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As one of the most important avian immunosuppressive and neoplastic diseases, Marek's disease (MD), caused by oncogenic Marek's disease virus (MDV), has caused huge economic losses worldwide over the past five decades. In recent years, MD outbreaks have occurred frequently in MD-vaccinated chicken flocks, but the key pathogenic determinants and influencing factors remain unclear. Herein, we analyzed the pathogenicity of seven newly isolated MDV strains from tumor-bearing chickens in China and found that all of them were pathogenic to chicken hosts, among which four MDV isolates, SDCW01, HNXZ05, HNSQ05 and HNSQ01, were considered to be hypervirulent MDV (HV-MDV) strains. At 73 days of the virus infection experiment, the cumulative incidences of MD were 100%, 93.3%, 90% and 100%, with mortalities of 83.3%, 73.3%, 60% and 86.7%, respectively, for the four viruses. The gross occurrences of tumors were 50%, 33.3%, 30% and 63.3%, respectively, accompanied by significant hepatosplenomegaly and serious atrophy of the immune organs. Furthermore, the immune protection effects of four commercial MD vaccines against SDCW01, CVI988, HVT, CVI988+HVT, and 814 were explored. Unexpectedly, during the 67 days of post-virus challenge, the protection indices (PIs) of these four MD vaccines were only 46.2%, 38.5%, 50%, and 28%, respectively, and the birds that received the monovalent CVI988 or HVT still developed tumors with cumulative incidences of 7.7% and 11.5%, respectively. To our knowledge, this is the first demonstration of the simultaneous comparison of the immune protection efficacy of multiple commercial MD vaccines with different vaccine strains. Our study revealed that the HV-MDV variants circulating in China could significantly break through the immune protection of the classical MD vaccines currently widely used. For future work, there is an urgent need to develop novel, more effective MD vaccines for tackling the new challenge of emerging HV-MDV strains or variants for the sustainable control of MD.

Marek's disease (MD) caused by pathogenic Marek's disease virus type 1 (MDV-1) is one of the most important neoplastic diseases of poultry. MDV-1-encoded unique Meq protein is the major oncoprotein and the availability of Meq-specific monoclonal antibodies (mAbs) is crucial for revealing MDV pathogenesis/oncogenesis. Using synthesized polypeptides from conserved hydrophilic regions of the Meq protein as immunogens, together with hybridoma technology and primary screening by cross immunofluorescence assay (IFA) on Meq-deleted MDV-1 viruses generated by CRISPR/Cas9-gene editing, a total of five positive hybridomas were generated. Four of these hybridomas, namely 2A9, 5A7, 7F9 and 8G11, were further confirmed to secrete specific antibodies against Meq as confirmed by the IFA staining of 293T cells overexpressing Meq. Confocal microscopic analysis of cells stained with these antibodies confirmed the nuclear localization of Meq in MDV-infected CEF cells and MDV-transformed MSB-1 cells. Furthermore, two mAb hybridoma clones, 2A9-B12 and 8G11-B2 derived from 2A9 and 8G11, respectively, displayed high specificity for Meq proteins of MDV-1 strains with diverse virulence. Our data presented here, using synthesized polypeptide immunization combined with cross IFA staining on CRISPR/Cas9 gene-edited viruses, has provided a new efficient approach for future generation of specific mAbs against viral proteins.

Over the past two decades, numerous non-coding RNAs (ncRNAs) have been identified in different biological systems including virology, especially in large DNA viruses such as herpesviruses. As a representative oncogenic alphaherpesvirus, Marek's disease virus (MDV) causes an important immunosuppressive and rapid-onset neoplastic disease of poultry, namely Marek's disease (MD). Vaccinations can efficiently prevent the onset of MD lymphomas and other clinical disease, often heralded as the first successful example of vaccination-based control of cancer. MDV infection is also an excellent model for research into virally-induced tumorigenesis. Recently, great progress has been made in understanding the functions of ncRNAs in MD biology. Herein, we give a review of the discovery and identification of MDV-encoded viral miRNAs, focusing on the genomics, expression profiles, and emerging critical roles of MDV-1 miRNAs as oncogenic miRNAs (oncomiRs) or tumor suppressor genes involved in the induction of MD lymphomas. We also described the involvements of host cellular miRNAs, lincRNAs, and circRNAs participating in MDV life cycle, pathogenesis, and/or tumorigenesis. The prospects, strategies, and new techniques such as the CRISPR/Cas9-based gene editing applicable for further investigation into the ncRNA-mediated regulatory mechanisms in MDV pathogenesis/oncogenesis were also discussed, together with the possibilities of future studies on antiviral therapy and the development of new efficient MD vaccines., (© 2023. Science China Press.)

