Rapid construction of infectious clones for distinct Newcastle disease virus genotypes

Zuhua Yu; Yuhao Zhang; Zedian Li; Qingzhong Yu; Yanyan Jia; Chuan Yu; Jian Chen; Songbiao Chen; Lei He

doi:10.3389/fvets.2023.1178801

Rapid construction of infectious clones for distinct Newcastle disease virus genotypes

Front Vet Sci. 2023 May 25:10:1178801. doi: 10.3389/fvets.2023.1178801. eCollection 2023.

Authors

Zuhua Yu^#¹, Yuhao Zhang^#¹, Zedian Li¹, Qingzhong Yu², Yanyan Jia¹, Chuan Yu³, Jian Chen¹, Songbiao Chen¹, Lei He¹

Affiliations

¹ The Key Lab of Animal Disease and Public Health/Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Henan University of Science and Technology, Luoyang, Henan, China.
² Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, Athens, GA, United States.
³ Animal Diseases and Public Health Engineering Research Center of Henan Province, Luoyang Polytechnic, Luoyang, Henan, China.

^# Contributed equally.

Abstract

The reverse genetics system of the Newcastle disease virus (NDV) has provided investigators with a powerful approach to understand 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. 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 with the conventional cloning approaches, the two-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.

Keywords: Newcastle disease virus; genome; genotype; rapid construction; reverse genetics.

Grants and funding

This research was supported by the Science and Technology Research Project of Henan Province (Nos. 222102110012 and 232102111036) and the provincial-level Science and Technology Innovation Platform Cultivation Project of the Henan University of Science and Technology (No. 2015SPT004).