Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV).

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9 Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

Major Histocompatibility Complex-B haplotype and ovarian graft response.

Gonadal tissue transfer is considered one of the best methods to preserve genetic variability. Poultry hosts can receive a gonad from a donor of a different genetic background, sustain the growth of this graft, and produce gametes from it. Unfortunately, the host's strong immune response may significantly reduce the gonadal graft's ability to reach maturity. Our study aimed to evaluate the influence of MHC-B alleles in rejecting a gonadal graft of similar or different genetic backgrounds. In the first experiment, ovarian tissue was transplanted to chicks of similar genetic backgrounds, either Lohmann White (LW) with variable MHC-B or Barred Rock (BR) with fixed MHC-B. The sustained growth of donor ovarian tissues occurred in (4/7 hosts) BR (MHC-B matched) hosts only-one of these graft-positive-BR hens produced eggs derived from the donor ovary. No grafts were recovered when the host and the donor had an LW background (0/9; MHC-B mismatched). In the second experiment, ovarian transplantation was done between chicks of either similar or different genetic backgrounds (Brown Leghorn [BL], BR, and BL/BR F1). The 2 pure lines contained only one MHC-B allele, whereas the F1 heterozygotes had both. All host birds were given a daily dose of an immunosuppressant (mycophenolate mofetil) until maturity. The success rate was assessed by microsatellite genotype confirmation of donor-derived ovaries plus physiological and histological analyses of ovarian grafts. In this second experiment, 11 out of 43 ovarian hosts laid eggs. However, all fertilized eggs from these hens were derived from the remnant host ovarian tissue, not from the donor ovaries. A necropsy assessment was done on all 43 host birds. Ten donor grafts were recovered from hosts having matched (6 hosts) and mismatched (4 hosts) MHC-B, and none were functional. Interestingly, 6 of them were enclosed by a serous membrane capsule filled with fluid and had various tissue growth. In addition, clusters of immune cells were observed in all recovered donor grafts. Our results demonstrated that genetic background could greatly influence the success of gonadal transfer in chickens.

Application of next-generation sequencing for the high-resolution typing of MHC-B in Korean native chicken.

The major histocompatibility complex-B (MHC-B) region of chicken is crucially important in their immunogenesis and highly diverse among different breeds, lines, and even populations. Because it determines the resistance/susceptibility to numerous infectious diseases, it is important to analyze this genomic region, particularly classical class I and II genes, to determine the variation and diversity that ultimately affect antigen presentation. This study investigated five lines of indigenous Korean native chicken (KNC) and the Ogye breed using next-generation sequencing (NGS) data with Geneious Prime-based assembly and variant calling with the Genome Analysis Toolkit (GATK) best practices pipeline. The consensus sequences of MHC-B (BG1-BF2) were obtained for each chicken line/breed and their variants were analyzed. All of the Korean native chicken lines possessed an excessive number of variants, including an ample amount of high-impact variants that provided useful information regarding modified major histocompatibility complex molecules. The study confirmed that next-generation sequencing techniques can effectively be used to detect MHC variabilities and the KNC lines are highly diverse for the MHC-B region, suggesting a substantial divergence from red junglefowl.

High Levels of Genetic Variation in MHC-Linked Microsatellite Markers from Native Chicken Breeds.

