A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek's Disease.

The chicken MHC is known to confer decisive resistance or susceptibility to various economically important pathogens, including the iconic oncogenic herpesvirus that causes Marek's disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The class I molecule BF2*1901 is better expressed and more thermostable than the closely related BF2*1501, but the peptide motif was not simpler as expected. In this study, we confirm for newly developed chicken lines that the chicken MHC haplotype B15 confers resistance to MD compared with B19. Using gas phase sequencing and immunopeptidomics, we find that BF2*1901 binds a greater variety of amino acids in some anchor positions than does BF2*1501. However, by x-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Although the self-peptides that bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows stronger binding and thus more peptides overall, correlating with the expected hierarchies for expression level, thermostability, and MD resistance. Our study provides a reasonable explanation for greater promiscuity for BF2*1501 compared with BF2*1901, corresponding to the difference in resistance to MD.

Efficient Identification of Tembusu Virus CTL Epitopes in Inbred HBW/B4 Ducks Using a Novel MHC Class I-Restricted Epitope Screening Scheme.

The identification of MHC class I-restricted CTL epitopes in certain species, particularly nonmammals, remains a challenge. In this study, we developed a four-step identification scheme and confirmed its efficiency by identifying the Anpl-UAA*76-restricted CTL epitopes of Tembusu virus (TMUV) in inbred haplotype ducks HBW/B4. First, the peptide binding motif of Anpl-UAA*76 was determined by random peptide library in de novo liquid chromatography-tandem mass spectrometry, a novel nonbiased, data-independent acquisition method that we previously established. Second, a total of 38 TMUV peptides matching the motif were screened from the viral proteome, among which 11 peptides were conserved across the different TMUV strains. Third, the conserved TMUV peptides were refolded in vitro with Anpl-UAA*76 and Anpl-ß2-microglobulin to verify the results from the previous two steps. To clarify the structural basis of the obtained motif, we resolved the crystal structure of Anpl-UAA*76 with the TMUV NS3 peptide LRKRQLTVL and found that Asp34 is critical for the preferential binding of the B pocket to bind the second residue to arginine as an anchor residue. Fourth, the immunogenicity of the conserved TMUV peptides was tested in vivo using specific pathogen-free HBW/B4 ducks immunized with the attenuated TMUV vaccine. All 11 conserved TMUV epitopes could bind stably to Anpl-UAA*76 in vitro and stimulate the secretion of IFN-γ and lymphocyte proliferation, and three conserved and one nonconserved peptides were selected to evaluate the CTL responses in vivo by flow cytometry and their tetramers. We believe that this new scheme could improve the identification of MHC class I-restricted CTL epitopes, and our data provide a foundation for further study on duck anti-TMUV CTL immunity.

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus.

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-ßeta (IFN-ß), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

An Invariant Arginine in Common with MHC Class II Allows Extension at the C-Terminal End of Peptides Bound to Chicken MHC Class I.

MHC molecules are found in all jawed vertebrates and are known to present peptides to T lymphocytes. In mammals, peptides can hang out either end of the peptide-binding groove of classical class II molecules, whereas the N and C termini of peptides are typically tightly bound to specific pockets in classical class I molecules. The chicken MHC, like many nonmammalian vertebrates, has a single dominantly expressed classical class I molecule encoded by the BF2 locus. We determined the structures of BF2*1201 bound to two peptides and found that the C terminus of one peptide hangs outside of the groove with a conformation much like the peptides bound to class II molecules. We found that BF2*1201 binds many peptides that hang out of the groove at the C terminus, and the sequences and structures of this MHC class I allele were determined to investigate the basis for this phenomenon. The classical class I molecules of mammals have a nearly invariant Tyr (Tyr84 in humans) that coordinates the peptide C terminus, but all classical class I molecules outside of mammals have an Arg in that position in common with mammalian class II molecules. We find that this invariant Arg residue switches conformation to allow peptides to hang out of the groove of BF2*1201, suggesting that this phenomenon is common in chickens and other nonmammalian vertebrates, perhaps allowing the single dominantly expressed class I molecule to bind a larger repertoire of peptides.

Protective T Cell Responses Featured by Concordant Recognition of Middle East Respiratory Syndrome Coronavirus-Derived CD8+ T Cell Epitopes and Host MHC.

