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To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF) cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101ΔMeq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA) test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the confirmed function of the bi-directional promoter provides better feasibilities to insert multiple foreign genes in MDV genome based vectors.

Construction of Recombinant Marek's Disease Virus (rMDV) Co-Expressing AIV-H9N2-NA and NDV-F Genes under Control of MDV's Own Bi-Directional Promoter