A Novel Approach For Expression Of Foreign Genes By Newcastle Disease Virus And The Evaluation Of Its Optimal Insertion Site For Foreign Genes Expression

Newcastle disease virus(NDV) is the causative agent of Newcastle disease(ND), one of the most important poultry diseases worldwide, affecting a wide variety of birds and causing significant economic loss in the poultry industry. NDV strains have been classified into lentogenic,mesogenic or velogenic pathotypes. Velogenic strains are further classified into viscerotropic strains and neurotropic velogenic strains. Viscerotropic velogenic strains produce lethal hemorrhagic lesions in the digestive tract with high mortality. Since the first NDV strain was successfully rescued, somel reverse genetics systems for different strains of NDV have been generated. The foreign genes are usually inserted into a non-coding region of the NDV genome as an independent transcription unit(ITU). Based on the well-accepted “stop-start” transcription mechanism for non-segmented, negative-stranded RNA viruses, the addition of ITU in the NDV genome would attenuate its downstream gene transcription, and subsequently interfere with virus replication and the level of foreign gene expression.For better understanding and systematically evaluating the optimal insertion sitefor NDV rLa Sota strain foreign gene expression, we developed a novel approach for foreign gene expression by NDV from a second open reading frame(2nd ORF) through an internal ribosomal entry site(IRES). We generated six NDV La Sota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein(RFP) gene in the NP, P, M, F, HN, or L gene using reverse genetics technology. The insertion of the 2nd ORF slightly attenuated the virus pathogenicity, but did not affect virus growth ability. Quantitative measurements of the RFP expression from recombinant virus infected DF-1 cells revealed that the abundance of viral m RNAs containing RFP and red fluorescence intensity were positively correlated with the gene order of NDV, 3’ NP-P-M-F-HN-L,proving the sequential transcription mechanism on NDV.In the meanwhile, the result of the growth curves showed that the all of the rescued viruses kept their reproduction characteristics. Quantification of RFP m RNA expression by real-time RT-PCR suggested that there is a gradient of m RNA abundance according to the gene position. In addition, the measurement of RFP fluorescence intensity from the infected DF-1 cells showed that the recombinant virus with RFP insertion at the the NP gene downstream non-coding region would be the best choice for the foreign gene insertion.The results suggested that the levelof foreign gene expression could be regulated by selecting the 2nd ORF insertion site relative to the 3’ end of the NDV vector to maximize the efficacy of vaccine and gene therapy.