Reglatory Roles Of The Meq-clustered MicroRNAs On The Pathogenic Phenotype Of Marek’s Disease Virus

Marek’s disease(MD) is a highly contagious, neoplastic, and immunosuppressivedisease of chicken caused by Marek’s disease virus (MDV). The tumorigenesis can beprevented effectively by immunization with attenuated or nonpathogenic forms ofMDV viruses. However, due to high-density poultry production and possible selectionpressure from vaccination, the virulence of MDV field strains has been seen to haveincreased. MD outbreaks happen sporadically every year in worldwide, and it hascaused huge losses in the poultry industry.MicroRNAs play important post-transcriptional regulatory roles in variousimportant biological processes. In recent years, a large number of viral miRNAs havebeen found to be encoded in the genomes of MDV, and these miRNAs may playcritical regulatory roles in infection, latency, and oncogenesis. Three distinct serotypesof MDV have been characterized, and the viral miRNAs are all located in the invertedrepeat sequences of MDV-1genomes and focused in three gene clusters. TheMeq-clustered miRNAs are sequentially located upstream from the Meq oncogene,and these miRNAs were hypothesized to play more important roles in MDVpathogenesis and oncogenesis.In order to further explore the molecular mechanism of MDV pathogenesis andtumorigenesis, we intend to construct a series of single-cluster-deleted orsingle-miRNA-deleted MDV strains using RecE/T homologous recombination basedon the BAC clones of vv MDV GX0101. Then the pathogenic or tumorigenicphenotypes would be evaluated by challenge SPF chicken with the miRNA-deletedmutants. The study will provide a means for further investigating the regulatoryfunctions of miRNAs in MDV pathogenic phenotypes and a basis for furtherelucidating the molecular regulation mechanism, and the research will also providereference for clarifying the regulation roles of miRNAs on biology, genetics, andimmunology of tumorigenesis.Firstly, the single Meq-clustered miRNAs and MDV1-miR-M2, MDV1-miR-M3, MDV1-miR-M4, MDV1-miR-M5, MDV1-miR-M9, MDV1-miR-M12were deletedrespectively based on the BAC clones of vv MDV GX0101by two rounds RecE/Tmutagenesis，then the existence of the GX0101genome and deletion of the individualMeq-clustered miRNAs were confirmed by PCR analysis using the different primersthat amplify different genes and by DNA sequence analysis. The results showed that aseries of BAC clones with the corresponding deletions of single Meq-clusteredmiRNAs and individual miRNA in Meq cluster were constructed successfully, namedGX0101Δ miR-M2-BAC, GX0101Δ miR-M3-BAC, GX0101Δ miR-M4-BAC,GX0101Δ miR-M5-BAC, GX0101Δ miR-M9-BAC, GX0101Δ miR-M12-BACand GX0101Δ Meq-miRs respectively.Secondly, the BAC DNA of GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9, GX0101ΔmiR-M12andGX0101ΔMeq-miRs were transfected into CEF respectively for the rescue of MDVvirus. Then the rescued viruses were confirmed by PCR analysis and indirectimmunofluorescence assay (IFA). The results indicated that GX0101ΔmiRNA viruseswere reconstituted successfully. To investigate the in vitro virus proliferation rates,real-time qPCR were performed on Meq and gB genes with the CEFs infectedGX0101BAC or GX0101ΔmiRNA viruses. The results showed that both the parentand the miRNAs-deleted mutant viruses have very similar replication kinetics,indicating that the six Meq-clustered miRNAs are not essential for MDV replicationin vitro.Finally, one-day-old white Leghorn SPF chickens were separately challengedwith CEFs containing MDV GX0101BAC, GX0101ΔmiR-M2, GX0101ΔmiR-M3,GX0101ΔmiR-M4, GX0101ΔmiR-M5, GX0101ΔmiR-M9，GX0101ΔmiR-M12orGX0101ΔMeq-miRs viruses by abdominal cavity inoculation, then the mortality,gross tumor incidences and in vivo replication of GX0101ΔmiRNA viruses, thegrowth rates, immune organs and microscopic tumorigenesis of birds wereexamined and analyzed. The results showed that the rates of cumulative mortality ofMDV GX0101ΔmiRNA viruses were all lower than the parent GX0101BAC, andMDV GX0101ΔmiR-M5was higher than that of GX0101ΔmiR-M3,GX0101ΔmiR-M9, GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M12andGX0101ΔMeq-miRs. By75dpi, the rates of cumulative gross tumor generation ofMDV GX0101ΔmiRNA viruses were all lower than that of the parent GX0101BAC. By90dpi, the rates of cumulative gross tumor generation of GX0101ΔMeq-miRswere lower than that of GX0101ΔmiR-M12, GX0101ΔmiR-M3, GX0101ΔmiR-M4,GX0101ΔmiR-M5, GX0101ΔmiR-M2and GX0101ΔmiR-M9. These results showedthat the pathogenicity and oncogenicity of the serial mutant viruses were stronglysuppressed when compared to the parental GX0101BAC virus; The Meq-clusteredmiRNAs were not essential for replication of MDV in vivo, and some effect of thedeletions in the Meq-cluster locus affecting the viral replication in vivo; There was nodifference in body weight of birds except GX0101ΔmiR-M3orGXΔmiR-M5-challenged birds whose body weights were lower at30dpi or at21to30dpi than that of the CEF controls. The ratios of bursa/body weight andthymus/body weight of birds inoculated with the mutant viruses were all significantlylower than that of the control birds at14and/or21dpi. However, there was noconsistent significant difference between the parental GX0101BAC and themiRNA-deleted mutants viruses.The rates of microscopic tumorigenesis in liver ofbirds showing no gross organic tumors showed that GX0101ΔmiRNA viruses were alllower that of the parental GX0101BAC, and the microscopic tumorigenesis ofGX0101Meq-miRs was the lowest, then followed by GX0101ΔmiR-M12,GX0101ΔmiR-M2, GX0101ΔmiR-M4, GX0101ΔmiR-M3, GX0101ΔmiR-M9andGX0101Δ miR-M5.In conclusion, these results suggest that the oncogeneicity was not completelyabolished in the mutant viruses the Meq-clustered miRNAs deleted but tumorprogression was hindered compared to that of the parental GX0101virus. Theseresults further indicate that the Meq-clustered miRNAs have important reglatory rolesin the pathogenic phenotype of Marek’s disease virus. The data suggests that in MDVoncogenesis, the Meq-clustered miRNAs possibly play different roles, soMDV1-miR-M12may be a more important regulator, followed by MDV1-miR-M2,MDV1-miR-M3, MDV1-miR-M4and MDV1-miR-M9，while MDV1-miR-M5maybe less significant. Further work will be needed to elucidate which other fundamentalmolecular mechanisms trigger the development of MD lymphoma.