The coordinated recognition of virus-derived T cell epitopes and MHC molecules by T cells plays a pivotal role in cellular immunity-mediated virus clearance. It has been demonstrated that the conformation of MHC class I (MHC I) molecules can be adjusted by the presented peptide, which impacts T cell activation. However, it is still largely unknown whether the conformational shift of MHC I influences the protective effect of virus-specific T cells. In this study, utilizing the Middle East respiratory syndrome coronavirus-infected mouse model, we observed that through the unusual secondary anchor Ile5, a CD8+ T cell epitope drove the conformational fit of Trp73 on the α1 helix of murine MHC I H-2Kd In vitro renaturation and circular dichroism assays indicated that this shift of the structure did not influence the peptide/MHC I binding affinity. Nevertheless, the T cell recognition and the protective effect of the peptide diminished when we made an Ile to Ala mutation at position 5 of the original peptide. The molecular bases of the concordant recognition of T cell epitopes and host MHC-dependent protection were demonstrated through both crystal structure determination and tetramer staining using the peptide-MHC complex. Our results indicate a coordinated MHC I/peptide interaction mechanism and provide a beneficial reference for T cell-oriented vaccine development against emerging viruses such as Middle East respiratory syndrome coronavirus.

Transcripts of antibacterial peptides in chicken erythrocytes infected with Marek's disease virus.

BACKGROUND: Chicken erythrocytes are involved in immunity through binding of toll-like receptors (TLRs) with their ligands to activate downstream signaling and lead to cytokine production in erythrocytes. Some avian ß-defensins (AvBDs) are constitutively expressed in tissues and some others can be induced by various bacteria and viruses. However, the expression of AvBDs in erythrocytes has not yet been studied extensively. RESULTS: The transcripts of eight AvBDs (AvBD1 to AvBD7, and AvBD9) and liver-expressed antimicrobial peptide-2 (LEAP-2) were found in normal chicken erythrocytes. The expression levels of AvBD2, 4 and 7 were significantly increased (P < 0.01), whereas the levels of AvBD1, 6 and 9 were significantly decreased (P < 0.01) after Marek's disease virus (MDV) infection. The mRNA expression level of LEAP-2 was not significantly changed after MDV infection. Highest viral nucleic acid (VNA) of MDV in the feather tips among the tested time points was found at 14 days post-infection (d.p.i.). In addition, 35 MD5-related gene segments were detected in the erythrocytes at 14 d.p.i. by transcriptome sequencing. CONCLUSIONS: These results suggest that the AvBDs in chicken erythrocytes may participate in MDV-induced host immune responses.

Autophagy activated by duck enteritis virus infection positively affects its replication.

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

[Advance in research of poultry major histocompatibility complex-structure].

The structure of poultry major histocompatibility complex(MHC) is closely associated with avianimmunology and avian disease control. This review summaried the structures of poultry MHC, including chicken, turkey, duck, goose, and quail. It was suggested that there were some common characteristics among these MHCs: all of them have conservative MHC region containing MHC I, MHC II, and unknown functional genes; they are simple and contracted; the lengths of introns of MHC I gene are shorter than those of mammals; all have 8 exons and 7 introns in MHC I genes in chicken, turkey, duck, and goose; all have 6 exons and 5 introns in MHC II genes; the structure patterns of BG genes in chicken, turkey, and quail are identical; and all have microsatellite repetitive motifs. However, there are differences among species: MHC I and MHC II genes are duplicated, while there are several copies in duck, goose, and quail; and the numbers of BG genes are different. It will be helpful to further study avian disease and avian immunologenetics through analysing MHC structures of the major poultrys.

Development of Microsatellite Marker System to Determine the Genetic Diversity of Experimental Chicken, Duck, Goose, and Pigeon Populations.

Poultries including chickens, ducks, geese, and pigeons are widely used in the biological and medical research in many aspects. The genetic quality of experimental poultries directly affects the results of the research. In this study, following electrophoresis analysis and short tandem repeat (STR) scanning, we screened out the microsatellite loci for determining the genetic characteristics of Chinese experimental chickens, ducks, geese, and pigeons. The panels of loci selected in our research provide a good choice for genetic monitoring of the population genetic diversity of Chinese native experimental chickens, ducks, geese, and ducks.

L-Glutamine Supplementation Alleviates Constipation during Late Gestation of Mini Sows by Modifying the Microbiota Composition in Feces.

Constipation occurs frequently in both sows and humans, particularly, during late gestation. The microbial community of the porcine gut, the enteric microbiota, plays a critical role in functions that sustain intestinal health. Hence, microbial regulation during pregnancy may be important to prevent host constipation. The present study was conducted to determine whether L-glutamine (Gln) supplementation improved intestinal function and alleviated constipation by regulation of enteric microbiota. 16S rRNA sequences obtained from fecal samples from 9 constipated sows (3 in the constipation group and 6 in the 1.0% Gln group) were assessed from gestational day 70 to 84. Comparative analysis showed that the abundance of intestinal-friendly microbiota, that is, Bacteroidetes (P = 0.007) and Actinobacteria (P = 0.037), was comparatively increased in the 1.0% Gln group, while the abundance of pernicious bacteria, Oscillospira (P < 0.001) and Treponema (P = 0.011), was decreased. Dietary supplementation with 1.0% Gln may ameliorate constipation of sows by regulated endogenous gut microbiota.

