Genome Analysis Of The Wild Strain Of Duck Plague Virus And Preliminary Study On The Function Of UL55 Gene

Duck plague virus(DPV), also known as duck enteritis virus(DEV), is the pathogen of the acute, contagious and lethal disease of ducks, geese and other anseriformes. Due to the high mortality and morbidity, DPV is currently one of the most serious infectious disease in the waterfowl industry all over the world. DPV belongs to the subfamily of alpha herpesvirinae and then was assigned to the Mardivirus genus. Due to the late starting of research, the molecular research progress of DPV is far behind that of its homologues in herpes virus family. Most of the researches related to this pathogen focused on epidemiology, prevention, diagnosis, and etc. for quite some time, while the genome structure, physical map, gene function of DPV and its replication and pathogenesis, virus infection and antiviral infection mechanisms are still unclear. So the purpose of the thesis are accomplishing genome analysis of the sequenced duck plague virus Chinese virulent strain; supplying techniques for DPV gene function study by generate a bacteria genetic operation platform; Besides, we hope to knock out UL55 gene by two step Red recombination based on this platform for interpretation UL55 gene function. The contents of our research are as follows:1 Genome analysis of duck plague virus Chinese virulent strainBioinformatic tools were used to analysis the sequenced DPV CHv genome in our lab. Its genome is a linear double-stranded DNA and has a typical D herpes virus genome structure:UL-IRS-US-TRS. DPV CHv genome is 162,175 bp in length, consist of 44.89%GC and owns 76 potential ORFs which may encoding functional proteins. We found Five strains of DPV shows high homologous to CHv strain through similarity analysis, which were classified according to their geography and virulence variation in the phylogenetic tree, DPV CHv located in the middle place of the tree. Scanning the coding region of these genome, we found there are 23 and 5 ORFs in UL and US region exhibit whole ORF deletion, partial insertion and deletion, respectively. We conclude that these mutations might be the reason of virulence and geography variation between strains. The rest 52 ORFs which exist no insertion of deletion shows high homologous on amino acid level and most of them are conserved genes in charge of viral DNA metabolism. Codon usage analysis of DPV CHv suggested DPV CHv genome prefer an A/T ending codon, and the codon usage bias was mainly determined by mutation pressure. Compared to homo species, E. coli and yeast, the codon usage pattern of DPV CHv is most close to that of the Yeast system.2 The bioinformatics analysis of duck plague virus UL55 geneDPV UL55 gene consists of 561 bp nucleotide, including a complete open reading frame, encoding a 186 amino acid protein whose mass is 20.7981 kDa. There is no signal peptide and transmembrane in UL55 protein. DPV UL55 gene prefers an A/T ending codon and the codon usage pattern of it is much more close to that of homo species. The UL55 protein is a hydrophilic protein and its antigen epitope are almost located in the corresponding hydrophilic region of random coil(Cc). The predicted function of UL55 protein is assembly, budding, mature and release in virus life cycle. Phylogenetic tree based on the nucleotide sequence of UL55 gene suggest DPV is the member of Marek’s disease genus.3 The cloning, expression and polyclonal antibody preparation of DPV UL55 geneWe obtained the fusion DPV UL55 protein in host E. coli BL21(DE) cells by cloning UL55 gene to prokaryotic vector pET32a(+). After optimization of the expression condition, the recombinant UL55 protein can produce large amounts of soluble form of inclusion body by 0.8mM IPTG inducing 4h at 37℃. Followed by inclusion body lytic, we performed purification and renaturation of UL55 protein for western blotting analysis with anti rabbit DPV CHv polyclonal antibody. The result of western-blot displayed high specificity which means the purified and renatured UL55 protein was similar to natural UL55 protein. Besides, the prepared anti rabbit UL55 polyclonal antibody reacts powerfully to purified UL55 protein.4 Establishment of an indirect ELISA for detecting DPV infected duck serum by coating prokaryotic expression UL55 protein as antigenThe indirect ELISA based on purified recombinant prokaryotic expression UL55 protein was used to detect DPV infected duck serum. This is a specific method which can recognize the DPV infected duck serum from other duck pathogens infected duck serum. Such as anti duck hepatitis virus (DHV), Riemerella anatipestifer serotype 1(RA), E. coli, Salmonella Enteritidis (SE), and influenza H5(AI), the indirect ELISA of which were all negative while the