Biological Characteristics Of Recombinant Marek’s Disease Virus (MDV) Serotype Ⅰ Co-expressing Different Exogenous Genes

Marek’s disease virus (MDV) is a member of the Alphaherpesvirinae subfamily, andits genome contains a linear double-stranded DNA of about180kb. MDV causes Marek’sdisease in chickens with lymphomas. GX0101is the first recombinant MDV field straincontaining the reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert isolatedfrom chicken both at home and abroad. In previous research in our laboratory, GX0101bacterial artificial chromosome and its mutant strain GX0101LTR with a deletion of theREV-LTR were constructed and demonstrated that REV-LTR insert increased thehorizontal transmission ability of GX0101. Mutant strain GX0101meq with a deletion ofmeq oncogene was demonstrated to have lost its pathogenicity in SPF chicken, and nolonger induced tumors and atrophy of thymus and bursa of fabricius. With the widespreaduse of molecular biology, it is promising for MD recombinant vaccine. MDV vaccine strainis considered as one of the most potential virus vectors, which could prevent poultry diseasethrough constructing multivalent live vaccine of exogenous gene antigen. The articlediscusses whether the expression of pp38/pp24heterodimers are the required activators forthe expression of the foreign genes. The confirmed function of the bi-directional promoterprovides better feasibilities to insert multiple foreign genes in MDV genome based vectorsk,and try to analyze rMDV replication ability in cells and animal bodies through serialpassage in vivo and in vitro. And discuss the stability of rearrange pieces in MDV genometo provide research direction for rearrange virus special biology activity. And study theimmune response ability of rMDV through inoculating SPF chickens, discuss synergisticeffect between inactivated vaccine rMDV and H9N9through co-inoculating chickens. Andprovide screening vaccine strain for the prevention of MDV and H9N9. And providetheoretical and technical support for constructing rearrange MDV virus vaccine in future.1. Absolute quantification of MDV dynamic loads and distributions in different tissuesusing duplex real-time PCR assayChickens infected with Marek’s disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors in vivoand increases its potential for horizontal transmission. There is a positive correlation betweenMDV loads and the incidence of malignant tumors and the level of viral transmission in thefeather follicle epithelia of chickens serving as the sole sources of infectious virus particles.The present study was aimed at developing a SYBR Green duplex real-time quantitative PCR(q-PCR) assay to detect and quantify MDV loads and distributions in different tissues,targeting the Meq gene of the virus and the house-keeping ovotransferrin (ovo) gene ofchickens. A bacterial artificial chromosome (BAC) clone of the MDV genome, BAC-GX0101,and a plasmid (pMD18T-ovo) bearing a fragment of the chicken ovo gene were used toquantify virus and host genomes, respectively. The assay had a dynamic range of8logs, amean inter-and intra-assay coefficient of variation (CV) of <2%and minimum detection limitof10MDV genome copies. The sensitivity of the assay was compared with that of bothconventional PCR and viremia assay. The q-PCR was validated using different tissue DNApreparations derived from chickens infected with1000plaque forming units (PFUs) of theSDWJ1302strain and sampled on days7,10,15,21,28,40,60and90post-infection (p.i.),along with uninfected chickens. The resulted indicated that the MDV genome was almostquantifiable in mmune organs of infected chickens by day7p.i., and the number of MDVgenome copies in the blood and different organs peaked by day28p.i., but then graduallydecreased by day40p.i.. The levels of viral loads in the lymphocytes, liver and spleen wereall higher than that in other organs, and that in the feather follicles was the highest at differentstages of MDV infection. The data provided further evidence of viral infection involvingmulti-organ distribution and mainly involving immune organ proliferation, resultingin immunosuppression and lymphocyte transformation. This is the first report of absolutequantification of MDV genomes in different tissues and organs during the early latentinfected and later incidence stages, and application of this assay could significantly furtherour understanding of the pathogenesis, spread, diagnosis, genetic resistance and vaccinalcontrol of Marek’s disease.2. Biological characteristics