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United States Department of Agriculture

Agricultural Research Service

Research Project: Intervention Strategies to Control and Prevent Enteric Viral Diseases of Poultry

Location: Endemic Poultry Viral Diseases Research Unit

Title: Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain

Authors
item Zhao, Wei -
item Hu, Haixia -
item Zsak, Laszlo
item Yu, Qingzhong
item Yang, Zengqi -

Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 19, 2013
Publication Date: August 6, 2013
Citation: Zhao, W., Hu, H., Zsak, L., Yu, Q., Yang, Z. 2013. Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain. Plasmid Journal. 70:314-320.

Interpretive Summary: Newcastle disease virus (NDV) can cause serious diseases and significant economic losses to the poultry industry worldwide. To understand the molecular biology and pathogenesis of NDV, infectious clones for different NDV strains have been made. However, the construction of infectious clone of the virus by conventional cloning methods is a time-consuming process and may take years to accomplish. In the present study, we proposed a novel and robust ligation-independent cloning (LIC) method for construction of an infectious clone. Using this method, we successfully generated a NDV minigenome construct within three weeks by two steps of LIC with the virus genomic terminal fragments and the green fluorescence protein (GFP) gene, as a reporter. Through detecting the reporter GFP expression in cell culture, we demonstrated that the NDV minigenome generated by the LIC method was able to replicate and express a foreign gene product. These results suggest that this LIC approach is a powerful tool for complex genetic manipulations, such as the construction of NDV infectious clone. This LIC approach may have a potential application for rapid development of gene therapy and recombinant vaccines.

Technical Abstract: Newcastle disease virus (NDV) can cause serious diseases and substantial economic losses to the poultry industry. To gain a better understanding of NDV pathogenesis, several reverse genetics systems for different NDV strains have been established. However, the construction of infectious cDNA clone by conventional restriction digestion/ligation cloning methods is a time-consuming process and has many drawbacks by its nature. To address the problems, we employed a novel and robust ligation-independent cloning (LIC) method for efficient assembly of multiple DNA fragments. Using this method, we successfully generated a NDV minigenome construct within three weeks by assembling RT-PCR products of the VG/GA strain genomic termini and a cDNA coding for the green fluorescence protein (GFP), as a reporter, into a modified pBluescript vector. Co-transfection of the NDV minigenome with three supporting plasmids expressing the N, P, and L proteins into MVA-T7 infected HEp-2 cells and followed by infection with NDV VG/GA resulted in the minigenome replication, transcription, and packaging as evidenced by the reporter gene GFP expression. These results suggest that this LIC approach is a powerful tool for all sequence-independent DNA cloning and multi-DNA fragment assembly, which has a potential application for rapid development of gene therapy and recombinant vaccines.

Last Modified: 10/1/2014
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