Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid

Abstract

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus

“…Tva is involved in TC-mediated Cbl uptake ( 29 ). Although Tva-deficient gene-edited chickens have been generated and could completely resist ALV-K infection, they exhibit specific Cbl metabolic disturbances due to the lack of Tva ( 42 ). In this study, E53, L55, H59, and G70 were identified as the key residues for Tva to interact with gp85 and mediate ALV-K entry into host cells.…”

Genetic Polymorphism of Avian Leukosis Virus Host Receptors in Korean Native Chickens and Establishment of Resistant Line

“…Recently reported data demonstrate that the avian TVA receptor protein belongs to the LDLR family, is an ortholog of mammalian CD320, and acts as a vitamin B 12 transporter as well as a viral receptor (Krchlikova et al, 2021). Further, tva knockout chickens generated using CRISPR/Cas9 technology are not only resistant to ALV-A and -K, but also exhibit vitamin deficiency (Koslova et al, 2021). Growth retardation due to vitamin deficiency was not observed in KO and KNC individuals resistant to ALV subgroups, and further studies are needed to determine whether there are other genes that can compensate for lack of vitamin uptake by the TVA receptor.…”

“…In addition, deletion of nucleotides in the intronic region of the chicken tva gene (tva r3 , tva r4 , tva r5 , and tva r6 ) affects the branch point signal involved in splicing, resulting in incomplete mRNA splicing and decreased susceptibility to ALV-A (Reinisova et al, 2012;Chen et al, 2015). Further, recent findings demonstrate that ALV-K shares the TVA receptor with ALV-A (Prikryl et al, 2019), and knockout of Tva in chicken using the CRISPR/Cas9 system confers resistance to both ALV-A and -K (Koslova et al, 2021).…”

Advances on genetic and genomic studies of ALV resistance

“…These deletions disrupt mRNA splicing of the Tva receptor gene and prematurely introduced the TGA stop codon, thereby reducing sensitivity to ALV-A [ 77 , 78 ]. Recent findings have demonstrated that ALV-A and ALV-K-resistant individuals can be produced after Tva knockout in chicken primordial germ cells (PGCs) using CRISPR/Cas9 gene editing technology [ 79 ].…”

“…In other words, the mutation NHE1 ΔW38 mediates the chicken resistance against HPRS-103, and the W38 deletion has no negative effects on chicken growth and health [ 175 – 177 ]. Furthermore, using the same method, a transgenic commercial chicken line resistant to ALV-A/K was obtained [ 79 ]. Those results shows that the viral receptor of ALV is not necessary in the growth cycle of chickens, and it can be eliminated to obtain resistance (Fig.…”