of rMDV lacking the meq oncogene and co-expressingAIV-H9N2-NA and NDV-F genes under control of MDV’s own bi-directionalpromoter Our previous studies reported a bi-directional promoter of only305bp between pp38geneand1.8kb mRNA family on Marek’s disease virus (MDV) genome, its transcriptionalfunction could be activated by pp38/pp24hetero-dimers as trans-acting transcriptional factors.To qualitatively analyze and evaluate its bi-directional transcriptional function in bothtransient and transgenic systems, several different plasmids were constructed and recombinantMDV type1strain GX0101was developed to co-express a NA gene from Avian InfluenzaVirus H9N2strain and a F gene from the Newcastle disease virus (NDV).The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmiddriven by the bidirectional promoter. NDV-F gene was located in the upstream of thepromoter, while the AIV-NA gene was located in the downstream of the promoter. To testwhether the expression of pp38/pp24heterodimers are the required activators for theexpression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containingexpression cassette for the two foreign genes was co-transfected with pp38/pp24expressionplasmid, pBud-pp38-pp24in chicken embryo fibroblast (CEF) cells. Alternatively, plasmidpPpp38-NA/1.8kb-F was transfected with GX0101-infected CEF which pp38/pp24wasexpressed via virus infection. Moreover, a recombinant MDV was constructed containing theexpression cassette of pPpp38-NA/1.8kb-F. To do so, a Meq-deleted MDV GX0101(GX0101Δmeq) was used as parental virus, the AIV-NA gene and NDV-F gene co-expression cassettewas inserted at the nonessential region of MDV US2to form a new recombinant MDV(MZC13NA/F) through homologous recombination. Indirect fluorescence antibody (IFA) test,ELISA and Western blot analyses indicated that F and NA genes were expressedsimultaneously under control of the bi-directional promoter, but in opposite directions. Theexpression of both foreign genes was activated by pp38/pp24dimers either via virus infection,or transient expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had noexpression. The data also indicated that the activity of the promoter in the1.8-kb mRNAtranscript direction was higher than that in the direction for the pp38gene. In vitro growthproperties of MZC13NA/F were also inspected and concluded that the MZC13F/NA had thesame growth kinetics in CEF cultures as its parental wild type virus GX0101.The pp38/pp24dimers expressed by pBud-pp38-pp24in co-transfected cells or byGX0101in infected cells were critical to activate the bi-directional promoter for expression of two foreign genes in two directions. The bi-directional promoter could be used as a potentialpromoter with some advantages in construction of recombinant MDVs to express more thanone foreign gene.3. Biological characteristics of rMDV lacking the meq oncogene and co-expressingAIV-H9N2-HA and NA genes under control of exogenous promotersTo develop a recombinant Marek’s disease virus (rMDV1) co-expressing thehemagglutinin gene (HA) and Neuramidinase gene (NA) from a low pathogenic AvianInfluenza virus (LPAIV) H9N2strain and lacking the Meq oncogene that shares homologywith the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101was used asparental virus, the HA and NA genes co-expression cassette under control of the CMV andSV40early promoters was inserted at two Meq sites of GX0101to form a new complete lackof oncogenicity of a Meq knock-out mutant MDV (MZC12HA/NA) through homologousrecombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDVDNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purifiedMZC12HA/NA was obtained after four rounds of plaque purification and proliferation. Invitro growth properties of recombinant virus were also inspected and concluded that theMZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virusGX0101. Southern bolt indicated that co-expression cassette was successfully inserted at twocopies sites of Meq gene, so two Meq genes were knocked-out completely. RT-qPCR showedtranscription and expression levels of the HA ana NA genes were both significantly higherthan that of GX0101own pp38gene. Indirect fluorescence antibody (IFA) test, and Westernblot analyses indicated that HA and NA genes were co-expressed simultaneously undercontrol of the different promoters but Meq genes were not. These results herald an new andeffective recombinant Meq-deleted MDV-based AIV-H9N2vaccine may be useful inprotecting chickens from very virulent MDV and H9N2challenges.