The apparent absence of numerous conserved vertebrate genes from avian genomes has puzzled researchers for over a decade. In recent years, a subset of these genes has been identified; however, their sequences are unusually problematic, often evading detection by standard sequencing technologies. This limitation has hindered detailed investigation of the phenomenon-until recent progress in long read technologies, which are more robust against sequencing biases. This enabled us to classify real gene losses extensively, which strikingly revealed that a large number of the genes residing on so-called dot chromosomes were indeed lost during avian evolution. We demonstrate that dot microchromosomes-small, repeat-dense avian chromosomes-harbor widespread gene attrition, with 29% of ohnologs (duplicates from ancestral genome doublings) eliminated, far exceeding rates on other chromosomes. Moreover, we reveal that genes retained on these dot chromosomes exhibit a previously undescribed form of dynamic genetic instability. This instability, which we term sequence stuttering, is characterized by a massive expansion of short sequences within intronic regions. Intriguingly, in some cases, the expanding sequences appear to originate from neighboring exons. As a result, intron lengths vary extensively among individual chickens, suggesting that these events are evolutionarily recent. Since this phenomenon has not been reported in any other vertebrate species, our findings lay the groundwork for future research into its underlying mechanisms, evolutionary implications, and potential identification of similar loci across vertebrate genomes.

The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development.

Regulatory T cells (Tregs), defined by the lineage-specific transcription factor FoxP3, are crucial for immune regulation and have been studied extensively in mammals. However, avian Tregs remain poorly characterized, leaving gaps in our understanding of their evolutionary conservation and unique features. In this study, we investigated the phenotype of chicken Tregs to define reliable markers for their identification and characterization. We analyzed CD4 + splenocytes sorted into CD25 negative , CD25 low , and CD25 high subpopulations using RNA sequencing. FOXP3 and other Treg-associated genes were expressed in both CD25 low and CD25 high populations, showing that CD25 expression alone is insufficient to distinguish chicken Tregs. To refine the marker profile, we evaluated additional markers, including CTLA-4 and GITR. Notably, we describe for the first time a chicken-specific CTLA-4 antibody, which uniquely stains CTLA-4 exclusively in intracellular (ic) compartments, distinguishing it from mammalian counterparts. Single-cell RNA sequencing further confirmed distinct FOXP3 + clusters enriched for expression of CTLA4 and TNFRSF18 (encoding GITR). While CTLA-4's ic expression limits usability in functional assays, the combination of CD4 + /CD25 + /CTLA-4 + /GITR + represents the most accurate characterization of putative chicken Tregs to date. These findings highlight evolutionary conservation and species-specific differences in Treg markers, providing the foundation for future studies on chicken Treg functionality.

Interferon regulatory factors (IRFs) are a family of transcription factors with important functions in immunity. The genomes of most vertebrates encode ten IRF genes. IRF3 and IRF9 have key roles in interferon (IFN) induction and signaling. Most of our knowledge about the IFN pathways originates from the study of the mammalian IFN system, and the description of the corresponding avian components is not as complete. Both IRF3 and IRF9 were considered missing from the chicken genome and from the genomes of other avian species. Here we describe multiple avian IRF3 and IRF9 genes, all with difficult GC-rich sequence context that prevented their earlier characterization. IRF3 orthologs are narrowly distributed and are present in the avian infraclass Palaeognathae. In contrast, IRF9 orthologs were found in most avian species, with the exception of the order Galliformes. In about half of the avian orders, IRF9 was located in noncanonical chromosomal positions, indicating past translocations. Phylogenetic analysis confirmed the correct orthology of all newly described IRFs. We further performed experiments using duck IRF9, confirming its role in the IFN pathway. IRF9 knockout in duck fibroblasts decreases the induction of IFN-stimulated genes (ISGs). Full induction of ISGs in duck cells requires both an intact IRF9 and a canonical IFN-stimulated response element. Lastly, intact IRF9 is needed for IFN-mediated protection of duck cells against the vesicular stomatitis virus-induced cytopathicity. The identification of avian IRFs fills an important gap in our understanding of avian immunology and brings new questions related to the evolution of the IRF family.

