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Marek's Disease (MD) has a significant impact on both the global poultry economy and animal welfare. The disease pathology can include neurological damage and tumour formation. Sexual dimorphism in immunity and known higher susceptibility of females to MD makes the chicken Z chromosome (GGZ) a particularly attractive target to study the chicken MD response. Previously, we used a Hy-Line F 6 population from a full-sib advanced intercross line to map MD QTL regions (QTLRs) on all chicken autosomes. Here, we mapped MD QTLRs on GGZ in the previously utilized F 6 population with individual genotypes and phenotypes, and in eight elite commercial egg production lines with daughter-tested sires and selective DNA pooling (SDP). Four MD QTLRs were found from each analysis. Some of these QTLRs overlap regions from previous reports. All QTLRs were tested by individuals from the same eight lines used in the SDP and genotyped with markers located within and around the QTLRs. All QTLRs were confirmed. The results exemplify the complexity of MD resistance in chickens and the complex distribution of p -values and Linkage Disequilibrium (LD) pattern and their effect on localization of the causative elements. Considering the fragments and interdigitated LD blocks while using LD to aid localization of causative elements, one must look beyond the non-significant markers, for possible distant markers and blocks in high LD with the significant block. The QTLRs found here may explain at least part of the gender differences in MD tolerance, and provide targets for mitigating the effects of MD.

Marek's disease (MD) was an immunosuppression disease induced by Marek's disease virus (MDV). MD caused huge economic loss to the global poultry industry, but it also provided an ideal model for studying diseases induced by the oncogenic virus. Alternative splicing (AS) simultaneously produced different isoform transcripts, which are involved in various diseases and individual development. To investigate AS events in MD, RNA-Seq was performed in tumorous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). In this study, 32,703 and 25,217 AS events were identified in TS and SS groups with NS group as the control group, and 1198, 1204, and 348 differently expressed (DE) AS events ( p -value < 0.05 and FDR < 0.05) were identified in TS vs. NS, TS vs. SS, SS vs. NS, respectively. Additionally, Function enrichment analysis showed that ubiquitin-mediated proteolysis, p53 signaling pathway, and phosphatidylinositol signaling system were significantly enriched ( p -value < 0.05). Small structural variations including SNP and indel were analyzed based on RNA-Seq data, and it showed that the TS group possessed more variants on the splice site region than those in SS and NS groups, which might cause more AS events in the TS group. Combined with previous circRNA data, we found that 287 genes could produce both circular and linear RNAs, which suggested these genes were more active in MD lymphoma transformation. This study has expanded the understanding of the MDV infection process and provided new insights for further analysis of resistance/susceptibility mechanisms.

The avian α-herpesvirus known as Marek's disease virus (MDV) linearly integrates its genomic DNA into host telomeres during infection. The resulting disease, Marek's disease (MD), is characterized by virally-induced lymphomas with high mortality. The temporal dynamics of MDV-positive (MDV + ) transformed cells and expansion of MD lymphomas remain targets for further understanding. It also remains to be determined whether specific host chromosomal sites of MDV telomere integration confer an advantage to MDV-transformed cells during tumorigenesis. We applied MDV-specific fluorescence in situ hybridization (MDV FISH) to investigate virus-host cytogenomic interactions within and among a total of 37 gonad lymphomas and neoplastic splenic samples in birds infected with virulent MDV. We also determined single-cell, chromosome-specific MDV integration profiles within and among transformed tissue samples, including multiple samples from the same bird. Most mitotically-dividing cells within neoplastic samples had the cytogenomic phenotype of 'MDV telomere-integrated only', and tissue-specific, temporal changes in phenotype frequencies were detected. Transformed cell populations composing gonad lymphomas exhibited significantly lower diversity, in terms of heterogeneity of MDV integration profiles, at the latest stages of tumorigenesis (>50 days post-infection (dpi)). We further report high interindividual and lower intraindividual variation in MDV integration profiles of lymphoma cells. There was no evidence of integration hotspots into a specific host chromosome(s). Collectively, our data suggests that very few transformed MDV + T cell populations present earlier in MDV-induced lymphomas (32-50 dpi), survive, and expand to become the dominant clonal population in more advanced MD lymphomas (51-62 dpi) and establish metastatic lymphomas.

Marek's disease (MD) represents a significant global economic and animal welfare issue. Marek's disease virus (MDV) is a highly contagious oncogenic and highly immune-suppressive α-herpes virus, which infects chickens, causing neurological effects and tumour formation. Though partially controlled by vaccination, MD continues to have a profound impact on animal health and on the poultry industry. Genetic selection provides an alternative and complementary method to vaccination. However, even after years of study, the genetic mechanisms underlying resistance to MDV remain poorly understood. The Major Histocompatability Complex (MHC) is known to play a role in disease resistance, along with a handful of other non-MHC genes. In this study, one of the largest to date, we used a multi-facetted approach to identify QTL regions (QTLR) influencing resistance to MDV, including an F 6 population from a full-sib advanced intercross line (FSIL) between two elite commercial layer lines differing in resistance to MDV, RNA-seq information from virus challenged chicks, and genome wide association study (GWAS) from multiple commercial lines. Candidate genomic elements residing in the QTLR were further tested for association with offspring mortality in the face of MDV challenge in eight pure lines of elite egg-layer birds. Thirty-eight QTLR were found on 19 chicken chromosomes. Candidate genes, miRNAs, lncRNAs and potentially functional mutations were identified in these regions. Association tests were carried out in 26 of the QTLR, using eight pure lines of elite egg-layer birds. Numerous candidate genomic elements were strongly associated with MD resistance. Genomic regions significantly associated with resistance to MDV were mapped and candidate genes identified. Various QTLR elements were shown to have a strong genetic association with resistance. These results provide a large number of significant targets for mitigating the effects of MDV infection on both poultry health and the economy, whether by means of selective breeding, improved vaccine design, or gene-editing technologies.