Glutamine Ameliorates Mucosal Damage Caused by Immune Responses to Duck Plague Virus.

The immune-releasing effects of L-glutamine (Gln) supplementation in duck plague virus (DPV)-infected ducklings were evaluated in 120 seven-day-old ducklings that were divided into 8 groups. The ducklings in control and DPV, 0.5Gln and DPV + 0.5Gln, 1.0Gln and DPV + 1.0Gln, and 2.0Gln and DPV + 2.0Gln received 0, 0.5, 1.0, and 2.0 g of Gln/kg feed/d by gastric perfusion, respectively. Then, the ducklings in control to 2.0Gln were injected with 0.2 mL of phosphate-buffered saline, while those in DPV to DPV + 2.0Gln were injected with DPV at 0.2 mL of 2000 TCID50 (50% tissue culture infection dose) 30 minutes after gavage with Gln, sampled at 12 hours and days 1, 2, 4, and 6. Glutamine supplementation under physiological conditions enhanced immune function and toll-like receptor 4 (TLR4) expressions in a dose-dependent manner. An increase in Gln supplementation under DPV-infected conditions enhanced growth performance, decreased immunoglobulin (Ig) release in plasma and secretory IgA in the duodenum, ameliorated plasma cytokine levels, and suppressed overexpressions of the TLR4 pathway in the duodenum. The positive effects of Gln on the humoral immunity- and intestinal inflammation-related damage should be considered a mechanism by which immunonutrition can assist in the recovery from DPV infection.

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.

Comments on duck circovirus (DuCV) genotype definition.

A standardised methodology has been used to define genotypes based on pairwise sequence comparisons (PASC). PASC is a widely accepted method in virus taxonomy, which is based on the histogram of pairwised differences among sequences. Recently, Zhang et al. (2013) concluded that the average p-distance of duck circovirus (DuCV) between genotypes 1 and 2 was 0.170, and subtype distance thresholds were 0.032 in DuCV-1 and 0.018 in DuCV-2, respectively. However, there might be some concerns on the methodology application to define the genotype of DuCV. Taking into account the concerns mentioned above, our authors conducted the PASC analyses of 54 capsid gene (ORF2) and genomic sequences including all the sequences from Zhang et al. (2013). Our results confirmed the existence of two DuCV genotypes (1 and 2) and, we suggest that DuCV ORF2 and genomic distance genotype thresholds were 0.061 and 0.038, respectively.

Characterization of swine leukocyte antigen (SLA) polymorphism by sequence-based and PCR-SSP methods in Chinese Bama miniature pigs.

The highly polymorphic swine leukocyte antigen (SLA) genes have been repeatedly shown to influence swine immune traits, disease resistance, vaccine responsiveness and tumour penetrance. Analysis of the SLA diversity in as many pig breeds as possible is important to clarify the relationships between SLA genes and diseases or traits, and develop these pigs as valuable animal models for biomedical research. The Chinese Bama miniature pig breed is an economically significant breed that is available at several research institutions in China. In this study, we identified a total of 32 alleles at five polymorphic SLA loci (SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing nine class I and seven class II haplotypes using the reverse transcription polymerase chain reaction (RT-PCR) sequence-based typing (SBT) method. The possible functional sites of the SLA genes were predicted and analyzed by comparison with those of the human and mouse. Based on the sequence information, we subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the progeny. In the studied cohort (2n = 562), the most prevalent Haplotype Hp-35.6 (SLA-1(∗)1201, SLA-1(∗)1301-SLA-3(∗)0502-SLA-2(∗)1001-DRB1(∗)0501-DQB1(∗)0801) was identified in 182 Bama pigs with a frequency of 32.38%. The presence of the duplicated SLA-1 locus was confirmed in five of the class I haplotypes. Moreover, we identified two crossovers within the class I region and one between the class I and class II regions, which corresponded to recombination frequencies of 0.36% and 0.18%, respectively. The information of this study is essential for an understanding of the SLA allelic architecture and diversity, and it will be helpful for studying the adaptive immune response and further developing the more effective vaccines in the context of SLA specificities.

Duck hepatitis A virus (DHAV) genotype definition: comment on the article by Cha et al.