Retinoic acid-inducible gene I (RIG-I) activates mitochondrial antiviral signaling proteins, initiating the antiviral response. RIG-I and RNF135, a ubiquitin ligase regulator, are missing in domestic chickens but conserved in mallard ducks. The chickens’ RIG-I loss was long believed to be linked to increased avian influenza susceptibility. We reinstated both genes in chickens and examined their susceptibility to infection with an H7N1 avian influenza virus. Uninfected RIG-I-expressing chickens exhibited shifts in T and B cells. At the same time, the H7N1 infection led to severe disease, persistent weight loss, and increased viral replication. The simultaneous expression of RIG-I and RNF135 potentiated the RIG-I activity and was associated with exacerbated inflammatory response and increased mortality without influencing virus replication. Additional animal infection experiments with two other avian influenza viruses validated these findings. They confirmed that the harmful effects triggered by RIG-I or RIG-I-RNF135-expression require a minimum degree of viral virulence. Our data indicate that the loss of RIG-I in chickens has likely evolved to counteract deleterious inflammation caused by viral infection and highlight an outcome of restoring evolutionary lost genes in birds.

The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development. ### Competing Interest Statement The authors have declared no competing interest.

Tetherin/BST2 is an antiviral restriction factor initially described in mammals. It is active against multiple enveloped viruses at the budding phase, where it is able to physically link the budding virions to the virus-producing cell. We and others have previously identified tetherin orthologs in birds, and characterized the antiviral activity and interferon-inducibility of chicken tetherin. In this work, we have generated an in vivo model of tetherin absence in chicken by CRISPR/Cas9 modification of chicken primordial germ cells (PGC). The modified PGCs were transplanted into roosters with suppressed endogenous spermatogenesis, and transgenic (tetherin knockout) progeny was obtained by further crosses. The viability and phenotype of tetherin knockout animals did not differ from wild type chicken. In more detailed investigation, flow cytometry based differential white blood cell count revealed an increased number of heterophils in tetherin knockouts. Upon challenge with avian sarcoma and leukosis virus (ASLV), a prototypic avian retrovirus, we detected increase in viremia at days 6 and 13 post infection in tetherin knockout animals. The increased virus susceptibility is consistent with absence of antiviral tetherin. In summary, we introduce a new in vivo knockout model of chicken antiviral gene tetherin. These animals can be used in further characterizations of avian antiviral defenses and also to define thus far unknown physiological effects of tetherin in birds. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT The restriction factor tetherin (bone marrow stromal cell antigen 2) is an interferon-inducible protein preventing the release of newly formed viral particles from infected cells. Tetherin displays antiviral activity against a broad range of enveloped viruses, including retroviruses. While tetherin orthologs have been identified in several mammalian species, little is known about its expression and activity in non-mammalian vertebrates, including birds. We have previously described antiviral activity of chicken ( Gallus gallus ) tetherin against the prototypical avian retrovirus avian sarcoma and leukosis virus (ASLV). Here, we report the loss of functional tetherin orthologs in several galliform birds, including turkey ( Meleagris gallopavo ) and Mikado pheasant ( Syrmaticus mikado ). In both species, the tetherin coding sequence acquired inactivating mutations, including an in-frame stop codon and frameshifting deletions. Similar to the chicken tetherin ortholog, reconstituted turkey and Mikado pheasant tetherins restricted ASLV and HIV-1 indicating antiviral activity of the ancestor protein. Previous work revealed the presence of the TMCC(aT ) gene in close proximity to tetherin, encoding a protein with tetherin-like structure in multiple vertebrates. Intriguingly, ectopic overexpression of chicken and turkey TMCC(aT) proteins in human cells decreased total HIV-1 yield. In contrast to tetherin, however, TMCC(aT) did not specifically restrict virion release, suggesting distinct antiviral mechanisms. In line with this, IFNα stimulation did not reduce the release of ASLV particles from infected turkey cells. Overall, our data describe the loss of functional tetherin genes in several galliform species and identify an antiviral activity of the related TMCC(aT) protein that is mechanistically different from that of tetherin. IMPORTANCE Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey ( Meleagris gallopavo ) and Mikado pheasant ( Syrmaticus mikado ). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Given that the majority of target sequences are not palindromic, the current model explains the symmetry by an overlap of the nonpalindromic motif present on one of the half-sites of the sequences. Here, we show that the implementation of multicomponent mixture models allows for different interpretations consistent with the existence of both palindromic and nonpalindromic submotifs in the sets of integration site sequences. We further show that the weak palindromic motifs result from freely combined site-specific submotifs restricted to only a few positions proximal to the site of integration. The submotifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identify HIV-1-favored palindromic sequences in Alu repeats serving as local hotspots for integration. The application of the novel statistical approach provides deeper insight into the selection of retroviral integration sites and may prove to be a valuable tool in the analysis of any type of DNA motifs.