The major histocompatibility complex (MHC) is a highly polymorphic gene region that regulates cellular communication in all specific immune responses. In this study, we investigated 11 microsatellite (MS) markers in the MHC-B region of chicken populations from four countries: Sri Lanka, Bangladesh, South Korea, and Nigeria. The MS markers were divided into two sets. Set 1 included five novel MS markers, which we assessed using 192 samples from 21 populations. Set 2 included six previously reported markers, which we assessed using 881 samples from 29 populations. The Set 1 MS markers had lower polymorphism (polymorphic information content (PIC) < 0.5) than the Set 2 markers (PIC = 0.4-0.9). In all populations, the LEI0258 marker was the most polymorphic, with a total of 38 alleles (PIC = 0.912, expected heterozygosity (He) = 0.918). Local populations from Sri Lanka, Bangladesh, and Nigeria had higher allele diversity and more haplotypes for Set 2 MS markers than Korean and commercial populations. The Sri Lankan Karuwalagaswewa village population had the highest MHC diversity (mean allele number = 8.17, He = 0.657), whereas the white leghorn population had the lowest (mean allele number = 2.33, He = 0.342). A total of 409 haplotypes (89 shared and 320 unique), with a range of 4 (Rhode Island red) to 46 (Karuwalagaswewa village (TA)), were identified. Among the shared haplotypes, the B21-like haplotype was identified in 15 populations. The genetic relationship observed in a neighbour-joining tree based on the DA distance agreed with the breeding histories and geographic separations. The results indicated high MHC diversity in the local chicken populations. The difference in the allelic pattern among populations presumably reflects the effects of different genotypes, environments, geographic variation, and breeding policies in each country. The selection of MHC allele in domestic poultry can vary due to intensification of poultry production. Preserved MHC diversity in local chicken provides a great opportunity for future studies that address the relationships between MHC polymorphisms and differential immune responses.

Chromosome level assembly reveals a unique immune gene organization and signatures of evolution in the common pheasant.

The common pheasant Phasianus colchicus, belonging to the order Galliformes and family Phasianidae, is the most widespread species. Despite a long history of captivity, the domestication of this bird is still at a preliminary stage. Recently, the demand for accelerating its transformation to poultry for meat and egg production has been increasing. In this study, we assembled high quality, chromosome scale genome of the common pheasant by using PacBio long reads, next-generation short reads, and Hi-C technology. The primary assembly has contig N50 size of 1.33 Mb and scaffold N50 size of 59.46 Mb, with a total size of 0.99 Gb, resolving most macrochromosomes into single scaffolds. A total of 23,058 genes and 10.71 Mb interspersed repeats were identified, constituting 30.31% and 10.71% of the common pheasant genome, respectively. Our phylogenetic analysis revealed that the common pheasant shared common ancestors with turkey about 24.7-34.5 million years ago (Ma). Rapidly evolved gene families, as well as branch-specific positively selected genes, indicate that calcium-related genes are potentially related to the adaptive and evolutionary change of the common pheasant. Interestingly, we found that the common pheasant has a unique major histocompatibility complex B locus (MHC-B) structure: three major inversions occurred in the sequence compared with chicken MHC-B. Furthermore, we detected signals of selection in five breeds of domestic common pheasant, several of which are production-oriented.

Genetic diversity of MHC-B in 12 chicken populations in Korea revealed by single-nucleotide polymorphisms.

This study used a single-nucleotide polymorphism (SNP) panel to characterise the diversity in the major histocompatibility complex B region (MHC-B) in 12 chicken populations in Korea. Samples were genotyped for 96 MHC-B SNPs using an Illumina GoldenGate genotyping assay. The MHC-B SNP haplotypes were predicted using 58 informative SNPs and a coalescence-based Bayesian algorithm implemented by the PHASE program and a manual curation process. In total, 117 haplotypes, including 24 shared and 93 unique haplotypes, were identified. The unique haplotype numbers ranged from 0 in Rhode Island Red to 32 in the Korean native commercial chicken population 2 ("Hanhyup-3ho"). Population and haplotype principal component analysis (PCA) indicated no clear population structure based on the MHC haplotypes. Three haplotype clusters (A, B, C) segregated in these populations highlighted the relationship between the haplotypes in each cluster. The sequences from two clusters (B and C) overlapped, whereas the sequences from the third cluster (A) were very different. Overall, native breeds had high genetic diversity in the MHC-B region compared with the commercial breeds. This highlights their immune capabilities and genetic potential for resistance to many different pathogens.

Assessing MHC-B diversity in Silkie chickens.