Duck hepatitis A virus (DHAV) is genetically divided into three different genotypes: the original type DHAV-1, a type recently isolated in Taiwan (DHAV-2), and a recently described type isolated in South Korea and China (DHAV-3). Recently, Cha et al. (2013) concluded that the existence that both DHAV-1 and DHAV-2 had been classified into one branch, with DHAV genotype 3 (DHAV-3) in another, and that the phylogenetic distance unit showed was 0.5, a tremendous value. However, there might be some concerns on the methodology application to define the genotypes of DHAV. Based on 110 genomic and 100 amino acid sequences of DHAV which included all the sequences from Cha et al. (2013) respectively, phylogenetic analysis in the present study showed a distinct and proposed DHAV genotype definition, that both DHAV-2 and DHAV-3 were clustered in one branch while DHAV-1 in another branch only, and that the phylogenetic distance unit of 0.02 was confirmed, which was much smaller than the value 0.5. Taking into account the genotype definition of DHAV, we also conducted the pairwise sequence comparisons (PASC) analysis of 110 genomic sequences, and proposed that the distance genotype definition threshold was 0.045.

[Preparation and characterization of polyclonal antibodies against haplotype chicken natural killer cell receptor B-NK19 and B-NK21].

OBJECTIVE: To express natural killer (NK) cell inhibitory receptor gene B-NK of B19 and B21 haplotype chicken, which show different susceptible to various avian diseases, using prokaryotic expression system, and to prepare B-NK19 and B-NK21-specific rabbit polyclonal antibodies. METHODS: B-NK genes of B19 (named as B-NK19) and B21 (named as B-NK21) chicken were amplified by reverse transcription PCR from peripheral blood lymphocytes of one B19 or B21 chicken, respectively, and then inserted into an expression vector of pET-30a. The recombinant plasmids of pET-B-NK19 and pET-B-NK21 were transformed into E.coli (DE3) and induced to express, respectively. Both His-tagged foreign proteins of B-NK19 and B-NK21 were seperated by SDS-PAGE and immunized to New Zealand rabbits to prepare polyclonal antibodies. The MHC-specificities of the sera were analyzed by Western blotting. RESULTS: The chicken NK cell inhibitory receptor gene B-NK from MHC haplotypes B19 and B21 were successfully amplified, cloned and expressed in E.coli after induction with 1.0 mmol/mL IPTG for 4 h at 37°C, respectively. Western blotting revealed that the prepared rabbit serum antibodies positively reacted to the expressed foreign proteins. CONCLUSION: Chicken NK cell inhibitory receptor proteins B-NK of B19 and B21 haplotypes could immunologically cross-react and their polyclonal antibodies are partially specific to B complex.

Specific TaqMan probed real-time quantitative RT-PCR methods and their application to differentiate the transcripts of duplicated BF or BLB genes in chicken MHC.

BF and BLB genes of chicken major histocompatibility complex (MHC) are responsible for classical antigen processing and presentation; therefore they play a central role in determining the genetic resistance or susceptibility of different MHC-B haplotypes to some infectious diseases. In this study, we developed specific TaqMan probed real-time quantitative reverse transcription PCR (TaqMan qRT-PCR) methods based on the diagnostic nucleotide polymorphisms present in duplicated BF or BLB genes in B2 and B19 haplotypes. The results showed very similar amplification efficiency but no cross-reaction between the duplicated BF or BLB genes of the same haplotype. Spleen mRNA samples of B2 and B19 chickens were used to validate these TaqMan qRT-PCR methods. We observed that BF2 or BLB2 gene was dominantly transcribed in all B2 and B19 chickens. Our findings verified the impact of diversified promoter sequences on the function of duplicated BF or BLB genes. Hence the principles adopted to establish these specific TaqMan qRT-PCR methods in this study can be applied to differentiate the transcripts of duplicated BF or BLB genes of other MHC-B haplotypes in chicken.

A duplex real-time reverse transcription polymerase chain reaction for the detection and quantitation of avian leukosis virus subgroups A and B.

Avian leukosis is a disease that is spreading widely in the world causing large economic losses to the poultry industry. In this study, a duplex quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify avian leukosis virus subgroups A and B (ALVA/B). The assay was optimised to measure viral gp85 and chicken housekeeping (ß-actin) genes. The result showed that the assay was specific for reference strains of ALVA/B subtype and no cross-reaction was detected with ALV subtypes E and J or with four other non-ALV viruses. The assay detected as few as 56 gp85 cDNA copies and was 100-fold more sensitive than a conventional RT-PCR. Seventy clinical blood samples were evaluated by both the qRT-PCR and the conventional RT-PCR assay, and the results show that 65 samples were positive by the qRT-PCR compared with 43 by the conventional RT-PCR. When this assay was used to quantify the viral load in ALV-inoculated embryos from three congenic chicken lines, the embryos from the B21 line showed the highest viral load, whereas the lowest load was found in the B5 line. This assay provides a powerful tool for quantitative detection of the ALVA/B and for the study of host genetic resistance to avian leukosis.