Comparisons Of Biological Characteristic Between BAC Cloned Marek's Disease Virus Field Strain GX0101 And It's Deletion Mutants

Marek's disease virus (MDV) is a member of the Alphaherpesvirinae subfamily of the Herpesviridae, and the genome of MDV(174kb) is double-stranded DNA, MDV can induce highly infectiousness and oncogenicity on non-vaccine chickens. The chickens infected with MDV can cause to death and the survival birds can releasing virus long term, so the virus exsited permantly in the surrounding environment. Reticuloendotheliosis virus(REV) is a"C"retrovirus and the genome of REV (8kb)is single-stranded RNA, the structure of the genome is analogous to a integrity transposon. REV is another tumor virus of chicken.Zhang et al(2004) first isolated the MDV strain integrated with REV-LTR from anticoagulated blood of tumor chickens naturally infected with MDV, named GX0101,GD0202, which demonstrated besides it can infected the same poultry with MDV simultaneously, REV can integrated into the MDV genome. This study further clarified the pathogenicity of the MDV recombination virus, particularly if the transmissibility or replication capacity in vivo can be enhanced due to the recombination. Based on above backgrounds, we Comparatived pathogenicity of the recombinant strain GX0101 and cloned it as an infectious bacterial artificial chromosome, on the base of it, we deleted the REV-LTR insertion to study the bionomics. Simultaneous, correlated investagation were carried out on two tumorigenesis gene (1.8kb mRNA,ICP4). Not only accumulated a large amount of data and make and verify a new point of view.1 To determination virulence of recombination field strain GX0101 on different species chickensThe determination pathogenicity of recombination field strain GX0101 were carried on Hy-line brown layer and SPF chickens. At 1 day of age these birds were vaccined with HVT and at 5 days of age these chickens were infected with either field strain GX0101 virus or vv vorulent rMd5 virus. The chickens were inspected daily and all the chickens that died during the experiment were evaluated for gross and histological lesions by necropsy. Experimental results show rMd5 infection more strongly inhibited growth rates of infected Hy-line brown layer than GX0101. There were 44.0% and 70.0% of mortality in Hy-line brown layer groups inoculated with GX0101 and rMd5(P<0.01). Also, 16.0% and 36.0% of birds demonstrated tumors in different tissues or organs in groups infected with GX0101 and rMd5(P<0.05). Both the mortality and oncogenicity of GX0101 strain were lower than the vvMDV strain rMd5. There were 28.24% and 64.63% of mortality in SPF chickens groups inoculated with GX0101 and rMd5(P<0.01). Also, 7.06% and 19.51% of SPF chickens demonstrated tumors in different tissues or organs in groups infected with GX0101 and rMd5(P<0.05). This thoroughly demonstrated that the virulence of GX0101 strain is lower than rMd5 strain. Zhang(2004) and Zhuang(2006) carried out artificial challenge experiment on SPF chickens, found that virulence of GX0101 strain is higher than GA strain. So the pathogenicity of GX0101 is between GA and rMd5. In view of this, the pathogenicity of GX0101 is not the competitive advantage to make it become a popular strain.2 Cloning of Marek's Disease Virus field strain With REV-LTR integration as an infectious BAC Clone and the Comparasion of pathogenicity2.1 Construction of Transfer Plasmid pDS-pHAI-US2For constructing the BAC transfer vector, two fragments (2.1 kb and 4.0 kb) on either side of the MDV US2 gene were used as the left- and right-sided homology arms. The left 2.1 kb homology arm was obtained from the plasmid pDS-pHAI by digesting with HindⅢand XhoⅠ. The right 4.0 kb homology arms came from plasmid pUS2-Partial-F by digesting with HindⅢand XhoⅠ. The transfer plasmid pDS-pHAI-US2 was constructed by ligating the two fragments.2.2 Cloning of GX0101 as an infectious BAC Clone and rescuing the recombinant virusTransfer vector pDSpHAI-US2 and GX0101 Total-DNA were cotransfected into CEF by LipfectamineTM2000 or calcium phosphate. Virus-containing cells were passaged four rounds in selection medium containing xanthine﹑hypoxanthine and mycophenolic acid to obtain puried recombinant viruses when the cells showed the CPE. Recombinant viral genomes was extracted and electroporated into E.coli DH10B. The second days, single colonies were picked and verified by digested with BamHI restriction enzyme or PCR ,recombinant virus were reconstituted from GX0101 BAC successfully. Further experiments indicated BAC-derived viruses had similar growth kinetics on CEF to the parental virus GX0101 in vitro.2.3 Comparison of the Pathogenicity of the GX0101 infectious BAC CloneAnimal experiments were carried out in 1-day-old specific-pathogen-free chicken.Further experiments indicated that there was no difference in growth ability and pathogenicity to chickens between the BAC derived virus and its parental virus. The BAC derived virus also maintained its oncogenicity and immunosuppressive effects. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.3 The Construction of infectious cloning of deleting REV-LTR and Comparisons of biological properties3.1 Deletion REV-LTR inserts from recombinant MDV strain GX0101In order to illustrate the change of biological properties caused by REV-LTR, gene mutation is a must procedure. Cloned GX0101 as infectious bacterial artificial chromosome. All the manipulations including DNA fragment deletion or insertion and point mutations can be carried out accurately in E. coli conveniently, and the mutated DNA can be used directly to reconstitute mutant viruses in transfected host cells. It would accelerate the understanding of the MDV gene functions and the relationships with pathogenesis and other biology properties.In this study, we constructed an identical REV-LTR deletion mutant of GX0101 BAC by Red/ET mutagenesis (Yu et al., 2000; Lee et al., 2001) .Using KanR cassette to instead of the REV-LTR inserts. KanR cassette flanked by FRT sites was amplified using primers LTR-kanaR-a and LTR-kanaR-b from the plasmid pKD13. KanaR selectable marker can be removed by using flanking Flpe target recognition sites(FRT). The recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF) by LipfectamineTM2000,named bac-GX0101△LTR.3.2 Characterization of Marek's Disease Virus GX0101 that lack REV-LTR inserts: REV-LTR inserts can increased horizontal transmission ability of MDVOn days 5 of age, SPF chickens vaccined HVT on day 1 of age were infected intra-abdominally with 1000 p.f.u. either bac-GX0101△LTR virus or bac-GX0101 virus. When SPF chickens were infected with bac-GX0101△LTR virus or bac-GX0101 virus, HI antibody titers against NDV, H5-AIV or H9-AIV after vaccination or growth ability were decreased at different levels compared to the control,relatively speaking, the pathogenicity of bac-GX0101△LTR was less than bac-GX0101.The result of detected MDV genome from feather tips by Dot-blot demonstrated, the time of detection MDV genome from non-inoculated chickens breeded with chickens inoculated bac-GX0101 in one cage was one week earlier than the time of detection MDV genome from non-inoculated chickens breeded with chickens inoculated bac-GX0101△LTR in one cage, and also with a higher detection ration. During 100 days after challenged at 1d of age with two MDVs, there were 28.13% and 43.75% of mortality in groups inoculated with bac-GX0101 and bac-GX0101△LTR respectively while no mortality in the control group. Also, 9.3% and 12.5% of chickens demonstrated tumors in different tissues or organs in groups infected with GX0101 and bac-GX0101.The result of animal experiment demonstrated the insertion of REV-LTR enhanced the horizontal transmission of GX0101 strain but not the virulence and the oncogenicity which made the GX0101 strain have some advantages in competitive selection and become an epidemic strain.4 The Deletion ORF A and C of MDV 1.8kbmRNA and Study of gene functionIn order to study the ORF's function of 1.8kbmRNA, we deleted the ORF(A+C) by Red/ET, and got the recovered virus bac-GX0101△(A+C) successfully.`Plasmid pP(pp38)-CAT and pP(1.8-kb)-CAT contained MDV upstream's bidirectional promoter were transfected after bac-GX0101△(A+C) and bac-GX0101 infected CEF respectly.48 hours later, in order to ascertain the influence to bidirectional promoter active of MDV 1.8-kb mRNA's ORF, we measured the CAT active in CEF lysate. The result demonstrated that the active of bidirectional promoter from two direction backdown significantly (P<0.01)because of the deleting of ORF A and C. The result certificated that 1.8kb mRNA could enhance the active of bidirectional promoter.SPF chickens were infected with bac-GX0101△(A+C) and bac-GX0101 strain respectively. The result of animal experiment demonstrated that the influence to weight was slight when deleteing ORF A and C of 1.8kbmRNA and the HI titre to NDV and AIV was higher than the group infected bac-GX0101. The time of tumor occurred on the chickens infected with bac-GX0101△(A+C) was 22 days later than infected with bac-GX0101,demonstrated the oncogenicity couldn't disappear only deleted the ORF A and C.5 The Deletion of MDV ICP4 gene and the Comparison of the PathogenicityIn this article,ICP4 gene were deleted and the recombinant virus bac-GX0101△ICP4 recovered sucessfully. SPF chickens were infected with bac-GX0101△ICP4 and bac-GX0101 respectively. The result of animal experiment demonstrated that the HI titre to NDV and AIV after deleting ICP4 gene was lower than control group, but there were no difference between bac-GX0101△ICP4 and bac-GX0101(P>0.05). The chickens infected with bac-GX0101△ICP4 can be detected with tumors 58 days after challenged which demonstrated ICP4 gene didn't participated in the forming tumor. But cell multiplication experiment demonstrated that ICP4 gene was correlation with the replication of viruses.