Avian leukosis virus (ALV), the prototypical alpharetrovirus, causes tumorigenesis, immunosuppression, and wasting disease in poultry. The ALV genus is classified into ten subgroups, which differ in their host range, cell tropism, and receptor usage. The subgroups A, B, K, and J cause significant economic losses worldwide. The most recently discovered subgroup, ALV-K, which is now widespread in China, has been shown to use the tva cell receptor and share it with ALV-A. However, the specific amino acid residues crucial for ALV-K host cell entry remain unknown. Using precise tva expression and chimeric tva receptors, we further elucidated the significance of the cysteine-rich domain in mediating interactions with both ALV-A and ALV-K. Through a comprehensive analysis of mutated tva receptor variants, we pinpointed tryptophan at position 33 (W33) as a pivotal amino acid residue essential for ALV-K virus binding and entry. Of note is the finding that the substitution of W33 induced resistance to ALV-K while preserving sensitivity to ALV-A. This study not only represents an advance in the understanding of the specificity of the tva receptor for ALV-K, but also offers a biotechnological strategy for the prevention of ALV-K infections in poultry.

• Keywords
• avian leukosis virus, chicken, guineafowl, tva receptor,
• MeSH
• Cell Line MeSH
• Virus Internalization * MeSH
• Chickens MeSH
• Poultry Diseases virology   MeSH
• Virus Attachment * MeSH
• Avian Leukosis virology   MeSH
• Amino Acid Substitution MeSH
• Receptors, Virus * genetics   metabolism   chemistry   MeSH
• Avian Leukosis Virus * physiology   genetics   classification   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Avian Proteins MeSH
• Tva receptor MeSH Browser
• Receptors, Virus * MeSH

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

• MeSH
• CRISPR-Cas Systems MeSH
• Gene Editing MeSH
• Fibroblasts virology   metabolism   MeSH
• Chickens * virology   MeSH
• Chick Embryo MeSH
• Evolution, Molecular MeSH
• Poultry Diseases virology   genetics   MeSH
• Disease Resistance genetics   MeSH
• Viral Envelope Proteins genetics   metabolism   MeSH
• Avian Leukosis * virology   genetics   MeSH
• Avian Leukosis Virus * genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Viral Envelope Proteins MeSH

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.

• Keywords
• avian leukosis virus subgroups A/K, gene editing in chicken, tva, vitamin B12/cobalamin,
• MeSH
• Gene Editing MeSH
• Gene Knockout Techniques MeSH
• Chickens virology   MeSH
• Chick Embryo MeSH
• Methylmalonic Acid blood   MeSH
• Frameshift Mutation MeSH
• Avian Proteins genetics   physiology   MeSH
• Receptors, Virus genetics   physiology   MeSH
• Avian Leukosis Virus classification   physiology   MeSH
• Vitamin B 12 metabolism   MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Methylmalonic Acid MeSH
• Avian Proteins MeSH
• Tva receptor MeSH Browser
• Receptors, Virus MeSH
• Vitamin B 12 MeSH