The major histocompatibility complex (MHC) is a highly polymorphic region on chromosome 16, which contains numerous immune response genes, and is known to influence disease susceptibility and resistance in chickens. Variability of MHC-B haplotypes in various well-known and commercially utilized breeds has previously been identified. This study aims to understand MHC-B diversity in the Silkie breed using a high-density SNP panel that encompasses the chicken MHC-B region. DNA was obtained from 74 females and 27 males from a commercial Silkie breeder colony that is maintained through minimal genetic selection practices. A previously described panel of 90 SNPs, all located within the MHC-B region, was used to evaluate MHC-B variability in the commercial Silkie breeder colony. MHC-B haplotypes identified from the individual SNP information in the Silkie colony were compared to published haplotypes from the same region. Of the 27 haplotypes identified in the Silkie population, 8 have been previously described. Nineteen haplotypes are unique to the Silkie population and include one novel recombinant and 2 additional possible novel recombinants. Six haplotypes were found at a frequency greater than 5% of the population, of which 4 are novel. Finally, Hardy Weinberg Equilibrium (HWE) was calculated for the observed haplotypes, which were found to be in HWE. This study shows considerable MHC-B diversity in the Silkie breed and adds further information on variability of the MHC-B region in the chicken.

Advances in methodologies for detecting MHC-B variability in chickens.

The chicken major histocompatibility B complex (MHC-B) region is of great interest owing to its very strong association with resistance to many diseases. Variation in the MHC-B was initially identified by hemagglutination of red blood cells with specific alloantisera. New technologies, developed to identify variation in biological materials, have been applied to the chicken MHC. Protein variation encoded by the MHC genes was examined by immunoprecipitation and 2-dimensional gel electrophoresis. Increased availability of DNA probes, PCR, and sequencing resulted in the application of DNA-based methods for MHC detection. The chicken reference genome, completed in 2004, allowed further refinements in DNA methods that enabled more rapid examination of MHC variation and extended such analyses to include very diverse chicken populations. This review progresses from the inception of MHC-B identification to the present, describing multiple methods, plus their advantages and disadvantages.

Mhc-B haplotypes in "Campero-Inta" chicken synthetic line.

The major histocompatibility complex-B (MHC-B) in chickens is a cluster of genes located on chromosome 16. The chicken MHC-B is known to be highly associated with resistance to numerous diseases caused by viruses, bacteria, and parasitic pathogens. Since the level of resistance varies with MHC-B haplotypes, identification and classification of different haplotypes within lines is important for sustaining lines. The "Campero-INTA" chicken breed is a meat-type free-range poultry breed that was developed specifically for small producers in Argentina. Campero-INTA was started by selection in populations produced by crosses between a variety of established lines. MHC-B variation was examined in 65 samples obtained in 2002 using the VNTR marker LEI0258, a marker for MHC-B region. These samples plus and an additional 55 samples from 2018 were examined for variation using the MHC-B specific SNP panel that encompasses ∼230,000 bp of the MHC-B region. Eleven MHC-B SNP haplotypes with 6 LEI0258 alleles were identified in the 120 samples representing the Campero-INTA AH (male) line. Seven haplotypes originate from the breeds originally used in the development of Campero-INTA AH line. Two appear to be recombinant haplotypes. The origin of the remaining 2 is not known, but may be associated with genes introduced from crosses with the Fayoumi breed conducted more recently to sustain the line.

MHC-B variability within the Finnish Landrace chicken conservation program.

The major histocompatibility complex (MHC) is a cluster of genes involved with immune responses. The chicken MHC has been shown to influence resistance to viruses, bacteria, and infections from both internal and external parasites. The highly variable chicken MHC haplotypes were initially identified by the use of haplotype-specific serological reagents. A novel SNP-based panel encompassing 210,000 bp of the MHC-B locus was developed to allow fine scale genetic analyses including rapid identification of novel haplotypes for which serological reagents are not available. The Finnish Landrace breed of chickens traces its origins to almost 1,000 years ago, with multiple lineages maintained as small populations in isolated villages. The breed is well adapted to the cooler Finnish climate and is considered to be an infrequent egg layer. Conservation efforts to protect this endangered breed were initiated by a hobby breeder in the 1960s. An official conservation program was established in 1998 and now 12 different populations are currently maintained by a network of volunteer hobbyist breeders. Variation in the MHC-B region in these populations was examined using a panel of 90 selected SNP. A total of 195 samples from 12 distinct populations (average of 15 individuals sampled per population) were genotyped with the 90 SNP panel specific for the MHC-B region, spanning 210,000 bp. There were 36 haplotypes found, 16 of which are a subset of 78 that had been previously identified in either commercially utilized or heritage breeds from North America with the remaining 20 haplotypes being novel. The average number of MHC-B haplotypes found within each Finnish Landrace population was 5.9, and ranged from one to 13. While haplotypes common to multiple populations were found, population-specific haplotypes were also identified. This study shows that substantial MHC-B region diversity exists in the Finnish Landrace breed and exemplifies the significance tied to conserving multiple populations of rare breeds.

