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

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.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene

“…In this study, a rapid and efficient method for the generation of recombinant FAdV-4 using the CRISPR/Cas9 and Cre-Loxp technique was first described. There are several approaches for targeted knock-in through CRISPR/Cas9 system, including homologous recombination (HR) (Bi et al, 2014;Zhang et al, 2017), non-homologous end joining (NHEJ) (Chang et al, 2017;Tang et al, 2018;Zai et al, 2022), microhomology-mediated end joining (MMEJ) (Nakade et al, 2014;Sakuma et al, 2016), and HMEJ (Yao et al, 2017). The HR requires a repair template with left and right homology arms (500-3,000 bp) that allowing precise insertion of exogenous genes.…”

Metabolomics analysis of CEF cells infected with avian leukosis virus subgroup J based on UHPLC-QE-MS

“…Chicken embryo fibroblast ( CEF ) cells were prepared from 9-day-old embryos as described previously ( Zai et al, 2022 ). CEF cells were grown in Dulbecco's Modified Eagle's Medium ( DMEM ) (Thermo Fisher Scientific, Shanghai, China) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific, Shanghai, China), 10% tryptone phosphate broth (Sigma, Shanghai, China), and 100 µg/mL of penicillin and streptomycin (Thermo Fisher Scientific, Shanghai, China) at 37°C in 5% CO 2 atmosphere.…”

Metabolomic profiling of Marek’s disease virus infection in host cell based on untargeted LC-MS

“…Nine-day-old specific-pathogen-free embryonated chicken embryos (purchased from Lihua) were used to generate chicken embryo fibroblasts (CEFs). As described in our previous report (Zai et al, 2022), CEFs were cultured with M199 medium (Gibco), supplemented with 5% fetal bovine serum (FBS; Lonsera), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (HAKATA). The cells were seeded into the culture flasks (SORFA) and placed at 37°C in a humidified atmosphere with 5% CO 2 .…”