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.

Putative roles as oncogene or tumour suppressor of the Mid-clustered microRNAs in Gallid alphaherpesvirus 2 (GaHV2) induced Marek's disease lymphomagenesis.

In the last decade, numerous microRNAs (miRNAs) have been identified in diverse virus families, particularly in herpesviruses. Gallid alphaherpesvirus 2 (GaHV2) is a representative oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts, namely Marek's disease (MD). In the GaHV2 genome there are 26 mature miRNAs derived from 14 precursors assembled into three clusters, namely the Meq-cluster, Mid-cluster and LAT-cluster. Several GaHV2 miRNAs, especially those in the Meq-cluster (e.g. miR-M4-5p), have been demonstrated to be critical in MD pathogenesis and/or tumorigenesis. Interestingly the downstream Mid-cluster is regulated and transcribed by the same promoter as the Meq-cluster in the latent phase of the infection, but the role of these Mid-clustered miRNAs in GaHV2 biology remains unclear. We have generated the deletion mutants of the Mid-cluster and of its associated individual miRNAs in GX0101 virus, a very virulent GaHV2 strain, and demonstrated that the Mid-clustered miRNAs are not essential for virus replication. Using GaHV2-infected chickens as an animal model, we found that, compared with parental GX0101 virus, the individual deletion of miR-M31 decreased the mortality and gross tumour incidence of infected chickens while the deletion individually of miR-M1 or miR-M11 unexpectedly increased viral pathogenicity or oncogenicity, similarly to the deletion of the entire Mid-cluster region. More importantly, our data further confirm that miR-M11-5p, the miR-M11-derived mature miRNA, targets the viral oncogene meq and suppresses its expression in GaHV2 infection. We report here that members of the Mid-clustered miRNAs, miR-M31-3p and miR-M11-5p, potentially act either as oncogene or tumour suppressor in MD lymphomagenesis.

The significance of the individual Meq-clustered miRNAs of Marek's disease virus in oncogenesis.

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.

In vivo expression patterns of microRNAs of Gallid herpesvirus 2 (GaHV-2) during the virus life cycle and development of Marek's disease lymphomas.

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.

Molecular characteristics and evolutionary analysis of field Marek's disease virus prevalent in vaccinated chicken flocks in recent years in China.

Marek's disease is a highly contagious, oncogenic, and immunosuppressive avian viral disease. Surveillance of newly registered Marek's disease virus (MDV) isolates is meaningful for revealing the potential factors involved in increased virulence. Presently, we have focused on the molecular characteristics of all available MDVs from China, including 17 new Henan isolates. Based on Meq, gE, and gI genes, we found that most Chinese isolates contain conserved amino acid point mutations in Meq, such as E(77), A(115), A(139), R(176), and A(217), compared to USA virulent MDVs. However, the 59-aa or 60-aa insertions are only found in a few mild MDVs rather than virulent MDVs in China. Further phylogenetic analysis has demonstrated that a different genotype of MDV has been prevalent in China, and for virulent MDVs, their recent evolution has possibly been geographically restricted. Our study has provided more detailed information regarding the field MDVs circulating in China.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge.

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Marek's disease virus-encoded analog of microRNA-155 activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-ß signaling pathway.

Marek's disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-ß binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-ß1, with suppression of TGF-ß signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

Virus-encoded miR-155 ortholog is an important potential regulator but not essential for the development of lymphomas induced by very virulent Marek's disease virus.

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.