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.

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.

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.