Comparative transcriptional activity of five promoters in BAC-cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus

•A Meq-deleted MDV was used as the vector, which was confirmed to have no pathogenicity.•The bi-directional promoter is a novel promoter used for the construction of recombination MDV.•This study turned our attention to the influence of different promoters for transcription of H9N2-AIV HA gene in recombination MDV.•Recombination MDVs were compared both at mRNA and protein level. On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters. These five promoters included three MDV endogenous promoters (the promoter for the gB gene and a bi-directional promoter in both directions for pp38 (ppp38) and 1.8kb RNA transcripts (p1.8kb)), and two exogenous promoters (CMV and SV40). Among these five promoters, the CMV promoter demonstrated the highest activity, followed by p1.8kb and SV40, which had a similar transcriptional activity level. Two of the MDV endogenous promoters showed much lower transcriptional activities, particularly the promoter ppp38, which had the lowest activity. The results of the in vivo experiment proved that none of the three recombinant viruses of rGX-CMV-HA, rGX-SV40-HA and rGX-p1.8kb-HA provided protection in SPF chickens. Chickens vaccinated with rGX-pPP38-HA induced 50% and rGX-gB-HA induced 25% protection against the challenge with H9N2, respectively.