Identification of a Novel Insertion Site HVT-005/006 for the Generation of Recombinant Turkey Herpesvirus Vector

Xusheng Zai; Bin Shi; Hongxia Shao; Kun Qian; Jianqiang Ye; Yongxiu Yao; Venugopal Nair; Aijian Qin

doi:10.3389/fmicb.2022.886873

Identification of a Novel Insertion Site HVT-005/006 for the Generation of Recombinant Turkey Herpesvirus Vector

Front Microbiol. 2022 May 25:13:886873. doi: 10.3389/fmicb.2022.886873. eCollection 2022.

Authors

Xusheng Zai^{1

2

3}, Bin Shi^{1

2

3}, Hongxia Shao^{1

2

3}, Kun Qian^{1

2

3}, Jianqiang Ye^{1

2

3}, Yongxiu Yao⁴, Venugopal Nair⁴, Aijian Qin^{1

2

3}

Affiliations

¹ Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou, China.
² Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China.
³ Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou, China.
⁴ The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Guildford, United Kingdom.

Abstract

Turkey herpesvirus (HVT) has been widely used as a successful live virus vaccine against Marek's disease (MD) in chickens for more than five decades. Increasingly, HVT is also used as a highly effective recombinant vaccine vector against multiple avian pathogens. Conventional recombination, or recombineering, techniques that involve the cloning of viral genomes and, more recently, gene editing methods have been used for the generation of recombinant HVT-based vaccines. In this study, we used NHEJ-dependent CRISPR/Cas9-based approaches to insert the mCherry cassette for the screening of the HVT genome and identifying new potential sites for the insertion of foreign genes. A novel intergenic site HVT-005/006 in the unique long (UL) region of the HVT genome was identified, and mCherry was found to be stably expressed when inserted at this site. To confirm whether this site was suitable for the insertion of other exogenous genes, haemagglutinin (HA) of the H9N2 virus was inserted into this site, and a recombinant HVT-005/006-HA was rescued. The recombinant HVT-HA can grow well and express HA protein stably, which demonstrated that HVT-005/006 is a promising site for the insertion of foreign genes.

Keywords: CRISPR/Cas9; H9N2; HVT vectored vaccine; hemagglutinin; influenza virus; insertion site.