Marek's disease virus (MDV) induces immunosuppression and neoplastic disease in chickens. The virus is controllable via an attenuated meq deletion mutant virus, which has the disadvantage of retaining the ability to induce lymphoid organ atrophy. To overcome this deficiency and produce more vaccine candidates, a recombinant MDV was generated from the highly virulent Md5BAC strain, in which both meq and a cytolytic replication-related gene, pp38 , were deleted. Replication of the double deletion virus, Md5BAC Δ meq Δ pp38 , was comparable with that of the parental virus in vitro . The double deletion virus was shown to be fully attenuated and to reduce lymphoid organ atrophy in vivo . Crucially, Md5BAC Δ meq Δ pp38 confers superior protection against highly virulent virus compared with a commercial vaccine strain, CVI988/Rispens. Transcriptomic profiling indicated that Md5BAC Δ meq Δ pp38 induced a different host immune response from CVI988/Rispens. In summary, a novel, effective, and safe vaccine candidate for prevention and control of MD caused by highly virulent MDV is reported. IMPORTANCE MDV is a highly contagious immunosuppressive and neoplastic pathogen. The virus can be controlled through vaccination via an attenuated meq deletion mutant virus that retains the ability to induce lymphoid organ atrophy. In this study, we overcame the deficiency by generating meq and pp38 double deletion mutant virus. Indeed, the successfully generated meq and pp38 double deletion mutant virus had significantly reduced replication capacity in vivo but not in vitro . It was fully attenuated and conferred superior protection efficacy against very virulent MDV challenge. In addition, the possible immunological protective mechanism of the double deletion mutant virus was shown to be different from that of the gold standard MDV vaccine, CVI988/Rispens. Overall, we successfully generated an attenuated meq deletion mutant virus and widened the range of potential vaccine candidates. Importantly, this study provides for the first time the theoretical basis of vaccination induced by fully attenuated gene-deletion mutant virus.

The avian immunosuppressive and neoplastic diseases caused by Marek's disease virus (MDV), avian leucosis virus (ALV), and reticuloendotheliosis virus (REV) are seriously harmful to the global poultry industry. In recent years, particularly in 2020-2022, outbreaks of such diseases in chicken flocks frequently occurred in China. Herein, we collected live diseased birds from 30 poultry farms, out of 42 farms with tumour-bearing chicken flocks distributed in central China, to investigate the current epidemiology and co-infections of these viruses. The results showed that in individual diseased birds, the positive infection rates of MDV, ALV, and REV were 69.5% (203/292), 14.4% (42/292), and 4.7% (13/277), respectively, while for the flocks, the positive infection rates were 96.7% (29/30), 36.7% (11/30), and 20% (6/30), respectively. For chicken flocks, monoinfection of MDV, ALV, or REV was 53.3% (16/30), 3.3% (1/30), and 0% (0/30), respectively, but a total of 43.3% (13/30) co-infections was observed, which includes 23.3% (7/30) of MDV+ALV, 10.0% (3/30) of MDV+REV, and 10.0% (3/30) of MDV+ALV+REV co-infections. Interestingly, no ALV+REV co-infection or REV monoinfection was observed in the selected poultry farms. Our data indicate that the prevalence of virulent MDV strains, partially accompanied with ALV and/or REV co-infections, is the main reason for current outbreaks of avian neoplastic diseases in central China, providing an important reference for the future control of disease.

Marek's disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek's disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant-GX0101Δpp38-and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas-namely, 4F9, 31G7, 34F2, and 35G9-were demonstrated to secrete specific antibodies against MDV-1's pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses-especially for large DNA viruses such as herpesviruses.

