Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism

Authors: ZHANG, ZHENYU - Northeast Agricultural University; ZHAO, WEI - Beijing Center For Diseases Prevention And Control; LI, DESHAN - Northeast Agricultural University; YANG, JINLONG - Chongqing Academy Of Animal Sciences; Zsak, Laszlo; Yu, Qingzhong

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2015
Publication Date: 8/1/2015
Publication URL: http://handle.nal.usda.gov/10113/61622
Citation: Zhang, Z., Zhao, W., Li, D., Yang, J., Zsak, L., Yu, Q. 2015. Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism. Journal of General Virology. 96:2028-2035. doi:10.1099/vir.0.000142.

Interpretive Summary: Virulent strains of Newcastle disease virus (NDV) are the causative agents of Newcastle disease (ND), affecting a wide variety of birds and causing significant economic losses to the poultry industry worldwide. Some of low virulent NDV strains, such as LaSota and B1, have been used as vaccines to protect poultry against ND worldwide and have also been developed as live vaccine vectors to express foreign genes for bivalent vaccine and gene therapy purposes. Usually the foreign gene is flanked by the NDV gene start and gene end sequences and inserted into the non-coding region of the NDV genome as an additional transcription unit, which may interfere with virus replication and result in a low level of the foreign gene expression. In the present study we developed a novel approach for foreign gene expression by NDV from a second open reading frame (ORF) through an internal ribosomal entry site (IRES). The results showed that the insertion of the second ORF slightly attenuated the virus virulence, but did not affect virus growth ability. Quantitative measurements of the foreign gene expression proved the sequential transcription mechanism on NDV. The results suggested that the level of foreign gene expression could be regulated by selecting the second ORF insertion site relative to the 3’ end of the NDV genome to maximize the efficacy of vaccine and gene therapy.

Technical Abstract: Newcastle disease virus (NDV) has been developed as a vector to express foreign genes for vaccine and gene therapy purposes. 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. In the present study, we developed a novel approach for foreign gene expression by NDV from a second open reading frame (ORF) through an internal ribosomal entry site (IRES). We generated six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene after the NP, P, M, F, HN, or L gene ORF 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 mRNAs 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. The results suggested that the level of 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.