Comparisons Of Biological Characteristic Between Recombinant Marek’s Disease Virus And Its Mutant Strains

Marek’s disease virus （MDV） is a member of the Alphaherpesvirinae subfamily, and itsgenome contains a linear double-stranded DNA of about180kb. MDV causes Marek’s diseasein chickens with lymphomas. GX0101is the first recombinant MDV field strain containingthe reticuloendotheliosis virus （REV） long terminal repeat （LTR） insert isolated from chickenboth at home and abroad. In previous research in our laboratory, GX0101bacterial artificialchromosome and its mutant strain GX0101LTR with a deletion of the REV-LTR wereconstructed and demonstrated that REV-LTR insert increased the horizontal transmissionability of GX0101. Mutant strain GX0101meq with a deletion of meq oncogene wasdemonstrated to have lost its pathogenicity in SPF chicken, and no longer induced tumors andatrophy of thymus and bursa of fabricius. On the basis of the above research, we furtherconstructed different mutants to study MDV related biological activities in-depth anddeveloped MDV gene-deleted vaccine.1Completed the sequencing analysis of the whole-genome of MDV strain GX0101Recombinant field strain GX0101possesses unique biological activities in many aspects,for instance, pathogenicity, immunogenicity, horizontal transmission ability and so on. Inorder to further carry on molecular virology research of GX0101, this study competed thesequencing analysis of the whole-genome of GX0101using its BAC clone combined withhigh-throughput sequencing technologies.The whole-genome sequence of GX0101consists of178,101nucleotides （nt）, and theterminal repeat long （TRL）, unique long （UL）, internal repeat long （IRL）, internal repeat short（IRS）, unique short （US）, and terminal repeat short （TRS） regions with the lengths of12,758,113,572,12,741,12,700,11,695, and13,134nt, respectively. The GX0101genome containsonly one REV-LTR insert located within SORF1gene in US region, at a site267nt upstreamof the SORF2gene. GX0101contains only one SORF2gene, which is different from rMd5.After homology comparison of nearly10MDV strains, GX0101has the highest identity totwo BAC clones pC12/130-10and pC12/130-15of United Kingdom strain C12/130withdistinct virulence. Besides the accessorial REV-LTR insert in GX0101genome, there are77ORFs in the compared190ORFs between different strains occur different degrees ofmutation. Among the mutated77ORFs, GX0101has50and47ORFs100%identical to PC12/130-10and PC12/130-15but only7to27ORFs100%identical to RB1B, Md5, GA,814and CVI988strains respectively. There are5consecutive repeats of a217-nt fragment in97.3-97.6ORF of the TRS region of GX0101, which is significantly more than other strains.There are3same repeats of a217-nt fragment in86.2-86.4ORF of the IRS region of GX0101,but only1or2copies repeats in other MDV strains.The comparative analysis of whole-genome sequence of GX0101will contribute toclarify genes relative to pathogenicity and transmissibility of MDV. It will also be useful inimplying the genetic variation and evolutionary relationship of MDV between differentregions.2Constructed the meq gene-deleted candidate vaccine strain SC9-1with betterprotective immunity than the most widely used CVI988/Rispens at home and abroad inSPF chicken experimentsThe recombinant virus GX0101meq appeared good protective immunity, but it cannotbe used as a vaccine as residues of Kanamycin resistance genes in the genome which do notconform to Regulations on Administration of Agricultural Genetically Modified OrganismsSafety.We knocked off Kanamycin resistance genes in GX0101meq and selected MDV strainSC9-1（GX0101meq kana） with good replication ability using the method designed in thestudy that partial induction of flp recombinase combined with multiple filters. MDV strainSC9-1was inoculated in SPF chickens and Highland brown layers with MDV maternalantibodies, there were no tumorigenicity and any pathogenicity such as immunosuppressionand growth retardation, and it can provide better protective immunity than CVI988/Rispensfor vaccinated chickens. Many times over repeat of the immune protection test of MDV strainSC9-1to SPF chicken by inoculating with500PFU of very virulent MDV strain rMd5demonstrated that SC9-1can provide100%protective immunity, significantly higher thanCVI988/Rispens with protection rate of80%to90%. When inoculating DNA of SC9-1infectious clone into1-day-old SPF chickens by intramuscular injection, SC9-1virus could beisolated from chickens14days post-inoculation, which provides a way to further discussBAC plasmid of SC9-1as DNA vaccine.MDV gene-deleted vaccine strain of SC9-1has won the ministry of agricultureenvironmental release approval book and finished six months of environmental release test in3000Highland brown layers with continuous observation for six months. SC9-1not onlyconforms to the agricultural