{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T03:13:05Z","timestamp":1792120385581,"version":"4.4.0"},"reference-count":29,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T00:00:00Z","timestamp":1758240000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science and Technology Program of Guangzhou City of China","award":["2023B03J1310"],"award-info":[{"award-number":["2023B03J1310"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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\u2014most notably the hexon gene (89% identity to HNF-70)\u2014suggesting altered immunogenicity. Using Red\/ET recombineering, we established a rapid (less than 3 weeks) reverse genetics platform and generated four E3-modified recombinants: \u0394E3-All-eGFP, \u0394E3-12.5K-eGFP, \u0394E3-12.5K+ORF4-eGFP, and E3-Insert-eGFP. Crucially, the \u0394E3-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 107.0 TCID50\/mL. Fluorescence intensity was inversely correlated with genome size, as the full-length E3-Insert-eGFP virus showed reduced expression compared with the \u0394E3 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.<\/jats:p>","DOI":"10.3390\/v17091270","type":"journal-article","created":{"date-parts":[[2025,9,19]],"date-time":"2025-09-19T12:33:32Z","timestamp":1758285212000},"page":"1270","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Isolation of Porcine Adenovirus Serotype 5 and Construction of Recombinant Virus as a Vector Platform for Vaccine Development"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-8129-5502","authenticated-orcid":false,"given":"Qianhua","family":"He","sequence":"first","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhilong","family":"Bian","sequence":"additional","affiliation":[{"name":"Artemis Shield Animal Health Co., Ltd., Guangzhou 510000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyun","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"570","DOI":"10.3181\/00379727-84-20714","article-title":"Isolation of a Cytopathogenic Agent from Human Adenoids Undergoing Spontaneous Degeneration in Tissue Culture","volume":"84","author":"Rowe","year":"1953","journal-title":"Proc. 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