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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Publications at this Location » Publication #403269
Research Project: Systems Biology Approaches to Develop Medical Countermeasures to Detect, Prevent, and Control Poultry Production Viral Diseases

Location: Endemic Poultry Viral Diseases Research

Title: Rapid construction of a reverse genetics rescue system for distinct Newcastle disease virus genotypes

Author
item YU, ZUHUA - Henan University Of Science And Technology
item ZHANG, YUHAO - Henan University Of Science And Technology
item LI, ZEDIAN - Henan University Of Science And Technology
item Yu, Qingzhong
item JIA, YANYAN - Henan University Of Science And Technology
item YU, CHUAN - Henan University Of Science And Technology
item CHEN, JIAN - Henan University Of Science And Technology
item CHEN, SONGBIAO - Henan University Of Science And Technology
item HE, LEI - Henan University Of Science And Technology

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2023
Publication Date: 5/25/2023
Citation: Yu, Z., Zhang, Y., Li, Z., Yu, Q., Jia, Y., Yu, C., Chen, J., Chen, S., He, L. 2023. Rapid construction of a reverse genetics rescue system for distinct Newcastle disease virus genotypes. Frontiers in Veterinary Science. 10. Article 1178801. https://doi.org/10.3389/fvets.2023.1178801.
DOI: https://doi.org/10.3389/fvets.2023.1178801

Interpretive Summary: Virulent strains of Newcastle disease virus (NDV) can cause a severe poultry disease, resulting in significant economic losses to the poultry industry worldwide. Since the 1990s, a new molecular technology, commonly called reverse genetics (RG), has been developed to study the molecular biology of the virus and develop recombinant vaccines against NDV or other targeted poultry diseases. This RG technology is involved in cloning a complete genome of NDV into a plasmid vector, which is difficult and time-consuming work due to the complexity and length of the NDV genome. To improve and simplify the NDV cloning procedure, in this study, we developed a two-step ligation-independent cloning (LIC) method to assemble a full-length cDNA clone of different genotype NDV strains and rescue an infectious NDV within three weeks. This two-step LIC approach significantly reduced the number of cloning steps and saved researchers substantial time constructing NDV infectious clones. Therefore, this novel cloning approach may apply to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.

Technical Abstract: The reverse genetics system of Newcastle disease virus (NDV) has provided investigators with a powerful approach to understanding viral molecular biology and vaccine development. It has been impressively improved with modified strategies since its first report, but it still poses some challenges. Most noteworthy, the genome complexity and length made full-length error-free cDNA assembly the most challenging and time-consuming step of NDV rescue. In the present study, we report a rapid full-length NDV genome construction with only a two-step ligation-independent cloning (LIC) strategy, which could be applied to distinct genotypes of NDVs. In this approach, the genome of NDV was divided into two segments, and the cDNA clones were generated by RT-PCR followed by LIC. Subsequently, the infectious NDVs were rescued by co-transfection of the full-length cDNA clones and supporting plasmids expressing the NP, P, and L proteins of NDV in BHK-21 cells. Compared to the conventional cloning approaches, the 2-step cloning method drastically reduced the number of cloning steps and saved researchers a substantial amount of time for constructing NDV infectious clones, thus enabling a rapid rescue of different genotypes of NDVs in a matter of weeks. Therefore, this two-step LIC cloning strategy may have an application to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.