Potential Roles Of The Mid-clustered MicroRNAs In The Pathogenesis Of Marek's Disease Virus

Marek's disease(MD)is one of the oncogenic herpesviruses and can finally leads to an important immunosuppressive and neoplastic disease of poultry caused by Marek's disease virus(MDV).Recently,the virulence of MDV field strains persistently increased and the MD outbreaks happened sporadically every year on a global scale.MDV infection provides the first demonstration of the efficacy of anti-viral vaccination in the control of cancer in any species.As a consequence,studies on MD provide an ideal model for investigating the biology,genetics,and immunology of virally-induced tumorigenesis.Micro RNA(miRNA)are a class of small non-coding RNA playing important post-transcriptional regulatory roles in multiple biological processes.In MDV-1 genome,the presence of 26 mature miRNA derived from 14 precursors assembled in three clusters,namely Meq-cluster,Mid-cluster and LAT-cluster,respectively.The Meq-cluster and the Mid-cluster are driven by a same promotor and represent similar expression patterns.Previously,many researches have revealed that most of the Meq-clustered miRNAs are critial for regulating the phenotype of MDV oncogenesis.However,roles of the Mid-clustered miRNAs involved in the pathogenesis and oncogenesis of MDV remains unknown.Therefore,we plan to construct a series of single-cluster-deleted or single-miRNA-deleted MDV strains,utilizing the bacterial artificial chromosomes(BAC)and Rec E/T homologous recombinant techniques,and to reveal the potential roles of the Mid-clustered miRNAs by animal challenge experiments.The present study will provides an important basis for further eluciating the molecular regulatory mechanisms mediated by the Mid-clustered miRNAs.Firstly,the single Mid-clustered miRNAs and mi R-M1,mi R-M11 and mi R-M31 were separately deleted based on the BAC clone of MDV-1 strain GX0101 by Rec E/T mutagenesis,then the existence of the GX0101 genome and deletion of the individual Mid-clustered miRNAs were confirmed by PCR amplification,DNA sequencing and RFLP analysis.The results showed that a series of BAC clones with the corresponding deletions of single Mid-cluster(GX?Mid-mi Rs-BAC)and individual miRNA(GX?mi R-M1-BAC,GX?mi R-M11-BAC and GX?mi R-M31-BAC)were successfully constructed by two rounds of Rec E/T mutagenesis.Secondly,the four BAC recombinants were seperately transfected into CEFs for the rescue of miRNA-deleted MDV viruses,then the genomic DNA of rescue MDV viruses were confirmed by PCR analysis and DNA sequencing.The q RT-PCR were performed to analyze the expressions of relevant miRNAs.To investigate the in vitro virus proliferation,real-time q PCR were performed on Meq and g B genes.The results showed that the Mid-clustered miRNA-deleted MDV viruses were successfully reconstituted,the adjacent miRNAs were normally expressed,and the expressions of related genes(meq?RLORF6?RLORF5a and RLORF4)were not affected.The miRNA-deleted mutants and the parental GX0101 virus have similar replication curves in vitro.Finally,one-day-old SPF chickens were separately challenged with CEFs containing GX0101,GX?mi R-M1,GX?mi R-M11,GX?mi R-M31 or GX?Mid-mi Rs viruses while the negative controls were inoculated with an equal volume of mock CEFs by abdominal cavity inoculation,then the replication of these miRNA-deleted MDV viruses,the body and immune organ weights of birds,the mortality and the gross tumor incidences were examined and analyzed.The results showed that the Mid-clustered miRNA-deleted MDV viruses and the parent MDV GX0101 virus have slightly different replication in vivo.From 14 dpi,the body and immune organ weights of the miRNA-deleted mutants and the GX0101-infected birds showed a similar effect,and all are lower than that of the CEF controls.Form 14 dpi,no death was observed in the CEF control group,while in the other groups there were a different number of deaths.At 75 dpi,all the birds of the GX?Mid-mi Rs virus group were dead,with a gross tumor incidence reached at 21.0%.By the end of 90 dpi,the mortalities of GX?mi R-M1,GX?mi R-M11 and GX?mi R-M31 virus groups had reached to 83.3%,71.8%and 69.2%,with the gross tumor incidences of 31.7%,45.0% and 31.7%,respectively.However,the mortality and cumulative gross tumor incidence of the parental GX0101 virus had reached 79.4% and 40.0%,respectively.In conclusion,the Mid-clustered miRNAs are not essential for MDV-1 replication and are not related to the immunosuppression caused by MDV-1 infection.However,the deletion of miRNAs significantly changed the mortality and the gross tumor incidences of MDV inordinately.Compared to parental GX0101 virus,the individual deletion of mi R-M1 and mi R-M11 separately increased the viral pathogenicity or oncogenecity while the individual deletion of mi R-M31 significantly decreased the mortality and gross tumour incidence of virus infected chickens.These results suggest that the Mid-clustered miRNAs possibly act either as tumour suppressor or potential oncogene in MD lymphomagenesis.Future works are needed to elucidate the miRNA-mediated fundamental molecular mechanisms triggering the development of MD lymphomas.