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.

LORF9 of Marek's disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development.

IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

Characterization of integrated Marek's disease virus genomes supports a model of integration by homology-directed recombination and telomere-loop-driven excision.

IMPORTANCE: Marek's disease virus (MDV) is a ubiquitous chicken pathogen that inflicts a large economic burden on the poultry industry, despite worldwide vaccination programs. MDV is only partially controlled by available vaccines, and the virus retains the ability to replicate and spread between vaccinated birds. Following an initial infection, MDV enters a latent state and integrates into host telomeres and this may be a prerequisite for malignant transformation, which is usually fatal. To understand the mechanism that underlies the dynamic relationship between integrated-latent and reactivated MDV, we have characterized integrated MDV (iMDV) genomes and their associated telomeres. This revealed a single orientation among iMDV genomes and the loss of some terminal sequences that is consistent with integration by homology-directed recombination and excision via a telomere-loop-mediated process.

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Novel lncRNA 803 related to Marek's disease inhibits apoptosis of DF-1 cells.

Marek's disease (MD) is a neoplastic disease that significantly affects the poultry industry. Long non-coding RNAs (lncRNAs) are crucial regulatory factors in various biological processes, including tumourigenesis. However, the involvement of novel lncRNAs in the course of MD virus (MDV) infection is still underexplored. Here, we present the first comprehensive characterization of differentially expressed lncRNAs in chicken spleen at different stages of MDV infection. A series of differentially expressed lncRNAs was identified at each stage of MDV infection through screening. Notably, our investigation revealed a novel lncRNA, lncRNA 803, which exhibited significant differential expression at different stages of MDV infection and was likely to be associated with the p53 pathway. Further analyses demonstrated that the overexpression of lncRNA 803 positively regulated the expression of p53 and TP53BP1 in DF-1 cells, leading to the inhibition of apoptosis. This is the first study to focus on the lncRNA expression profiles in chicken spleens during MDV pathogenesis. Our findings highlight the potential role of the p53-related novel lncRNA 803 in MD pathogenesis and provide valuable insights for decoding the molecular mechanism of MD pathogenesis involving non-coding RNA.RESEARCH HIGHLIGHTS Differentially expressed lncRNAs in spleens of chickens infected with Marek's disease virus at different stages were identified for the first time.The effects of novel lncRNA 803 on p53 pathway and apoptosis of DF-1 cells were reported for the first time.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

Marek's Disease Virus Requires Both Copies of the Inverted Repeat Regions for Efficient In Vivo Replication and Pathogenesis.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of chickens. The MDV genome consists of two unique regions that are both flanked by inverted repeat regions. These repeats harbor several genes involved in virus replication and pathogenesis, but it remains unclear why MDV and other herpesviruses harbor these large sequence duplications. In this study, we set to determine if both copies of these repeat regions are required for MDV replication and pathogenesis. Our results demonstrate that MDV mutants lacking the entire internal repeat region (ΔIRLS) efficiently replicate and spread from cell-to-cell in vitro However, ΔIRLS replication was severely impaired in infected chickens and the virus caused significantly less frequent disease and tumors compared to the controls. In addition, we also generated recombinant viruses that harbor a deletion of most of the internal repeat region, leaving only short terminal sequences behind (ΔIRLS-HR). These remaining homologous sequences facilitated rapid restoration of the deleted repeat region, resulting in a virus that caused disease and tumors comparable to the wild type. Therefore, ΔIRLS-HR represents an excellent platform for rapid genetic manipulation of the virus genome in the repeat regions. Taken together, our study demonstrates that MDV requires both copies of the repeats for efficient replication and pathogenesis in its natural host.IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes losses in the poultry industry of up to $2 billion per year. The virus is also widely used as a model to study alphaherpesvirus pathogenesis and virus-induced tumor development in a natural host. MDV and most other herpesviruses harbor direct or inverted repeats regions in their genome. However, the role of these sequence duplications in MDV remains elusive and has never been investigated in a natural virus-host model for any herpesvirus. Here, we demonstrate that both copies of the repeats are needed for efficient MDV replication and pathogenesis in vivo, while replication was not affected in cell culture. With this, we further dissect herpesvirus genome biology and the role of repeat regions in Marek's disease virus replication and pathogenesis.

Distinct polymorphisms in a single herpesvirus gene are capable of enhancing virulence and mediating vaccinal resistance.