In recent years, outbreaks of Marek's disease (MD) have been frequently reported in vaccinated chicken flocks in China. Herein, we have demonstrated that four Marek's disease virus (MDV) isolates, HN502, HN302, HN304, and HN101, are all pathogenic and oncogenic to hosts. Outstandingly, the HN302 strain induced 100% MD incidence, 54.84% mortality, and 87.10% tumor incidence, together with extensive atrophy of immune organs. Pathotyping of HN302 was performed in comparison to a standard very virulent (vv) MDV strain Md5. We found that both CVI988 and HVT vaccines significantly reduced morbidity and mortality induced by HN302 or Md5 strains, but the protection indices (PIs) provided by these two vaccines against HN302 were significantly lower (27.03%) or lower (33.33%) than that against Md5, which showed PIs of 59.89% and 54.29%, respectively. These data suggested that HN302 possesses a significant higher virulence than Md5 and at least could be designated as a vvMDV strain. Together with our previous phylogenetic analysis on MDV-1 meq genes, we have presently suggested HN302 to be a typical highly virulent MDV variant belonging to an independent Chinese branch. To our knowledge, this is the first report to provide convincible evidence to identify a pathogenic MDV variant strain with a higher virulence than Md5 in China, which may have emerged and circulating in poultry farms in China for a long time and involved in the recent MD outbreaks.

Marek's disease (MD) is a neoplastic disease of chickens caused by an avian alphaherpesvirus, Marek's disease virus (MDV, also known as Gallid alphaherpesvirus 2 [GaHV2]). A total of 14 microRNA (miRNA) precursors and 26 mature miRNAs have been identified in MDV genome, which were grouped in three distinct clusters. In recent years, our studies revealed the role of MDV encoded cluster 3 miRNAs (or miR-M8-M10) and the specific function of its three members, miR-M6, miR-M7 and miR-M10, in regulating MDV replication and pathogenesis. In this study, we characterized the unique function of the other two members, miR-M8 and miR-M13, in cluster 3 miRNAs. Our results show that miR-M8 and miR-M13 are not important for MDV plaque formation and genome replication in vitro. Animal experiment results show that deletion of miR-M8-5p and miR-M13-5p eliminates the bursa atrophy, but not thymus atrophy, of MDV inoculated chickens. In addition, we found that the survival curve and MD incidences were not affected by disruption of miR-M8 and miR-M13. Taken together, this study uncovers the unique role of miR-M8 and miR-M13 in MDV replication and pathogenesis, which filled the gap in the research of MDV encoded miRNAs., (Copyright © 2022 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are a class of approximately 22 nucleotides long non-coding RNAs, and virus-encoded miRNAs play an important role in pathogenesis. Marek's disease virus (MDV) is an oncogenic avian alphaherpesvirus that causes immunosuppression and tumors in its natural host, chicken. In the MDV genome, 14 miRNA precursors and 26 mature miRNAs were identified, thus MDV has been used as a model to study the function of viral miRNAs in vivo. Recently, a cluster of miRNAs encoded by MDV, Cluster 3 miRNAs (miR-M8-M10), has been shown to restrict early cytolytic replication and pathogenesis of MDV. In this study, we further analyzed the role of miR-M6 and miR-M10, members of cluster miR-M8-M10, in MDV replication and pathogenicity. We found that, compared to parental MDV, deletion of miR-M6-5p significantly enhanced the replication of MDV in cell culture, but not in chickens. The replication of miR-M6-5p deletion MDV was restored once the deleted sequences were re-inserted. Our results also showed that deletion of miR-M10-5p did not affect the replication of MDV in vitro and in vivo. In addition, our animal study results showed that deletion of miR-M6-5p or miR-M10-5p did not alter the pathogenesis of MDV. In conclusion, our study shows that both miR-M6 and miR-M10 are dispensable for MDV replication and pathogenesis in chickens, while also suggests a repressive role of miR-M6 in MDV replication in cell culture., (Copyright © 2021 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are a class of ∼22 nucleotides non-coding RNAs that are encoded by a wide range of hosts. Viruses, especially herpesviruses, encode a variety of miRNAs that involved in disease progression. Recently, a cluster of virus-encoded miRNAs, miR-M8-M10, have been shown to restrict early cytolytic replication and pathogenesis of Marek's disease virus (MDV), an oncogenic avian alphaherpesvirus that causes lymphoproliferative disease in chickens. In this study, we specifically dissected the role of miR-M7, a member of cluster miR-M8-M10, in regulating MDV replication and pathogenesis. We found that deletion of miR-M7-5p did not affect the virus plaque size and growth in cell culture. However, compared to parental virus, infection of miR-M7-5p deletion virus significantly increased MDV genome copy number at 5 days post infection, suggesting that miR-M7 plays a role to restrict MDV replication during early cytolytic phase. In addition, our results showed that infection of miR-M7-5p deletion virus significantly enhanced the mortality of chickens, even it induced lymphoid organ atrophy similar to parental and revertant viruses. Taken together, our study revealed that the miR-M7 acts as a repressive factor of MDV replication and pathogenesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV) induces severe immunosuppression and lymphomagenesis in the chicken, its natural host, and results in a condition that investigated the pathogenesis of MDV and have begun to focus on the expression profiling of circular RNAs (circRNAs). However, little is known about how the expression of circRNAs is referred to as Marek's disease. Previous reports have is regulated during MDV replication. Here, we carried out a comprehensive profiling analysis of N6-methyladenosine (m 6 A) modification on the circRNA transcriptome in infected and uninfected chicken embryonic fibroblast (CEF) cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that m 6 A modification was highly conserved in circRNAs. Comparing to the uninfected group, the number of peaks and conserved motifs were not significantly different in cells that were infected with MDV, although reduced abundance of circRNA m 6 A modifications. However, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses revealed that the insulin signaling pathway was associated with the regulation of m 6 A modified circRNAs in MDV infection. This is the first report to describe alterations in the transcriptome-wide profiling of m 6 A modified circRNAs in MDV-infected CEF cells.