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.

Linker for activation of T cells (LAT) plays a key role in T-cell antigenic signaling in mammals. Accordingly, LAT orthologues were identified in the majority of vertebrates. However, LAT orthologues were not identified in most birds. In this study, we show that LAT gene is present in genomes of multiple extant birds. It was not properly assembled previously because of its GC-rich content. LAT expression is enriched in lymphoid organs in chicken. The analysis of the coding sequences revealed a strong conservation of key signaling motifs in LAT between chicken and human. Overall, our data indicate that mammalian and avian LAT genes are functional homologues with a common role in T-cell signaling.

Retinoic acid-inducible gene I ( RIG-I ) activates mitochondrial antiviral signaling proteins, initiating the antiviral response. RIG-I and RNF135 , a ubiquitin ligase regulator, are missing in domestic chickens but conserved in mallard ducks. It was long believed that chickens’ RIG-I loss was linked to increased avian influenza susceptibility. We reinstated both genes in chickens and examined their susceptibility to the avian influenza virus H7N1. Uninfected RIG-I -expressing chickens exhibited shifts in T and B cells, while the H7N1 infection led to severe disease, persistent weight loss, and increased viral replication. Conversely, the co-expression of RIG-I and RNF135 reduced the viral replication and was associated with high inflammatory response. Our data indicate that the loss of RIG-I in chickens likely evolved to counteract deleterious inflammation caused by viral infection. We highlight the effects of restoring evolutionary lost genes in birds and suggest a new immunological approach to reduce viral replication and prevent infection. Graphical abstract

In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta (IL1B) and 6 (IL6)) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

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(+) Foxp3(high) 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.

In vertebrates, an ancient duplication in the genes for cannabinoid receptors (CNRs) allowed the evolution of specialised endocannabinoid receptors expressed in the brain (CNR1) and the periphery (CNR2). While dominantly conserved throughout vertebrate phylogeny, our comparative genomic analysis suggests that certain taxa may have lost either the CNR1 regulator of neural processes or, more frequently, the CNR2 involved in immune regulation. Focussing on conspicuous CNR2 pseudogenization in parrots (Psittaciformes), a diversified crown lineage of cognitively-advanced birds, we highlight possible functional effects of such a loss. Parrots appear to have lost the CNR2 gene at at least two separate occasions due to chromosomal rearrangement. Using gene expression data from the brain and periphery of birds with experimentally-induced sterile inflammation, we compare CNR and inflammatory marker (interleukin 1 beta, IL1B ) expression patterns in CNR2 -deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2 -intact passerines (represented by the zebra finch, Taeniopygia guttata ). Though no significant changes in CNR expression were observed in either parrots or passerines during inflammation of the brain or periphery, we detected a significant up-regulation of IL1B expression in the brain after stimulation with lipopolysaccharide (LPS) only in parrots. As our analysis failed to show evidence for selection on altered CNR1 functionality in parrots, compared to other birds, CNR1 is unlikely to be involved in compensation for CNR2 loss in modulation of the neuroimmune interaction. Thus, our results provide evidence for the functional importance of CNR2 pseudogenization for regulation of neuroinflammation.

All vertebrate genomes have been colonized by retroviruses along their evolutionary trajectory. Although endogenous retroviruses (ERVs) can contribute important physiological functions to contemporary hosts, such benefits are attributed to long-term coevolution of ERV and host because germline infections are rare and expansion is slow, and because the host effectively silences them. The genomes of several outbred species including mule deer (Odocoileus hemionus) are currently being colonized by ERVs, which provides an opportunity to study ERV dynamics at a time when few are fixed. We previously established the locus-specific distribution of cervid ERV (CrERV) in populations of mule deer. In this study, we determine the molecular evolutionary processes acting on CrERV at each locus in the context of phylogenetic origin, genome location, and population prevalence. A mule deer genome was de novo assembled from short- and long-insert mate pair reads and CrERV sequence generated at each locus. We report that CrERV composition and diversity have recently measurably increased by horizontal acquisition of a new retrovirus lineage. This new lineage has further expanded CrERV burden and CrERV genomic diversity by activating and recombining with existing CrERV. Resulting interlineage recombinants then endogenize and subsequently expand. CrERV loci are significantly closer to genes than expected if integration were random and gene proximity might explain the recent expansion of one recombinant CrERV lineage. Thus, in mule deer, retroviral colonization is a dynamic period in the molecular evolution of CrERV that also provides a burst of genomic diversity to the host population.

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.

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.