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek's disease virus in vitro.

BACKGROUND: The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek's disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells. RESULTS: Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection. CONCLUSIONS: In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

MicroRNA profiling in the bursae of Marek's disease virus-infected resistant and susceptible chicken lines.

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include bursal/thymic atrophy, neurologic disorders, and T cell lymphomas. MiRNAs play key roles in regulation of gene expression by targeting translational suppression or mRNA degradation. MDV encodes miRNAs that are associated with viral pathogenicity and oncogenesis. In this study, we performed miRNA sequencing in the bursal tissues, non-tumorous but viral-induced atrophied lymphoid organ, from control and infected MD-resistant and susceptible chickens at 21 days post infection. In addition to some known miRNAs, a minimum of 300 novel miRNAs were identified in each group that mapped to the chicken genome with no sequence homology to existing miRNAs in chicken miRbase. Comparative analysis identified 54 deferentially expressed miRNAs between the chicken lines that might shed light on underlying mechanism of bursal atrophy and resistance or susceptibility to MD.

The Herpesviridae Conserved Multifunctional Infected-Cell Protein 27 (ICP27) Is Important but Not Required for Replication and Oncogenicity of Marek's Disease Alphaherpesvirus.

The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

Inhibition of DNA-Sensing Pathway by Marek's Disease Virus VP23 Protein through Suppression of Interferon Regulatory Factor 7 Activation.

The type I interferon (IFN) response is the first line of host innate immune defense against viral infection; however, viruses have developed multiple strategies to antagonize host IFN responses for efficient infection and replication. Here, we report that Marek's disease virus (MDV), an oncogenic herpesvirus, encodes VP23 protein as a novel immune modulator to block the beta interferon (IFN-ß) activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) in chicken fibroblasts and macrophages. VP23 overexpression markedly reduces viral DNA-triggered IFN-ß production and promotes viral replication, while knockdown of VP23 during MDV infection enhances the IFN-ß response and suppresses viral replication. VP23 selectively inhibits IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) activation. Furthermore, we found that VP23 interacts with IRF7 and blocks its binding to TANK-binding kinase 1 (TBK1), thereby inhibiting IRF7 phosphorylation and nuclear translocation, resulting in reduced IFN-ß production. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.IMPORTANCE Despite widespread vaccination, Marek's disease (MD) continues to pose major challenges for the poultry industry worldwide. MDV causes immunosuppression and deadly lymphomas in chickens, suggesting that this virus has developed a successful immune evasion strategy. However, little is known regarding the initiation and modulation of the host innate immune response during MDV infection. This study demonstrates that the cGAS-STING DNA-sensing pathway is critical for the induction of the IFN-ß response against MDV infection in chicken fibroblasts and macrophages. An MDV protein, VP23, was found to efficiently inhibit the cGAS-STING pathway. VP23 selectively inhibits IRF7 but not NF-κB activation. VP23 interacts with IRF7 and blocks its binding to TBK1, thereby suppressing IRF7 activation and resulting in inhibition of the DNA-sensing pathway. These findings expand our knowledge of DNA sensing in chickens and reveal a mechanism through which MDV antagonizes the host IFN response.

Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3.

Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek's disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication.IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

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.

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing.

BACKGROUND: Marek's disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. RESULTS: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. CONCLUSIONS: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Genome-wide analysis of circular RNAs involved in Marek's disease tumourigenesis in chickens.

Marek's disease (MD), induced by Marek's disease virus (MDV), is a lymphotropic neoplastic disease and causes huge economic losses to the poultry industry. Non-coding RNAs (ncRNAs) play important regulatory roles in disease pathogenesis. To investigate host circular RNA (circRNA) and microRNA (miRNA) expression profile, RNA sequencing was performed in tumourous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). A total of 2,169 circRNAs were identified and more than 80% of circRNAs were derived from exon. The flanking introns of 1,744 exonic circRNAs possessed 579 reverse complementary matches (RCMs), which mainly overlapped with chicken repeat 1 family (CR1F). It suggested that CR1F mediated the cyclization of exons by intron pairing. Out of 2,169 circRNAs, 113 were differentially expressed circRNAs (DECs). The Q-PCR and Rnase R digestion experiments showed circRNA possessed high stability compared with their linear RNAs. Integrated with previous transcriptome data, we profiled regulatory networks of circRNA/long non-coding RNA (lncRNA)-miRNA-mRNA. Extensive competing endogenous RNA (ceRNA) networks were predicted to be involved in MD tumourigenesis. Interestingly, circZMYM3, an intronic circRNA, interacted with seven miRNAs which targeted some immune genes, such as SWAP70 and CCL4. Gga-miR-155 not only interacted with circGTDC1 and circMYO1B, but also targeted immune-related genes, such as GATA4, which indicated the roles of non-coding RNAs played to mediate immune responsive genes. Collectively, this is the first study that integrated RNA expression profiles in MD model. Our results provided comprehensive interactions of ncRNAs and mRNA in MD tumourigenesis.