Marek's disease (MD) is a T cell lymphoma disease induced by Marek's disease virus (MDV), a highly oncogenic α herpesvirus primarily affecting chickens. MD is a chronic infectious disease that threatens the poultry industry. However, the mechanisms of genetic resistance for MD are complex and not completely understood. In this study, to identify high-confidence candidate genes of MD genetic resistance, high throughput sequencing (RNA-seq) was used to obtain transcriptomic data of CD4 + T cells isolated from MDV-infected and non-infected groups of two reciprocal crosses of individuals mating by two highly inbred chicken lines (6 3 MD-resistant and 7 2 MD-susceptible). After RNA-seq analysis with two biological replicates in each group, we identified 61 and 123 single nucleotide polymorphisms (SNPs) (false discovery rate (FDR) < 0.05) annotated in 39 and 132 genes in intercrosses 6 3 × 7 2 and 7 2 × 6 3 , respectively, which exhibited allele-specific expression (ASE) in response to MDV infection. Similarly, we identified 62 and 79 SNPs annotated in 66 and 96 genes in infected and non-infected groups, respectively. We identified 534 and 1543 differentially expressed genes (DEGs) (FDR < 0.05) related to MDV infection in intercrosses 6 3 × 7 2 and 7 2 × 6 3 , respectively. We also identified 328 and 20 DEGs in infected and non-infected groups, respectively. The qRT-PCR using seven DEGs further verified our results of RNA-seq analysis. The qRT-PCR of 11 important ASE genes was performed for gene functional validation in CD4 + T cells and tumors. Combining the analyses, six genes ( MCL1 , SLC43A2 , PDE3B , ADAM33 , BLB1 , and DMB2 ), especially MCL1 , were highlighted as the candidate genes with the potential to be involved in MDV infection. Gene-set enrichment analysis revealed that many ASE genes are linked to T cell activation, T cell receptor (TCR), B cell receptor (BCR), ERK/MAPK, and PI3K/AKT-mTOR signaling pathways, which play potentially important roles in MDV infection. Our approach underlines the importance of comprehensive functional studies for gaining valuable biological insight into the genetic factors behind MD and other complex traits, and our findings provide additional insights into the mechanisms of MD and disease resistance breeding in poultry.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IR L /TR L ) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.

Marek's Disease is a lymphoproliferative disease of chickens caused by Marek's Disease Virus. Similar to other herpesviruses, Marek's Disease Virus (MDV) encodes its own small non-coding regulatory RNAs termed microRNAs (miRNAs). We previously found that the expression profile of these viral miRNAs is affected by vaccination with Herpesvirus of Turkeys (HVT). To further characterize miRNA-mediated gene regulation in MDV infections, in the current study we examine the impact of HVT vaccination on cellular miRNA expression in MDV-infected specific-pathogen-free (SPF) chickens. We used small RNA-seq to identify 24 cellular miRNAs that exhibited altered splenic expression in MDV infected chickens (42 dpi) compared to age-matched uninfected birds. We then used Real Time-quantitative PCR (RT-qPCR) to develop expression profiles of a select group of these host miRNAs in chickens receiving the HVT vaccine and in vaccinated chickens subsequently infected with MDV. As was seen with viral miRNA, host miRNAs had unique splenic expression profiles between chickens infected with HVT, MDV, or co-infected birds. We also discovered a group of transcription factors, using a yeast one-hybrid screen, which regulates immune responses and cell growth pathways and also likely regulates the expression of these cellular miRNAs. Overall, this study suggests cellular miRNAs are likely a critical component of both protection from and progression of Marek's Disease.

Despite successful control by vaccination, Marek's disease (MD) has continued evolving to greater virulence over recent years. To control MD, selection and breeding of MD-resistant chickens might be a suitable option. MHC-congenic inbred chicken lines, 6₁ and 7₂, are highly resistant and susceptible to MD, respectively, but the cellular and genetic basis for these phenotypes is unknown. Marek's disease virus (MDV) infects macrophages, B-cells, and activated T-cells in vivo. This study investigates the cellular basis of resistance to MD in vitro with the hypothesis that resistance is determined by cells active during the innate immune response. Chicken bone marrow-derived macrophages from lines 6₁ and 7₂ were infected with MDV in vitro. Flow cytometry showed that a higher percentage of macrophages were infected in line 7 2 than in line 6₁. A transcriptomic study followed by in silico functional analysis of differentially expressed genes was then carried out between the two lines pre- and post-infection. Analysis supports the hypothesis that macrophages from susceptible and resistant chicken lines display a marked difference in their transcriptome following MDV infection. Resistance to infection, differential activation of biological pathways, and suppression of oncogenic potential are among host defense strategies identified in macrophages from resistant chickens., Competing Interests: The authors declare no conflicts of interest and no competing financial interests.