genetically modified organisms safety, and renders good immuneprotection effect, making it an ideal new type of vaccine strain. In addition, it has been transferred to Beijing Lingyu Bio-tech for subsequent related productive experiment.Preparation of pilot product certificates that SC9-1can provide better protective immunitythan the most widely used vaccine CVI988/Rispens at home and abroad.3Tracking test of consecutive virus identification and quantification of GX0101andGX0101LTR after the mixed infection in chickens demonstrated that REV-LTR insertimproved the horizontal transmission ability of GX0101and was the competitiveselection pressure advantage of making it a popular strainIn order to simulate natural infection transmission mode to compare and confirm thecompetitive selection pressure of GX0101with its mutant strain GX0101LTR in horizontaltransmission ability in chickens, comparative study on the primers, probes and reactionsystem of dual fluorescence quantitative PCR was carry out to differentially quantify genomeof GX0101and GX0101LTR virus, which were further successfully used to compare thevirus viremia dynamic of different viruses in the process of continuously passage afterco-infection of the above two viruses in chickens.The results show that after inoculating SPF chicken with the same dose of GX0101andGX0101LTR, GX0101can be detected from blank SPF chickens used for contacting withchallenged chickens14days post contact infection, while GX0101LTR can be detected28days post contact infection. GX0101can be detected from the second generation used tocontact with the first generation chickens both21and28days post contact infection, whileGX0101LTR can be detected from only one chicken28days post contact infection.Similarly, GX0101can be detected from the third generation used to contact with the secondgeneration chickens both21and28days post contact infection, while GX0101LTR can notbe detected. When part of SPF chickens were co-infected with GX0101significantly lowerthan GX0101LTR（100PFU GX0101and2000PFU GX0101LTR per chicken）and kept inthe same isolator to infect blank chickens by horizontal transmission. After two consecutivepassages, although GX0101LTR can be detected from part of chickens （6in10） used forcontacting with challenged chickens and is still the dominant strain in individual chicken,GX0101containing REV-LTR can be checked out from all of the chickens used for contactingwith challenged chickens. Also, GX0101can be detected, while GX0101LTR can not, frompart of chickens （4in10）.The above results demonstrated that GX0101containing REV-LTR shows a significantcompetitive selection pressure advantage compared with GX0101LTR with a deletion of itsREV-LTR in natural infection and transmission process in chickens. This can explain thebiological mechanisms why GX0101containing REV-LTR with a low rate of recombination between MDV and REV in chickens can evolve from a very low proportion into a popularstrain easily isolated from chickens.4Comparison of biological activity of GX0101SORF2gene-deleted strain demonstratethat SORF2gene expression product is closely related with horizontal transmissionability of different MDV strainsREV-LTR fragment possesses eukaryotic promoter and enhancer activity, which canenhance expression of genes downstream of insertion site in MDV. REV-LTR locatesupstream of SORF2gene in GX0101genome and it is likely to have a significant impact ontranscription and expression of SORF2gene. To study the function of SORF2gene inrecombinant virus GX0101in-depth, SORF2gene-deleted strain GX0101sorf2wasconstructed on the basis of infectious clone GX0101-BAC.The results show that SORF2is not the essential gene for MDV replication andtumorigenicity. SORF2gene-deleted strain GX0101sorf2exhibited the same replicationdynamics as GX0101on cell culture. Compared with GX0101, the mortality and tumor rate ofGX0101sorf2in chickens were lower but not significantly different. The horizontaltransmission rate of GX0101sorf2was lower than GX0101. The REV-LTR insert in GX0101enhances the expression of SORF2protein, demonstrated that the relative strong horizontaltransmission ability of GX0101may be related with the molecular mechanism of a greatamount of SORF2expression.5Construction of recombinant MDV with ALV-LTR insert and comparative study on itsbiological activityReticuloendotheliosis virus （REV） and avian leukosis virus （ALV） are avian retroviruses.Co-infection of ALV-J and MDV in chickens is very common. To study on the stability ofALV-LTR insert at the recombinant site of GX0101genome and the influence of ALV-LTRinsert on biological activity of MDV, recombinant virus GX0101-ALV-LTR containingALV-LTR fragment was constructed by completely replacing REV-LTR of bac-GX0101withLTR fragment of ALV-J China field strain NX0101at the same site using homologousrecombination, based on