Detection of p53 mutation and serum monitoring alert caused by Marek's disease virus in poultry.

BACKGROUND: Marek's disease (MD) is a chicken neoplastic disease, which brings huge economic losses to the global poultry industry. The wild type p53, a tumor suppressor gene, plays a key role in blocking cell cycle, promoting apoptosis, and maintaining the stability of the genome. However, the mutant p53 losses its tumor inhibitory role and become an oncogene when a mutation has happened. RESULTS: The mutation rate of p53 was 60% in the experimentally and naturally infected chickens. The mutations included point-mutations and deletions, and mostly located in the DNA-binding domain. The mutated p53 was expressed in various tumor tissues in an infected chicken. The mutant P53 proteins were notably accumulated in the cytoplasm due to the loss in the function of nuclear localization. Unlike the study on human cancer, the concentrations of P53 in the serums of MD infected chicken were significantly lower than the control group. CONCLUSIONS: The p53 mutations were apparent in the development of MD. P53 and P53 antibody level in serum could be a useful marker in the diagnosis and surveillance of MD.

Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek's disease virus (MDV) transformed cell line MSB1 by targeting RORA.

BACKGROUND: Marek's disease (MD) is caused by the oncogenic Marek's disease virus (MDV), and is a highly contagious avian infection with a complex underlying pathology that involves lymphoproliferative neoplasm formation. MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in most cancers. The gga-miR-155 is downregulated in the MDV-infected chicken tissues or lymphocyte lines, although its exact role in tumorigenesis remains unclear. The aim of this study was to analyze the effects of gga-miR-155 on the proliferation, apoptosis and invasiveness of an MDV-transformed lymphocyte line MSB1 and elucidate the underlying mechanisms. RESULTS: The expression level of gga-miR-155 was manipulated in MSB1 cells using specific mimics and inhibitors. While overexpression of gga-miR-155 increased proliferation, decreased the proportion of G1 phase cells relative to that in S and G2 phases, reduced apoptosis rates and increased invasiveness. However, its downregulation had the opposite effects. Furthermore, gga-miR-155 directly targeted the RORA gene and downregulated its expression in the MSB1 cells. CONCLUSION: The gga-miR-155 promotes the proliferation and invasiveness of the MDV-transformed lymphocyte line MSB1 and inhibits apoptosis by targeting the RORA gene.

Marek's Disease Virus Infection Induced Mitochondria Changes in Chickens.

Mitochondria are crucial cellular organelles in eukaryotes and participate in many cell processes including immune response, growth development, and tumorigenesis. Marek's disease (MD), caused by an avian alpha-herpesvirus Marek's disease virus (MDV), is characterized with lymphomas and immunosuppression. In this research, we hypothesize that mitochondria may play roles in response to MDV infection. To test it, mitochondrial DNA (mtDNA) abundance and gene expression in immune organs were examined in two well-defined and highly inbred lines of chickens, the MD-susceptible line 72 and the MD-resistant line 63. We found that mitochondrial DNA contents decreased significantly at the transformation phase in spleen of the MD-susceptible line 72 birds in contrast to the MD-resistant line 63. The mtDNA-genes and the nucleus-genes relevant to mtDNA maintenance and transcription, however, were significantly up-regulated. Interestingly, we found that POLG2 might play a potential role that led to the imbalance of mtDNA copy number and gene expression alteration. MDV infection induced imbalance of mitochondrial contents and gene expression, demonstrating the indispensability of mitochondria in virus-induced cell transformation and subsequent lymphoma formation, such as MD development in chicken. This is the first report on relationship between virus infection and mitochondria in chicken, which provides important insights into the understanding on pathogenesis and tumorigenesis due to viral infection.