DPV infected duck serum are positive. Repeat trials showed the variation coefficient inside or between coated plate are both less than 10%. This diagnose method can detected the 1:6400 diluted positive serum of DPV attenuated vaccine strain infected ducks. Comparison analysis of it with classic neutralization test and DPV virion coated ELISA for 50 DPV infected duck samples found the positive detection rate of UL55-ELISA is in the middle place of all. Considering the advantage of the indirect ELISA, such as easy preparation, high specificity and sensitivity and safe to the environment, the UL55-ELISA has a good prospect of application and be made of detection kits.5 Transcriptional and dynamic expression analysis of DPV UL55 geneTranscriptional analysis of UL55 gene in DPV infected host cells in vitro was performed by relative fluorescence quantitative method using P-actin gene as an internal control. The result indicated that the transcription product of UL55 gene was at a relative low level in the first 0-8h post transcription(h.p.t.), the transcription product of UL55 gene increased fast after 12h.p.t. until it reached the peak at 36h.p.t. After that, it began to decline gradually, a lot of transcription product of UL55 gene still can be detected after 60h.p.t. The transcriptional process can be inhibited by nucleic acid inhibitors which means the UL55 genes is a y2 gene because the transcription of it is strictly dependent on DNA synthesis. The dynamic expression analysis of UL55 protein by Western-blot displayed the same rule of above research, further support the conclusion that UL55 gene is a real late gene(y2).6 Construction of rescued bacterial artificial chromosome recombinant duck plague virus platformWe obtained the recombinant virus DPV CHv-BAC-G by co-transfection DPV CHv DNA with recombinant DPV transfer vector pUC18/EGFP-TKAB-BAC11 which consist of TK gene homology arm, reporting gene EGFP and the core functional components of BAC. Then the recombinant DPV was purified by 8 rounds of plaque purification using EGFP as reporter gene.Extracting the circled DPV CHv-BAC-G and electric shocking it to the DH10B cells for producing recombinant UL55 clone which can replicate in bacteria. Then we rescued the recombinant DPV CHv-BAC-G by transfecting the virus clone plasmid pBAC-DPV into host DEF cells. The rescued recombinant virus can survival as bacteria and virus at the same time and can replicate in both system. It will be helpful for using the mature gene manipulation techniques in prokaryotic system to study DPV which only can be learned in cell level before. Make the study of DPV no longer restricted by technology and also constructed a new DPV research platform in our lab.7 Construction of DPV UL55 deletion strain and its molecular characteristic researchDPV UL55 deletion and its revertant were generated by two step of Red recombination based on the constructed rescued BAC recombinant DPV virus platform above. The rescued UL55 deletion revertant can generate same CPE as the parental virus did and exhibit no difference in RFLP analysis, which means it is a real revertant. Plaque formation analysis, one step growth curve and pathogenic analysis indicated the constructed UL55 deletion and its revertant were constructed as we expected, because they can produce the similar CPE and plaques, exhibit same propagation cycle in infected cells and infected the host ducks as their parental virus DPV CHv-BAC-G did.8 Intracellular analysis of DPV UL55 gene in DPV infected host cells in vitroIndirect immunofluorescence assay(IFA) was used for detecting the dynamic distribution of UL55 protein in infected cells by using anti rabbit UL55 polyclonal antibody as the first antibody. The result shows that the UL55 protein expressed in cytoplasm as early as 5.5 hours post infection(h.p.i) and increased gradually as times move on until it reached the peak at 22.5 h.p.i. After that, the expression of UL55 protein declined gradually and started to forming fluorescent spot and transferred to the nuclear membrane peripheral, most of them distribute near the two poles of nuclear. After that, the fluorescence of UL55 protein dismissed gradually with time move on, suggested the protein disappeared following the ending of virus replication cycle and disruption of cells.9 Co-intracellular analysis of DPV UL55 and UL26.5 gene in DPV infected host cells in vitroCo-intracellular analysis of UL55 and UL26.5 proteins indicated that the UL55 and UL26.5 protein was adjacent and overlapped in infected cells. When the virus doesn’t express UL55 protein, there was no change in the location of UL26.5 which means the distribution of UL26.5 will not be affected by UL55 protein and its downstream operation can performed without the participation of UL55 protein.