Transcriptome Analysis Of Chicken Spleen Infected With Marek's Disease Virus And The Pathogenesis Of Marek's Disease Regulated By MiR-M4-5p

Marek's disease(MD)is a major infectious disease affecting poultry health worldwide and is responsible for approximate annual global economic losses of$2 billion.MD is characterized in the early stages by transient neurological signs and immunosuppression,followed by lymphoma formation in susceptible breeds in various visceral organs.The causative agent,Gallid alphaherpesvirus 2(GaHV2),commonly known as Marek's disease virus type 1(MDV1),is an oncogenic avian herpesvirus belonging to the subfamily Alphaherpesvirinae.In the past several decades the virulence of MDV1 has persistently increased due possibly to highly intensive farming or to imperfect immunization by MD vaccines.Outbreaks of MD caused by highly virulent MDV1 strains may be a greater threat thus it is important to reveal the underlying molecular mechanisms that trigger the virus-host interactions in MDV-1 pathogenesis and oncogenesis.MicroRNAs(miRNAs)are small single-stranded RNA species of approximately 20-24bases in length,and they regulate gene expression through post transcriptional mechanisms.MiRNAs plays crucial roles in diverse of biological processes covering cellular differentiation,apoptosis,development and proliferation.In the viral genome of MDV1,a total of 26 mature microRNAs focused into three gene clusters.Previous studies have demonstrated that some of these miRNAs play important potential roles in MDV1 biology,among which miR-M4-5p has been characterized as a viral analog of cellular miR-155.It is well known that miR-155 has multiple regulatory roles in human cancers and growing evidence indicates that miR-M4-5p may be directly involved in MD lymphomagenesis.Further studies on miR-M4-5p will contribute a lot to understanding of MD molecular mechanisms,especially the mechanisms of tumorigenesis1.Transcriptome analysis of spleen tissues infected with very virulent(vv)MDV strain GX0101Herein,utilizing RNA-seq,we have performed a comprehensive sequencing of GX0101-infected chicken spleen.After filtering,the clean reads accounted for more than 97.5%of the raw reads and at least 89.7%of the clean reads mapped to reference genome.The DEGs in GX0101-infected birds compared to the mock controls were further analyzed.Totals of 1,567,1,342,2,503,3,517,3,810 and 1,351 genes were significantly differentially expressed at 3,7,14,21,30 and 60 dpi,respectively.Among these DEGs,736,812,1,507,2,250,2,390 and 571 genes were observed to be up-regulated during viral infection at 3 to 60dpi,along with the simultaneous down-regulation of 831,530,996,1,267,1,420 and 780genes.The RNA-seq data was well supported by the qRT-PCR confirmation allowing it to provide the basis for the following analysis.Innate immune responses and inflammatory responses were established at early cytolytic phase and persisted until lymphoma formation.Humoral immunity began to play a role firstly in the intestinal system starting at late cytolytic phase.During the proliferative phase many pathways associated with transcription and/or translation was significantly enriched.Based on the high-throughput technology,the dynamic and differential gene expression profiles obtained from GX0101-infected chicken spleens will provide an important basis and more valuable information for future studies on the molecular mechanisms of MD pathogenesis and tumorigenesis.2.MiR-M4-5p promotes proliferations of chicken embryo fibroblast and DF-1 cells by targeting hnRNPABTo reveal the regulatory mechanisms mediated by miR-M4-5p in MDV-1 infection,bioinformatics predictions were first performed and a total of 7 candidate chicken mRNA genes containing potential binding sites in the 3'UTRs were obtained.It includes the EGR1(early growth response 1),CTGF(connective tissue growth factor),FN1(fibronectin 1),IGJ(immunoglobulin J chain),hnRNPAB(heterogeneous nuclear ribonucleoprotein AB),PTPN5(protein tyrosine phosphatase,non-receptor type 5)and COL12A1(collagen,type XII,alpha1).Firstly,bioinformatics predictions were performed based on down-regulated genes.The site-directed DLRA was performed to investigate whether miR-M4-5p directly binds to the3'UTRs of candidate genes.The results shows the repression effect of hnRNPAB-3'UTR was specifically dependent on the predicted binding site or seed sequences.The expression level of hnRNPAB mRNA in CEFs over-expressing miR-M4-5p or GX0101-infected was significantly reduced,and western blot analysis confirmed the reduction of hnRNPAB at the protein level in CEFs.When the miR-M4-5p was over-expressed or hnRNPAB knocked-down in CEFs,the cell viability of CEFs significantly increased,and it had no influence on apoptosis of CEFs.The same results were obtained in DF-1 cells.This study shows the recognition and down-regulation of hn RNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas.3.Development and characterization of specific monoclonal antibodies against chicken hnRNPAB proteinOur recent work suggests that chicken hnRNPAB is a putative biological target for miR-M4-5p.There is no commercial poly-or monoclonal antibodies available for chicken hnRNPAB protein.We constructed recombiant pET28a-hnRNPAB,pET30a-hnRNPAB and pET32a-hnRNPAB proteins.The recombinant proteins were purified and used as antigens to immunize mice.Two monoclonal antibodies,5H5-H1 and 5H5-G2,were further developed and the titer was 8.19×10~5.Western blot analysis demonstrated that they both specifically react with the native chicken hnRNPAB protein.Furthermore,the immunofluorescence and immunocytochemistry assays demonstrated that in addition to chicken hnRNPAB protein,these mAbs also recognize the native hnRNPAB proteins derived from different mammalian sources.This study provides material for researches on hnRNPAB protein levels.