First step in characterization of cis-acting sequences involved in fowl adenovirus 1 (CELO) packaging and its effect on the development of a helper-dependent vector strategy.

Adenovirus-based vectors are widely developed for potential utilization as vectors in vaccine and gene therapy strategies. We focused on developing a helper-dependent adenoviral (HD-Ad) vector for the potential use of CELO, a member of the Aviadenovirus genus, in avian species vaccination. Our aim was to localize sequences which could play an essential role in CELO genome encapsidation and, when deleted, was unable to produce viruses to develop a helper CELO virus. A panel of 6 mutants with deletions between nt 80 and 350 of the CELO genome was constructed and characterized for its ability to produce viable virus. To develop a helper-dependent adenoviral vector derived from CELO, a helper virus was developed by inserting loxP sequences around the region containing the identified putative packaging sequences. A LMH (Leghorn Male Hepatocarcinoma) cell line expressing Cre recombinase was developed to allow the excision of this region. We demonstrated that the region from nt 200 to 250 was important and the region from nt 250 to 300 at the left end of the CELO genome was essential for virus encapsidation. We also showed that the loxP-flanked region was efficiently removed in a Cre expressing cell line to produce a candidate helper virus.