Establishment of six homozygous MHC-B haplotype populations associated with susceptibility to Marek's disease in Chinese specific pathogen-free BWEL chickens.

The highly polymorphic chicken major histocompatibility complex (MHC) is associated with different levels of immunologic responses to certain avian pathogens. MHC-B haplotype chickens are an important genetic resource for studying the genetic determination of pathogen resistance and susceptibility. The BWEL chicken population is the only specific pathogen-free (SPF) chickens bred and developed by the State Center of Poultry Genetic Resources of Laboratory Animals in China. In this study, we successfully established six homozygous MHC-B haplotype populations from the BWEL chickens using microsatellite marker technology, named as BW/G(1, 2, 3, 5, 6, 7) lines, and their molecular genotypes were matched to six serologically defined MHC-B haplotypes, B13, B15, B2, B5, B21 and B19, respectively. The sequences of BF genes exons 2 and 3 from four successive generations (F1-F4) of the BW/G(n) lines were completely consistent with those of serologically defined MHC-B haplotypes. Subsequently, six BW/G(n) line specific allo-antisera were prepared by immunization with red blood cells (RBCs) and hemagglutination tests results showed the BW/G(n) SPF chickens could be serologically differentiated. Additionally, susceptibility to Marek's disease (MD) in the BW/G3 (B2 haplotype) and BW/G7 (B19 haplotype) lines were determined by comparing mortality, macroscopic and histopathological lesions, and viral loads in feather pulp. The BW/G7 line showed greater genetic susceptibility to the very virulent MD virus (MDV) strain than the BW/G3 line. The establishment of MHC-B haplotype chicken populations associated with susceptibility to MD will be helpful for studying host immune responses and further developing the more effective vaccines in the context of MHC specificities, and they are also very useful for an understanding of MHC genes architecture and function.

Genetic variability, evidence of potential recombinational event and selection of LEI0258 in chicken.

The chicken major histocompatibility complex (MHC) plays an important role in the immune response, disease resistance, productivity, and other important economic traits of the chicken. Therefore, a description of the polymorphisms of this region is crucial for understanding the genetic pattern of the MHC. The tandem repeat LEI0258 is located within the B region of the chicken MHC and is surprisingly strongly associated with serology. This marker has been used worldwide to provide a picture of the core area of the chicken MHC-B region and to categorize chicken MHC haplotypes. Thus, insight into the evolutionary pattern of LEI0258 may be useful for understanding MHC diversity. In the current study, 30 alleles of LEI0258 from 12 populations were screened and sequenced, and alleles that have previously been published in GenBank were also analyzed. The resulting 124 alleles were classified into four clusters according to the SNPs and indels found within the sequences flanking the repeats. Furthermore, a recombination region was identified between -30 and +43 that suggests that recombination may have played a role in the evolution of this MHC. Finally, strong evidence regarding the selection and evolutionary dynamics of the LEI0258 region is presented. Generally speaking, microsatellite is a classic anonymous marker which changes by genetic drift rather than by direct selection. Although, the genotypes of LEI0258 in MHC-B correlate with serology, its mechanism of inheritance and evolution was unclear. This study not only establishes a framework of further diversity or association studies in LEI0258, but also unraveling the reason what driving force and formulate the evolutionary dynamics of this region.