Early Cytolytic Phase Research Articles

Virus-encoded microRNA-M7 restricts early cytolytic replication and pathogenesis of Marek’s disease virus

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.

The role of Meq-vIL8 in regulating Marek's disease virus pathogenesis.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

Deletion of LORF9 but not LORF10 attenuates Marek’s disease virus pathogenesis

Marek’s disease virus (MDV) genome contains a number of uncharacterized long open reading frames (LORF) and their role in viral pathogenesis has not been fully investigated. Among them, LORF9 (MDV069) and LORF10 (MDV071) are locate at the right terminus of the MDV genome unique long region (UL). To investigate their role in MDV pathogenesis, we generated LORF9 or LORF10 deletion and revertant viruses. In vitro growth kinetics results show that both LORF9 and LORF10 are not essential for virus growth in cell culture. However, LORF9, but not LORF10, is involved in MDV early cytolytic replication in vivo, as evidenced by limited viral antigen expression in lymphoid organs of LORF9 deletion virus inoculated chickens. MDV genome copy number data further confirmed that LORF9 is important for MDV replication in spleen during early cytolytic phase. Deletion of LORF9 also partially impairs the replication of MDV in feather follicle epithelium (FFE); however, it can still establish latency and transformation. In addition, pathogenesis studies show that deletion of LORF9, but not LORF10, result in significant reduction of MDV induced mortality and slightly reduce MDV associated tumors of inoculated chickens. Importantly, we confirmed these results with the generation of LORF9 and LORF10 revertant viruses that fully restore the phenotypes of parental MDV. In conclusion, our results show that deletion of LORF9, but not LORF10, significantly impair viral replication in lymphoid organs during early cytolytic phase and attenuate Marek’s disease virus pathogenesis.

Transcriptional Profiles Associated with Marek's Disease Virus in Bursa and Spleen Lymphocytes Reveal Contrasting Immune Responses during Early Cytolytic Infection.

Marek’s disease virus (MDV), an alpha herpes virus, causes a lymphoproliferative state in chickens known as Marek’s disease (MD), resulting in severe monetary losses to the poultry industry. Because lymphocytes of bursa of Fabricius and spleen are prime targets of MDV replication during the early cytolytic phase of infection, the immune response in bursa and spleen should be the foundation of late immunity induced by MDV. However, the mechanism of the MDV-mediated host immune response in lymphocytes in the early stage is poorly understood. The present study is primarily aimed at identifying the crucial genes and significant pathways involved in the immune response of chickens infected with MDV CVI988 and the very virulent RB1B (vvRB1B) strains. Using the RNA sequencing approach, we analyzed the generated transcriptomes from lymphocytes isolated from chicken bursa and spleen. Our findings validated the expression of previously characterized genes; however, they also revealed the expression of novel genes during the MDV-mediated immune response. The results showed that after challenge with CVI988 or vvRB1B strains, 634 and 313 differentially expressed genes (DEGs) were identified in splenic lymphocytes, respectively. However, 58 and 47 DEGs were observed in bursal lymphocytes infected with CVI988 and vvRB1B strains, respectively. Following MDV CVI988 or vvRB1B challenge, the bursal lymphocytes displayed changes in IL-6 and IL-4 gene expression. Surprisingly, splenic lymphocytes exhibited an overwhelming alteration in the expression of cytokines and cytokine receptors involved in immune response signaling. On the other hand, there was no distinct trend between infection with CVI988 and vvRB1B and the expression of cytokines and chemokines, such as IL-10, IFN-γ, STAT1, IRF1, CCL19, and CCL26. However, the expression profiles of IL-1β, IL-6, IL8L1, CCL4 (GGCL1), and CCL5 were significantly upregulated in splenic lymphocytes from chickens infected with CVI988 compared with those of chickens infected with vvRB1B. Because these cytokines and chemokines are considered to be associated with B cell activation and antigenic signal transduction to T cells, they may indicate differences of immune responses initiated by vaccinal and virulent strains during the early phase of infection. Collectively, our study provides valuable data on the transcriptional landscape using high-throughput sequencing to understand the different mechanism between vaccine-mediated protection and pathogenesis of virulent MDV in vivo.

Dynamic Changes in the Splenic Transcriptome of Chickens during the Early Infection and Progress of Marek\u2019s Disease

Gallid alphaherpesvirus 2 (GaHV2) is an oncogenic avian herpesvirus inducing Marek’s disease (MD) and rapid-onset T-cell lymphomas. To reveal molecular events in MD pathogenesis and tumorigenesis, the dynamic splenic transcriptome of GaHV2-infected chickens during early infection and pathogenic phases has been determined utilizing RNA-seq. Based on the significant differentially expressed genes (DEGs), analysis of gene ontology, KEGG pathway and protein-protein interaction network has demonstrated that the molecular events happening during GaHV2 infection are highly relevant to the disease course. In the ‘Cornell Model’ description of MD, innate immune responses and inflammatory responses were established at early cytolytic phase but persisted until lymphoma formation. Humoral immunity in contrast began to play a role firstly in the intestinal system and started at late cytolytic phase. Neurological damage caused by GaHV2 is first seen in early cytolytic phase and is then sustained throughout the following phases over a long time period. During the proliferative phase many pathways associated with transcription and/or translation were significantly enriched, reflecting the cell transformation and lymphoma formation. Our work provides an overall view of host responses to GaHV2 infection and offers a meaningful basis for further studies of MD biology.

