Construction And Biological Activity Study Of Marek's Disease Virus GX0101 Strain With Deletion Of Latent Associated Transcript Cluster MiRNAs

Marek's disease virus(MDV)can establish and maintain lifelong latent infection after infecting the host,causing immunosuppression and seriously jeopardizing the development of poultry industry.In recent years,studies have found that some miRNAs encoded by MDV play a key role in regulating latent virus infection and tumorigenesis.Among them,the Meq gene cluster and Mid gene cluster miRNAs have been confirmed to have oncogenic and tumor-suppressive effects.However,the regulatory functions of MDV-encoded latencyassociated transcript(LAT)cluster miRNAs in the pathogenesis of MDV infection remain unclear.In order to study the role of LAT-cluster miRNAs,this experiment constructed MDV LAT-cluster miRNAs deletion strain and miR-M10 deletion strain and compared the biological activity of gene deletion strains.First,based on the supervirulent MDV GX0101 BAC clone,two rounds of Red/ET homologous recombination technology were used to knock out miR-M10 in the MDV LATcluster and the miRNAs in the entire LAT-cluster,respectively.Clones were identified by PCR and DNA sequenced.The results showed that after two rounds of Red/ET homologous recombination,GX0101?LAT-miRs BAC and GX0101?miR-M10 BAC clones were successfully constructed that lack the entire LAT-cluster miRNAs and the miR-M10 single gene in the LAT-cluster.Secondly,GX0101?miR-M10 BAC and GX0101?LAT-miRs BAC plasmids were extracted and transfected into CEF cells by calcium phosphate transfection method,and the rescued viruses were identified by indirect immunofluorescence.The results showed that the GX0101?miR-M10 and GX0101?LAT-miRs viruses were successfully rescued.The copy numbers of GX0101?miR-M10,GX0101?LAT-miRs and GX0101 viruses were detected at different times after infection of CEF by probe-based fluorescence quantitative PCR,and the replication levels of each virus in vitro were analyzed and compared.In vitro studies showed that the in vitro replication ability of GX0101?LAT-miRs was significantly increased compared with the parental strain GX0101 within 144 h after CEF infection;the in vitro proliferation curve of GX0101?miR-M10 was similar to that of the parental strain GX0101,with no significant difference in replication ability.Furthermore,animal experiments were used to study the biological activities of GX0101?miR-M10 and GX0101?LAT-miRs in vivo,such as replication,spread,and pathogenesis.GX0101?miR-M10,GX0101?LAT-miRs and GX0101 viruses were infected with 1-day-old SPF chickens,respectively,and the mortality,tumor rate,transmission ability,replication ability in challenge chickens,chicken growth performance,immune organ development and immunosuppression of each virus strain were observed.Detection and analysis,etc.The results showed that the inhibitory effect of GX0101?LAT-miRs on chicken growth was significantly reduced compared with GX0101.The inhibitory effect of GX0101?miR-M10 on chicken growth was attenuated in the early stage.The inhibitory effect of GX0101?LAT-miRs on immune organ development was significantly attenuated,and the inhibitory effect of GX0101?miR-M10 on bursa development was enhanced.The replication capacity of GX0101?LAT-miRs was decreased in vivo,and the replication capacity of GX0101?miR-M10 was enhanced in vivo.The three viruses,GX0101,GX0101?miR-M10,and GX0101?LAT-miRs,could infect SPF chickens in a short time.Compared with GX0101,GX0101?LAT-miRs could significantly attenuate the suppression of cellular and humoral immunity,while GX0101?miR-M10 attenuated the late humoral immunosuppression of SPF chickens.The mortality and tumor rates of GX0101?LAT-miRs and GX0101?miR-M10 at90 days were lower than those of GX0101,and the mortality rate and tumor rate of GX0101 knocking out LAT-cluster miRNAs were the lowest.GX0101?LAT-miRs and GX0101?miR-M10 mutants were less pathogenic than GX0101.However,there is still a gap between GX0101?miR-M10 and GX0101?LAT-miRs,so other miRNAs in the LAT cluster may play more important regulatory functions.In this study,the MDV LAT-cluster miRNAs deletion strain was constructed and it was found that the deletion of the LAT-cluster miRNAs would reduce the pathogenicity of MDV,but there was a gap between GX0101?miR-M10 and GX0101?LAT-miRs,indicating that miR-M10 did not play a leading role.Other miRNAs in the cluster may play more important regulatory functions.The regulatory role of other miRNAs in the LAT-cluster on the pathogenicity of MD can be further studied,which lays a good experimental foundation for identifying the key miRNAs that play the main regulatory function.