Dual-expression-site fowl adenovirus 4 (FAdV-4) with high viral titer: a promising viral vector for multivalent poultry vaccines

Abstract

Fowl adenovirus serotype 4 (FAdV-4) is a nonenveloped double-stranded DNA virus with a 43–45 kb genome, making it a promising viral vector for developing multivalent poultry vaccines. However, limited genomic studies have hindered the design of strategies for generating recombinant FAdV-4 capable of expressing multiple foreign proteins while maintaining high viral titers. In this study, three deletion mutants (rD14AB, rD2042, rD19A) were generated to assess their roles in viral replication and pathogenicity. Only rD2042 showed impaired replication and virulence compared with wild-type FAdV-4. Foreign gene expression assays demonstrated that the D14AB site had a maximum insertion capacity of 2349 bp, and an SV40 poly(A) signal was required for efficient protein expression and viral propagation at this locus. In contrast, the D19A site tolerated insertions of up to 1257 bp in the absence of this signal. Using the insertion strategy optimized for wild-type CH_JS_2017, we modified the previously reported nonpathogenic rON1 strain (harboring the Hexon gene from the attenuated ON1 strain) to co-express mCherry, eGFP, and mTagBFP2 at the two independent sites. SPF chicken trials verified the safety of the engineered virus at immunization age and the genetic stability of exogenous genes. The recombinant virus maintained wild-type-comparable titers while carrying 3606 bp of total foreign sequences, highlighting its dual-site advantage and potential as a multivalent poultry vaccine vector.