Histone modifications induced by MDV infection at early cytolytic and latency phases.

BackgroundMarek’s disease (MD) is a highly contagious, lymphomatous disease of chickens induced by a herpesvirus, Marek’s disease virus (MDV) that is the cause of major annual losses to the poultry industry. MD pathogenesis involves multiple stages including an early cytolytic phase and latency, and transitions between these stages are governed by several host and environmental factors. The success of vaccination strategies has led to the increased virulence of MDV and selective breeding of naturally resistant chickens is seen as a viable alternative. While multiple gene expression studies have been performed in resistant and susceptible populations, little is known about the epigenetic effects of infection.ResultsIn this study, we investigated temporal chromatin signatures induced by MDV by analyzing early cytolytic and latent phases of infection in the bursa of Fabricius of MD-resistant and –susceptible birds. Major global variations in chromatin marks were observed at different stages of MD in the two lines. Differential H3K27me3 marks were associated with immune-related pathways, such as MAP kinase signaling, focal adhesion and neuroactive ligand receptor interaction, and suggested varying degrees of silencing in response to infection. Immune-related microRNAs, e.g. gga-miR-155 and gga-miR-10b, bore chromatin signatures, which suggested their contribution to MD-susceptibility. Finally, several members of the focal adhesion pathway, e.g. THBS4 and ITGA1, showed marked concordance between gene expression and chromatin marks indicating putative epigenetic regulation in response to MDV infection.ConclusionOur comprehensive analysis of chromatin signatures, therefore, revealed further clues about the epigenetic effects of MDV infection although further studies are necessary to elucidate the functional implications of the observed variations in histone modifications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1492-6) contains supplementary material, which is available to authorized users.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek’s disease virus

BackgroundAn immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek’s disease virus (MDV), which induces immunosuppression in infected chickens.ResultsA chicken T cell line, Lee1, which constitutively produces IFN-γ was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-γ expression level was analyzed in the cell line by real-time RT-PCR. The IFN-γ expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field.ConclusionsWe demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host.

Gene Expression Profiling in rMd5- and rMd5Δmeq-Infected Chickens

Marek's disease (MD) is a lymphoproliferative disorder of domestic chickens caused by a highly contagious and oncogenic alpha-herpesvirus, Marek's disease virus (MDV). MD is characterized by bursal-thymic atrophy and rapid onset of T-cell lymphomas that infiltrate lymphoid tissues, visceral organs, and peripheral nerves with severe clinical signs that include transient paralysis, anemia, weight loss, and neurologic disorders. Using overlapping cosmids- and BAC-cloned MDV, it has been shown that MDV-encoded vIL-8, pp38, vTR, vLIP, RLORF4, and meq are among the many essential genes that play critical roles in viral pathogenesis. Of all the genes investigated so far, only meq has been shown to be consistently expressed in all MDV-derived tumors and lymphoblastoid cell lines. Meq is a basic leucine-zipper protein that shares homology with the jun/fos family of transcriptional factors. There are two copies of meq gene within the MDV genome that are only present in the serotype-1 strains. It has been shown conclusively that deletion of meq results in loss of transformation of T cells in chickens, with no effect on the early cytolytic phase of infection in lymphoid organs, which is essential for induction of innate and adaptive immunity. The goal of this study was to investigate 1) the effect of the meq oncogene on the expression pattern of select chicken immune and nonimmune-related genes, and 2) its potential role in MDV-induced apoptosis. We used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profiling of a panel of chicken genes in rMd5- and rMd5deltameq-infected chickens at 5, 14, 21, and 35 days postinfection (dpi). Although the transcriptional activities of several immune-related genes, including IL-6, IL-10, cMGF, GM-CSF, iNOS, IFNbeta, and INFgamma, were higher in rMd5deltameq-infected chickens at 5 dpi when compared to the rMd5-infected birds, the differences in expression levels of the tested genes between the two viral constructs were not significant. In addition, a reduction in the transcriptional activity of Bdcl2 in recombinant fowlpox virus (rFPV)+meq-infected chicken embryonic fibroblasts suggested that meq alone did not impede FPV-induced apoptosis. The likely suppressive nature and anti-inflammatory function of the meq oncogene and its possible role in virus-induced cell death is discussed.

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Marek's disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek's disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

Further characterization of Marek's disease virus-infected lymphocytes. I. In vivo infection.

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.