Background: The newly discovered reversible N6-methyladenosine (m 6 A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek's disease virus (MDV) replication but how m 6 A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m 6 A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells., Results: Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m 6 A modification is highly conserved with MDV infection increasing the expression of lncRNA m 6 A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m 6 A modifications were highly associated with signaling pathways associated with MDV infection., Conclusions: In this study, the alterations seen in transcriptome-wide m 6 A 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 m 6 A modification in lncRNAs of MDV-infected CEF cells.

Marek's disease virus serotype 1 (MDV-1) is an important oncogenic α-herpesvirus that induces immunosuppressive and rapid-onset T-cell lymphomatous disease in poultry commonly referred to as Marek's disease (MD). As an excellent biomodel for the study of virally-induced cancers in natural hosts, MDV-1 encoded microRNAs (miRNAs) have been previously demonstrated with the potential roles to act as critical regulators in virus replication, latency, pathogenesis and especially in oncogenesis. Similar to the oncogenic γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV), miR-M4-5p, the cellular microRNA-155 (miR-155) ortholog encoded by MDV-1, is also involved in MD oncogenesis. In lymphoblastoid cell lines derived from MDV-induced T-cell lymphomas, miR-M4-5p has been shown to be highly expressed and participate in inducing MD lymphomagenesis by regulating multiple signal pathways. Herein we report the new identification of the host WW domain-containing oxidoreductase (WWOX) as a biological target for miR-M4-5p. Further experiments revealed that as a critical oncomiRNA, miR-M4-5p promotes the proliferations of both chicken embryo fibroblast (CEF) and MSB-1 cells via suppressing cell apoptosis by targeting WWOX, a well-known tumor suppressor. Our data presents a novel insight in elucidating the regulatory mechanisms mediated by the viral analog of miR-155 that potentially contribute to MD tumorigenesis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV), a lymphotropic α-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek's disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 ( pp38 ), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek's disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38 -deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.

