Location: Endemic Poultry Viral Diseases ResearchTitle: 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
|ZHANG, ZHENYU - Northeast Agricultural University, China|
|ZHAO, WEI - Beijing Centers For Disease And Prevention, Department Of Pest Inspection|
|LI, DESHAN - Northeast Agricultural University, China|
|YANG, JINLONG - Chongqing Academy Of Animal Sciences|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/18/2017
Publication Date: 7/17/2017
Citation: Yu, Q., Zhang, Z., Zhao, W., Li, D., Yang, J., Zsak, L. 2017. 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 [abstract]. The 17th International Congress of Virology, July 17-21, 2017, Singapore. p. 21.
Technical Abstract: Objectives: Newcastle disease virus (NDV), a member of the Paramxoviridae family, 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), which potentially attenuates its downstream gene transcription, and subsequently interferes with virus replication and the level of foreign gene expression. To overcome the drawback of the IUT method, in the present study, 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). Methods: The IRES sequence and a red fluorescence protein (RFP) reporter gene were inserted behind the NP, P, M, F, HN, or L gene ORF in a NDV LaSota strain-based infectious clone as a 2nd ORF. Six NDV recombinant viruses vectoring the IRES/RFP gene at each of the six viral genes were rescued using reverse genetics technology. The pathogenicity and growth dynamics of theses six viruses were compared with their parental virus in chicks, embryonating eggs, and cell cultures. The viral mRNAs containing the RFP gene obtained from recombinant virus infected DF-1 cells were detected by quantitative RT-PCR, and the RFP fluorescence intensities in the infected cells were measured by using a fluorescence microplate reader. Results: 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 the red fluorescence intensity were positively correlated with the gene order of NDV 3’ NP>P>M>F>HN>L. The IRES/RFP gene inserted into the 3’ proximal NP gene expressed the highest level of RFP, whereas that inserted into the 5’ proximal L gene expressed the lowest level of RFP. Conclusion: The results provide direct proof for a sequential transcription mechanism on NDV. The novel NDV vector could be used to express a foreign gene in a controlled manner by selecting a 2nd ORF insertion site relative to the 3’ end of the NDV vector to maximize the efficacy of vaccine and gene therapy.