Streptomycin negative screening system. GX0101-ALV-LTR can beused as a model for further study of recombination between ALV and MDV.GX0101-ALV-LTR exhibited the same replication dynamics as GX0101at the cellularlevel. PCR proved that the ALV-LTR insert remained stable in GX0101-ALV-LTR genomefollowing20passages on cell culture. GX0101-ALV-LTR was inoculated in1-day-old SPFchickens and ALV-LTR was proved to remain stable in GX0101-ALV-LTR genome6-8weekspost-inoculation by PCR analysis. ALV-LTR still remains stable inheritance in the genome of virus by contact infection. Mortality and tumor rate of GX0101-ALV-LTR were55%and20%respectively90days post-inoculation. Compared with GX0101LTR, IFA and Western blotresults demonstrated that SORF2protein expression was increased significantly inGX0101-ALV-LTR, and Northern blot results demonstrated that the amount of transcripts ofSORF2, US1, US10were increased significantly.GX0101-ALV-LTR is the first recombinant MDV containing LTR derived from ALV athome and abroad. It has been demonstrated that ALV-LTR fragment integrated at the samesite as REV-LTR in MDV also inherits stably and displays similar biological characteristics.6Comparative analysis of the influence of LTR insert fragment on different geneexpression level of recombinant virus using gene chipCompare and analyze virus gene expression differences on CEF infected with GX0101containing REV-LTR, its LTR deletion strain GX0101LTR and ALV-LTR alternative strainGX0101-ALV-LTR using the customized Agilent expression profile chip. The results ofmicroarray analysis indicate: among differentially expressed gene（sP<0.05）between GX0101and GX0101LTR virus, the expression level of75MDV genes in GX0101are significantlyup-regulated, there are14genes with an up-regulation fold change more than10, and theup-regulation fold change of SORF2, US1and US10genes directly downstream of REV-LTRis more than30, in which SORF2gene with the highest up-regulation fold change of399; thetranscription level of UL38in GX0101lower with a down-regulation fold change of8.3.GX0101-ALV-LTR is much similar to GX0101. Compared with GX0101LTR, theup-regulation fold change of SORF2, US1and US10gene also located directly downstreamof ALV-LTR is more than30, in which SORF2gene with the highest up-regulation foldchange; the down-regulation fold change of UL38in GX0101-ALV-LTR is6.7. Northern blotand Western blot results demonstrated that the amount of transcripts and protein expression ofSORF2in recombinant virus with LTR insert are both increased significantly.The results of comparative study will contribute to further analyze what gene expressionproducts are exactly associated with the increased horizontal transmission ability ofrecombinant MDV that the LTR insert confers.7Comparative analysis of the influence of LTR insert fragment on different geneexpression level of cell genome infected by recombinant MDV using gene chipCompare and analyze cell genome gene expression differences of CEF infected withGX0101containing REV-LTR, its LTR deletion strain GX0101LTR and ALV-LTRalternative strain GX0101-ALV-LTR using the customized Agilent expression profile chip.Analysis of expression differences between41,765chicken genes indicate: compared with GX0101LTR, transcription level of12genes are significantly higher in cell genome ofCEF infected by GX0101with a fold-change of more than20, in which the up-regulation foldchange of ChEST1024a11（GenBank:BU288902.1） and SKOR2（NCBI Reference Sequence:XM₀01233569.2）is190and165respectively; transcription level of4genes are significantlylower with a fold-change of more than20, in which the down-regulation fold change ofChEST445i1（GenBank: CR390488.1） is105. Compared with GX0101LTR, transcriptionlevel of5genes are significantly higher in cell genome of CEF infected byGX0101-ALV-LTR with a fold-change of more than20, in which the up-regulation foldchange of ChEST1024a11（GenBank:BU288902.1） and SKOR2（NCBI Reference Sequence:XM₀01233569.2）is203and83respectively; the fold-change of down-regulation genes areall less than20.The significantly different gene screened from gene chip expression profile willcontribute to further explore the relationship between different biological activity ofrecombinant MDV containing LTR insert with the host gene transcription level.This study not only demonstrates that REV-LTR insert improves the horizontaltransmission ability of GX0101virus in chickens significantly by simulating natural infectiontransmission mode, which is the epidemiology mechanism of GX0101evolving from ahandful of mutant strains into a popular strain, but also partly illustrates the molecularmechanism of REV-LTR improving the horizontal transmission ability of GX0101. Inaddition, GX0101is converted into a new candidate vaccine strain with better protectiveimmunity than CVI988/Rispens using BAC clone technology.