Construction and characterization of novel fowlpox virus shuttle vectors

• Fowlpox virus (FPV) loci FPV150 and FPV193 were chosen to new insert sites.
• Two FPV shuttle vectors with a triple-gene expression cassette were constructed.
• Recombinant FPV were generated and purified using the eGFP reporter gene.
• Three exogenous genes were expressed simultaneously and stably in multiple passages.
• Foreign gene insertion did not affect recombinant FPV morphogenesis or replication.

Viral vectors are important vehicles in vaccine research. Avipoxviruses including fowlpox virus (FPV) play major roles in viral vaccine vector development for the prevention and therapy of human and other veterinary diseases due to their immunomodulatory effects and safety profile. Recently, we analyzed the genomic and proteomic backgrounds of the Chinese FPV282E4 strain. Based on analysis of the whole genome of FPV282E4, the FPV150 and FPV193 loci were chosen as insertion sites for foreign genes, and two shuttle vectors with a triple-gene expression cassette were designed and constructed. Homologous recombination between the FPV virus genome and sequences within the shuttle plasmids in infected cells was confirmed. The recombinants were obtained through several rounds of plaque purification using enhanced green fluorescent protein as a reporter and evaluated for the correct expression of foreign genes in vitro using RT-PCR, real-time PCR and Western blotting. Morphogenesis and growth kinetics were assayed via transmission electron microscopy and viral titering, respectively. Results showed that recombinant viruses were generated and correctly expressed foreign genes in CEF, BHK-21 and 293T cells. At least three different exogenous genes could be expressed simultaneously and stably over multiple passages. Additionally, the FPV150 mutation, FPV193 deletion and insertion of foreign genes did not affect the morphogenesis, replication and proliferation of recombinant viruses in cells. Our study contributes to the improvement of FPV vectors for multivalent vaccines.