Isolation And Identification Of Marek's Disease Virus And Analysis Of The Dynamic Viral Transcriptome Of GX0101 Strain

Marek's disease(MD)is an kind of important immunosuppressive and neoplastic avian disease caused by Marek's disease virus.In recent years,with the long-term and extensive application of vaccines,the virulence of MDV is increased and pandemic strain is evolving.And it brought a huge economic losses to the poultry industry because of the epidemic of MDV in China.Henan province,as the biggest Poultry breeding base in China,to understand the latest MDV epidemiological characteristics in Henan chicken groups has important practical significance.In addition,the huge viral genome of MDV encodes hundreds of proteins,but most are remains unclear.The task to deeply study the unknow genes function and potential pathogenic factors is still arduous.To investigate the molecular evolution characteristics of Marek's disease virus(MDV)and provide more useful information for the efficient control of MD,The suspected clinical cases of MD in the local chicken breeds collected from the area of Jiaozuo in Henan province were diagnosed and confirmed by polymerase chain reaction(PCR)analysis and DNA sequencing.Three prevalent viruses,namely HNJZ101,HNJZ102 and HNJZ103,were finally isolated through chicken embryo fibroblast(CEF)cultivation and primarily identified by indirect immunofluorescence assay(IFA).Alignment of meq,gE and gI analysis showed that the three isolates HNJZ101,HNJZ02 and HNJZ103 are possibly virulent MDV-1 strains and their pathogenicities and pathotypes deserve to be further studied.To a further study the infection,pathogenic and tumor mechanism of MDV applied by GX0101,one-day-old specific pathogen free(SPF)chickens were challenged by GX0101 to establish the animal model of MD.Then,the kinetic expression levels of 10 representative MDV protein-coding genes at different time and space expression were determined by RT quantitative PCR(RT-qPCR).Our previous and present data indicates that the ‘Cornell Model' of MD caused by GX0101 are as blow: the early cytolytic phase occurs at 1-7 dpi,the latent phase establishes at about10 dpi and the late cytolytic and immunosuppressive phase appears at 14-21 dpi,while the T cell transformation and lymphomagenesis possibly starts at about 14 dpi.To further study the transcriptomics of the MDV encoding genes,the spleens infected by GX0101 collected at different times points were analyzed by RNA-Seq.The results showed that all the viral encoding genes of GX0101 were detected,and finally got the dynamic spectrum of MDV encoding genes expressed in different disease stages.Totals of 57,33,70,68 and 33 genes were identified as DEGs between3 dpi and 7,14,21,30 or 60 dpi,respectively.Among those DEGs,33,28,37,29 and15 genes were observed to be up-regulated during viral infection at 7 to 60 dpi,along with the simultaneous down-regulation of 24,5,33,39 and 18 genes,and the qPCR experiments exhibited consistent results to those of RNA-seq.Based on the data from RNA-seq,large numbers of MDV encoding genes were found to show a change in their level of expression,which provides a basis for further studying their potential fuctions.