Transcriptomics And Immunosuppression Mechanism Of Reticuloendotheliosis Virus Infection

Reticuloendotheliosis virus (REV) is classified as a member of the genus Gamma retrovirus in the family Retroviridae and causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. Recently, the co-infection of REV with other avian viruses has been reported, potentially representing additional dangers to the poultry industry. The enhancement of avian reticuloendotheliosis virus co-infection is most likely a consequence of its immunosuppression. However, the mechanism of REV-induced tumourigenesis and immunosuppression has not yet been fully characterised.This study observed the different cell lines infected with REV HA 1101 strain, and analyzed the gene transcripts of host cell using gene expression profile microarray. The findings of this study would provide important data and clues for a better understanding the host immunity and pathogenic mechanism of REV.1. The development and application of multiple real-time PCR assay for detecetion of reticuloendotheliosis virusTo establish a rapid, sensitive and specific method to detect Reticuloendotheliosis virus (REV), and evaluate replication kinetics for REV in chicken embryo fibroblasts (CEF), a multiple real-time PCR assay was developed. The method using SYBR Green I and three pairs of primers specific to the conserved region of gag, env and LTR gene was contacted and measured copies of viral gene at different post infection days. The results showed real-time PCR assay was linear in the range of 108 to 10’copies/ul, which detection limit was as little as 101 copies/μl. We found that the copy numbers of three viral genes decreased first, and then increased over time. REV in CEF demonstrated some suitable stage at its early infection and immediately followed by the massive virus replication. In conclusion, this study provided new method for REV detection and its mechanisms of infection and pathogenesis.2. Transcriptional profiling of host gene expression in chicken embryo fibroblasts infected with reticuloendotheliosis virusReticuloendotheliosis virus, a member of the genus Gamma retrovirus in the family retroviridae, causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. To better understand host interactions at the transcriptional level, microarray datas analysis was performed at 1,3,5, and 7 days post infection with REV infected chicken embryo fibroblasts. The present study showed that 1785 differentially expressed genes were classified into a number of function groups that included signal transduction, immune response, biological adhesion and endocytosis. Significant differences were observed in expression of some genes involved in immune response, specifically later in post-infection. It was identified that the major genes responding to REV infection were associated with MAPK, JAK-STAT, and TLR receptors pathways. These results revealed that differentially expressed genes, IL-6、STAT-1、Myd-88、TLRs、NF-κB、IRFs, and ISGs play an important role in the pathogenicity of REV infection. Our research is the first microarray experiment to investigate REV, and the findings can provide insightinto the underlying mechanism of the host antiviral response and the molecular basis of viral pathogenesis.3. Replication of the reticuloendotheliosis virus in different chicken cell linesAs we know, REV can be cultured in chicken fibroblasts. However, immune organ of chicken is the target of REV infection. Up to now, there are only few reports in viral life cycle, immunosuppression and pathogenesis mechanism of REV. In order to make clear the characterization of REV in different kind of cells, chicken embryo fibroblast cell line DF1, chicken macrophage cell line HD11, chicken T lymphocyte cell line MSB1 and chicken B lymphocyte cell line DT40 were used for virus infection. Our results showed that copy numbers of REV infection in different cell lines were continuously increasing over time. Moreover, replication efficiency of REV in DF1 cell lines was different from that in immune cell lines. The indirect immunofluorescence assay (IFA) resulted that REV could not be detected until 96h in the virus infected DF1 cell, while at 48h in infected HDI1, MSB1, DT40 cells. In addition, morphological changes were found in infected immune cell lines through microscopic examination which differ from the infected fibroblast cell line DF1, which showed changes of increased cellular debris, reduced cell density, shrinkage, refraction abate. This study firstly described replication characteristic of REV infection in immune cell lines, and this provides a platform for further studying REV pathogenic mechanism.4. Apoptosis inducted by reticuloendotheliosis virus infection in immune cell lines in vitroInfection with REV, a highly contagious and immunosuppressive disease in birds, can result in immunosuppression and subsequent increased susceptibility to other pathogens infections. In addition, REV suppresses of immune cells proliferative responses. Though REV infection-induced host cell apoptosis has been established in vivo, the related mechanism is still unclear. In this study, we investigated the ability of REV to induce apoptosis in immune cell lines. We found that REV could induce apoptosis in various immune cell lines, including HD11 cells, MSB1 cells, and DT40 cells determined by cell viability assays, TUNEL assay, and flow cytometry assays. The expression of caspase-3,-8, and-9, genes were up-regulated in immune cells infected with REV by Real-time PCR. These results demonstrated that REV infection induced apoptosis of immune cells by overexpression of caspase-3 genes. Therefore, the results in this study will help us to understand the immunosuppression mechanism and host immune response to virus pathogenesis.5. Suppression of the innate immune response induced b\reticuloendotheliosis virus infection in immune cell linesThe innate immune response to viral pathogens is critical in host immune system. Cells of the innate immune system detect viral infection largely through germ line-encoded pattern recognition receptors (PRRs) present either on the cell surface or within distinct intracellular compartments.However, it is unclear whether REV induced cell innate immunity is associated with PRRs mediated signalling pathway. In this study, the expression of PPRs involved in REV infection mediated immunity in HD11, MSB1, DT40 cells is differ from the infected DF1 cells. Our results demonstrated that some host immune-related genes were down-regulated duringREV infection in immune cells. TLR-3 and MDA5 have the most significant impact by viral infection inhibition. In addition, the ligands LPS and ploy (I:C) stimulation treatment had inhibitory effect on REV infection and replication but not the MyD88-dependent TLR pathway. Our study expanded the present knowledge of host immune responses against REV infection and might provide a clue about that REV may interfere with innate immune response of immune cells through PRRs signals.