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

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.

• Klíčová slova
• avian leukosis virus subgroups A/K, gene editing in chicken, tva, vitamin B12/cobalamin,
• MeSH
• editace genu MeSH
• genový knockout MeSH
• kur domácí virologie   MeSH
• kuřecí embryo MeSH
• kyselina methylmalonová krev   MeSH
• posunová mutace MeSH
• ptačí proteiny genetika   fyziologie   MeSH
• virové receptory genetika   fyziologie   MeSH
• virus ptačí leukózy klasifikace   fyziologie   MeSH
• vitamin B 12 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina methylmalonová MeSH
• ptačí proteiny MeSH
• Tva receptor MeSH Prohlížeč
• virové receptory 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.

• Publikační typ
• časopisecké články MeSH

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

• MeSH
• lidé MeSH
• Retroviridae klasifikace   genetika   izolace a purifikace   fyziologie   MeSH
• retrovirové infekce virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodní články MeSH
• úvodníky 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

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.

• Klíčová slova
• CRISPR/Cas9, avian leukosis virus, retrovirus receptor, virus-resistance in chicken,
• MeSH
• buněčné linie MeSH
• CRISPR-Cas systémy * MeSH
• editace genu * MeSH
• genetické techniky MeSH
• genetické vektory genetika   MeSH
• kur domácí MeSH
• odolnost vůči nemocem genetika   MeSH
• ptačí leukóza genetika   virologie   MeSH
• sekvence nukleotidů MeSH
• virové geny MeSH
• virové receptory genetika   metabolismus   MeSH
• virus ptačí leukózy fyziologie   MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• virové receptory MeSH
• vodící RNA, systémy CRISPR-Cas MeSH

The extent of virus transmission among individuals and species is generally determined by the presence of specific membrane-embedded virus receptors required for virus entry. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor is the first and crucial step in determining host specificity. Using a well-established retroviral model-avian Rous sarcoma virus (RSV)-we analyzed changes in an RSV variant that had repeatedly been able to infect rodents. By envelope gene (env) sequencing, we identified eight mutations that do not match the already described mutations influencing the host range. Two of these mutations-one at the beginning (D32G) of the surface Env subunit (SU) and the other at the end of the fusion peptide region (L378S)-were found to be of critical importance, ensuring transmission to rodent, human, and chicken cells lacking the appropriate receptor. Furthermore, we carried out assays to examine the virus entry mechanism and concluded that these two mutations cause conformational changes in the Env variant and that these changes lead to an activated, or primed, state of Env (normally induced after Env interaction with the receptor). In summary, our results indicate that retroviral host range extension is caused by spontaneous Env activation, which circumvents the need for original cell receptor. This activation is, in turn, caused by mutations in various env regions.

• Klíčová slova
• Rous sarcoma virus, envelope glycoprotein, receptor-independent entry, retrovirus, virus entry,
• MeSH
• genetické vektory * genetika   metabolismus   MeSH
• genové produkty env * genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kur domácí MeSH
• lidé MeSH
• missense mutace * MeSH
• nádorové buněčné linie MeSH
• substituce aminokyselin MeSH
• transdukce genetická * MeSH
• virus Rousova sarkomu * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env * 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.

• Klíčová slova
• ALV-J, Na+/H+ exchanger, New World quail, antiretroviral resistance, retroviral receptor,
• MeSH
• aminokyseliny MeSH
• exprese genu MeSH
• fylogeneze MeSH
• genetické lokusy MeSH
• hostitelská specificita MeSH
• interakce hostitele a patogenu MeSH
• interakční proteinové domény a motivy MeSH
• křepelky a křepelovití * MeSH
• kultivované buňky MeSH
• molekulární evoluce MeSH
• Na(+)-H(+) antiport chemie   genetika   metabolismus   MeSH
• náchylnost k nemoci * MeSH
• odolnost vůči nemocem genetika   MeSH
• polymorfismus genetický MeSH
• ptačí leukóza genetika   metabolismus   virologie   MeSH
• replikace viru MeSH
• sekvence aminokyselin MeSH
• virus ptačí leukózy klasifikace   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• Na(+)-H(+) antiport MeSH

This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.

• Klíčová slova
• cell transformation, nonpermissiveness to virus infection, virus integration, virus rescue,
• MeSH
• buněčné linie MeSH
• fúze buněk * MeSH
• genom virový genetika   MeSH
• genové produkty env genetika   MeSH
• krysa rodu Rattus MeSH
• kur domácí virologie   MeSH
• onkogenní protein pp60(v-src) genetika   MeSH
• proviry genetika   růst a vývoj   MeSH
• virová transformace buněk MeSH
• virus Rousova sarkomu genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• onkogenní protein pp60(v-src) 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
• divoká zvířata * MeSH
• genetická variace * MeSH
• hospodářská zvířata * MeSH
• kultivované buňky MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• molekulární sekvence - údaje MeSH
• Na(+)-H(+) antiport chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Na(+)-H(+) antiport MeSH