) reverse genetics platform and generated four E3-modified recombinants: ΔE3-All-eGFP, ΔE3-12.5K-eGFP, ΔE3-12.5K+ORF4-eGFP, and E3-Insert-eGFP. Crucially, the ΔE3-All-eGFP construct (complete E3 deletion) failed to be rescued, while constructs preserving the 12.5K open reading frame (ORF) yielded replication-competent viruses with sustained eGFP expression over three serial passages and titers over 10(7.0) TCID(50)/mL. Fluorescence intensity was inversely correlated with genome size, as the full-length E3-Insert-eGFP virus showed reduced expression compared with the ΔE3 variants. Our work identifies the 12.5K ORF as essential for PAdV-5 replication and provides an optimized vaccine engineering platform that balances genomic payload capacity with replicative fitness.