Marek's disease virus type 1 (MDV-1) is a representative oncogenic Alpha herpesvirus that causes an immunosuppressive and neoplastic lymphoproliferative avian disease, namely Marek's disease (MD). The rapid-onset T-cell lymphoma in chickens induced by MDV-1 has been historically regarded as an ideal natural model for herpesvirus-related cancer research. As a viral analog of cellular miR-155, the MDV-1-encoded miR-M4-5p has been shown to be crucial for the virally-induced MD tumorigenesis. Our previous studies demonstrated that miR-M4-5p induces an over-expression of oncogene c-Myc by targeting LTBP1 and suppressing the TGF-β signaling pathway during MDV-1 infection. We have now further identified the chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) as a new cellular biological target for miR-M4-5p. Suppression of hnRNPAB expression mediated by miR-M4-5p promotes the proliferation, but not the apoptosis, of both primary chicken embryo fibroblasts (CEFs) and transformed chicken fibroblast DF-1 cell line. HnRNPAB is a member of the hnRNP family of proteins that play important roles in normal biological processes as well as cancer development. Our data suggests that the recognition and down-regulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the past decade, a large number of microRNAs (miRNAs) have been identified in the viral genome of Gallid herpesvirus 2 (GaHV-2), which is historically known as Marek's disease virus type 1. The biological role of most GaHV-2 miRNAs remains unclear. In the present study, we have performed an overall gene expression profile of GaHV-2 miRNAs during the virus life cycle at each phase of the developing disease, a highly contagious, lymphoproliferative disorder, and neoplastic immunosuppressive disease of poultry known as the Marek's disease. According to their distinct in vivo expression patterns, the GaHV-2 miRNAs can be divided into three groups: 12 miRNAs in group I, including miR-M4-5p, displayed a typical expression pattern potentially correlated to the latent, late cytolytic, and/or the proliferative phases in the cycle of GaHV-2 pathogenesis; group II consisting of another 12 miRNAs with expression correlated to the early cytolytic and/or latent phases in GaHV-2's life cycle; while the other two miRNAs in group III showed no identical expression features. Our findings may provide meaningful clues in the search for further potential functions of viral miRNAs in GaHV-2 biology.

Marek's disease virus (MDV) is an important oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts. The Meq-clustered miRNAs encoded by MDV have been suggested to play potentially critical roles in the induction of lymphomas. Using the technique of bacterial artificial chromosome mutagenesis, we have presently constructed a series of specific miRNA-deleted mutants and demonstrate that these miRNAs are not essential for replication of MDV and have no effects on the early cytolytic or latent phases of the developing disease. However, compared to the parental GX0101, mortality of birds infected with the mutants GXΔmiR-M2, GXΔmiR-M3, GXΔmiR-M5, GXΔmiR-M9 and GXΔmiR-M12 was reduced from 100 % to 18 %, 30 %, 48 %, 24 % and 14 %, coupled with gross tumour incidence reduction from 28 % to 8 %, 4 %, 12 %, 8 % and 0 %, respectively. Our data confirm that except for mdv1-miR-M4, the other Meq-clustered miRNAs also play critical roles in MDV oncogenesis. Further work will be needed to elucidate the miRNA-mediated regulatory mechanisms that trigger the development of MD lymphomas., (© 2015 The Authors.)

The microRNA (miRNA) mdv1-miR-M4, a functional miR-155 ortholog encoded by oncogenic Marek's disease virus (MDV), has previously been suggested to be involved in MDV pathogenesis. Using the technique of bacterial artificial chromosome mutagenesis, we have presently evaluated the potential role of mdv1-miR-M4 in the oncogenesis of the very virulent (vv) MDV strain GX0101. Unexpectedly, deletions of the Meq-cluster or mdv1-miR-M4 alone from the viral genome strongly decreased rather than abolished its oncogenicity. Compared to GX0101, mortalities of mutants GXΔmiR-M4 and GXΔMeq-miRs were reduced from 100% to 18% and 4%, coupled with the gross tumor incidence reduction from 28% to 22% and 8%, respectively. Our data suggests that the mdv1-miR-M4 is possibly an important regulator in the development of Marek's disease (MD) lymphomas but is not essential for the oncogenicity of vvMDV. In addition, some of the other Meq-clustered miRNAs may also play potentially critical roles in vvMDV induction of lymphomas., (© 2013 Published by Elsevier Inc.)