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
• Avian Proteins 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

The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl).Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV.IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.

• Publication type
• Journal Article MeSH

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

• Keywords
• CRISPR/Cas9 genome editing in chicken, Na+/H+ exchanger type 1, avian leukosis virus subgroup J, disease resilience in poultry, primordial germ cells,
• MeSH
• CRISPR-Cas Systems MeSH
• Gene Editing MeSH
• Animals, Genetically Modified genetics   immunology   virology   MeSH
• Chickens MeSH
• Poultry Diseases genetics   immunology   virology   MeSH
• Disease Resistance MeSH
• Avian Leukosis genetics   immunology   virology   MeSH
• Avian Proteins genetics   immunology   MeSH
• Sodium-Hydrogen Exchanger 1 genetics   immunology   MeSH
• Avian Leukosis Virus classification   genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Avian Proteins MeSH
• Sodium-Hydrogen Exchanger 1 MeSH

It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].

• MeSH
• Humans MeSH
• Retroviridae classification   genetics   isolation & purification   physiology   MeSH
• Retroviridae Infections virology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Introductory Journal Article MeSH
• Editorial MeSH

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

• Keywords
• Tva, avian leukosis virus K, host range, resistance/susceptibility to retrovirus, retrovirus receptor, superinfection interference,
• MeSH
• Cell Line MeSH
• Species Specificity MeSH
• Fibroblasts cytology   metabolism   virology   MeSH
• Virus Internalization MeSH
• Mesocricetus MeSH
• Chickens MeSH
• Disease Susceptibility MeSH
• Avian Leukosis genetics   metabolism   virology   MeSH
• Avian Proteins genetics   metabolism   MeSH
• Receptors, Virus genetics   metabolism   MeSH
• Avian Leukosis Virus classification   pathogenicity   physiology   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

Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.

• Keywords
• CRISPR/Cas9, avian leukosis virus, retrovirus receptor, virus-resistance in chicken,
• MeSH
• Cell Line MeSH
• CRISPR-Cas Systems * MeSH
• Gene Editing * MeSH
• Genetic Techniques MeSH
• Genetic Vectors genetics   MeSH
• Chickens MeSH
• Disease Resistance genetics   MeSH
• Avian Leukosis genetics   virology   MeSH
• Base Sequence MeSH
• Genes, Viral MeSH
• Receptors, Virus genetics   metabolism   MeSH
• Avian Leukosis Virus physiology   MeSH
• RNA, Guide, CRISPR-Cas Systems MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Virus MeSH
• RNA, Guide, CRISPR-Cas Systems MeSH

UNLABELLED: The J subgroup of avian leukosis virus (ALV-J) infects domestic chickens, jungle fowl, and turkeys. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na+/H+ exchanger 1. The resistance to avian leukosis virus subgroup J in a great majority of galliform species has been explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na+/H+ exchanger 1. Because there are concerns of transspecies virus transmission, we studied natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to infection with avian leukosis virus subgroup J, and the cloned Na+/H+ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new avian leukosis virus subgroup J variants but resistant to subgroups A and B and weakly susceptible to subgroups C and D of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the avian leukosis virus subgroup J could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential for further evolution. IMPORTANCE: Since its spread in broiler chickens in China and Southeast Asia in 2000, ALV-J remains a major enzootic challenge for the poultry industry. Although the virus diversifies rapidly in the poultry, its spillover and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the evolution of the virus and its potential host range in wild birds. Because resistance to avian retroviruses is due particularly to receptor incompatibility, we studied Na+/H+ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible with virus entry, and we confirmed the susceptibilities of four New World quail species in vitro We propose that a prospective molecular epidemiology study be conducted to identify species with the potential to become reservoirs for ALV-J.

• Keywords
• ALV-J, Na+/H+ exchanger, New World quail, antiretroviral resistance, retroviral receptor,
• MeSH
• Amino Acids MeSH
• Gene Expression MeSH
• Phylogeny MeSH
• Genetic Loci MeSH
• Host Specificity MeSH
• Host-Pathogen Interactions MeSH
• Protein Interaction Domains and Motifs MeSH
• Quail * MeSH
• Cells, Cultured MeSH
• Evolution, Molecular MeSH
• Sodium-Hydrogen Exchangers chemistry   genetics   metabolism   MeSH
• Disease Susceptibility * MeSH
• Disease Resistance genetics   MeSH
• Polymorphism, Genetic MeSH
• Avian Leukosis genetics   metabolism   virology   MeSH
• Virus Replication MeSH
• Amino Acid Sequence MeSH
• Avian Leukosis Virus classification   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids MeSH
• Sodium-Hydrogen Exchangers MeSH

J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey and enters the host cell through a receptor encoded by tvj locus and identified as Na+/H+ exchanger 1 (NHE1). The resistance to ALV-J in a great majority of examined galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of NHE1, and genetic polymorphisms around this site predict the susceptibility or resistance of a given species or individual. In this study, we examined the NHE1 polymorphism in domestic chicken breeds and documented quantitative differences in their susceptibility to ALV-J in vitro. In a panel of chicken breeds assembled with the aim to cover the maximum variability encountered in domestic chickens, we found a completely uniform sequence of NHE1 extracellular loop 1 (ECL1) without any source of genetic variation for the selection of ALV-J-resistant poultry. In parallel, we studied the natural polymorphisms of NHE1 in wild ducks and geese because of recent reports on ALV-J positivity in feral Asian species. In anseriform species, we demonstrate a specific and highly conserved critical ECL1 sequence without any homologue of tryptophan 38 in accordance with the resistance of duck cells to prototype ALV-J. Last, we demonstrated that the new Asian strains of ALV-J have not evolved their envelope glycoprotein to the entry the duck cells. Our results contribute substantially to the current discussion of possible heterotransmission of ALV-J and its spill-over into the wild ducks and geese.

• MeSH
• Animals, Wild * MeSH
• Genetic Variation * MeSH
• Animals, Domestic * MeSH
• Cells, Cultured MeSH
• Chickens MeSH
• Chick Embryo MeSH
• Molecular Sequence Data MeSH
• Sodium-Hydrogen Exchangers chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Animals MeSH
• Check Tag
• Chick Embryo MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Hydrogen Exchangers MeSH