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.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are key RNA virus sensors belonging to the RIG-I-like receptor (RLR) family. The activation of the RLR inflammasome leads to the establishment of antiviral state, mainly through interferon-mediated signaling. The evolutionary dynamics of RLRs has been studied mainly in mammals, where rare cases of RLR gene losses were described. By in silico screening of avian genomes, we previously described two independent disruptions of MDA5 in two bird orders. Here, we extend this analysis to approximately 150 avian genomes and report 16 independent evolutionary events of RIG-I inactivation. Interestingly, in almost all cases, these inactivations are coupled with genetic disruptions of RIPLET/RNF135, an ubiquitin ligase RIG-I regulator. Complete absence of any detectable RIG-I sequences is unique to several galliform species, including the domestic chicken (Gallus gallus). We further aimed to determine compensatory evolution of MDA5 in RIG-I-deficient species. While we were unable to show any specific global pattern of adaptive evolution in RIG-I-deficient species, in galliforms, the analyses of positive selection and surface charge distribution support the hypothesis of some compensatory evolution in MDA5 after RIG-I loss. This work highlights the dynamic nature of evolution in bird RNA virus sensors.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3. We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4−CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.

Two key cytosolic receptors belonging to the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family sense the viral RNA-derived danger signals: RIG-I and melanoma differentiation-associated protein 5 (MDA5). Their activation establishes an antiviral state by downstream signaling that ultimately activates interferon-stimulated genes (ISGs). While in rare cases RIG-I gene loss has been detected in mammalian and avian species, most notably in the chicken, MDA5 pseudogenization has only been detected once in mammals. We have screened over a hundred publicly available avian genome sequences and describe an independent disruption of MDA5 in two unrelated avian lineages, the storks (Ciconiiformes) and the rallids (Gruiformes). The results of our RELAX analysis confirmed the absence of negative selection in the MDA5 pseudogene. In contrast to our prediction, we have shown, using multiple dN/dS-based approaches, that the MDA5 loss does not appear to have resulted in any compensatory evolution in the RIG-I gene, which may partially share its ligand-binding specificity. Together, our results indicate that the MDA5 pseudogenization may have important functional effects on immune responsiveness in these two avian clades.