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 systémy MeSH
• editace genu MeSH
• fibroblasty virologie   metabolismus   MeSH
• kur domácí * virologie   MeSH
• kuřecí embryo MeSH
• molekulární evoluce MeSH
• nemoci drůbeže virologie   genetika   MeSH
• odolnost vůči nemocem genetika   MeSH
• proteiny virového obalu genetika   metabolismus   MeSH
• ptačí leukóza * virologie   genetika   MeSH
• virus ptačí leukózy * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny virového obalu 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.

• Klíčová slova
• Tva, avian leukosis virus K, host range, resistance/susceptibility to retrovirus, retrovirus receptor, superinfection interference,
• MeSH
• buněčné linie MeSH
• druhová specificita MeSH
• fibroblasty cytologie   metabolismus   virologie   MeSH
• internalizace viru MeSH
• křeček rodu Mesocricetus MeSH
• kur domácí MeSH
• náchylnost k nemoci MeSH
• ptačí leukóza genetika   metabolismus   virologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• virové receptory genetika   metabolismus   MeSH
• virus ptačí leukózy klasifikace   patogenita   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ptačí proteiny MeSH
• Tva receptor MeSH Prohlížeč
• virové receptory MeSH

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

• MeSH
• Alpharetrovirus genetika   fyziologie   MeSH
• genetická predispozice k nemoci * MeSH
• introny MeSH
• kur domácí genetika   metabolismus   virologie   MeSH
• molekulární sekvence - údaje MeSH
• nemoci drůbeže genetika   metabolismus   virologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• ptačí sarkom genetika   metabolismus   virologie   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční delece * MeSH
• sestřih RNA * MeSH
• virové receptory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ptačí proteiny MeSH
• Tva receptor MeSH Prohlížeč
• virové receptory MeSH

The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.

• MeSH
• butyrofiliny MeSH
• klonování DNA MeSH
• kultivované buňky MeSH
• kur domácí MeSH
• membránové glykoproteiny fyziologie   MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• terminační kodon MeSH
• virové receptory chemie   genetika   fyziologie   MeSH
• virus ptačí leukózy fyziologie   MeSH
• viry ptačího sarkomu fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• butyrofiliny MeSH
• membránové glykoproteiny MeSH
• terminační kodon MeSH
• virové receptory MeSH

The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tva(s) encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tva(s) cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tva(s) cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 7(2). Line C tva(r) contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of Tva(R) for the ASLV(A) envelope glycoproteins. Line 7(2) tva(r2) contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.

• MeSH
• křepelky a křepelovití MeSH
• kultivované buňky MeSH
• kur domácí imunologie   virologie   MeSH
• kuřecí embryo MeSH
• molekulární sekvence - údaje MeSH
• mutace * MeSH
• ptačí leukóza imunologie   virologie   MeSH
• ptačí proteiny MeSH
• ptačí sarkom imunologie   virologie   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• virové receptory chemie   genetika   metabolismus   MeSH
• virus ptačí leukózy metabolismus   patogenita   MeSH
• viry ptačího sarkomu metabolismus   patogenita   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• ptačí proteiny MeSH
• Tva receptor MeSH Prohlížeč
• virové receptory MeSH