Study On Partial Function Of ORF1, ORF3 And ORF5 Genes Of Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2) is the main pathogen to cause post-weaningmultisystemic wasting syndrome(PMWS) in piglets, which can't be prevented at presentbecause there is no vaccine available. The complete genome sequence of PCV2 is 1767～1768bp with eleven open reading frames(ORFs), in which the function of many ORFs is notvery clear. In this study, the complete genome of PCV2 was amplified directly from pig'spathological sample and cloned to the plasmid vector, then the mutational strains of PCV2with deletion in open reading frame 1 (ORF1), open reading frame 3(ORF3) and open readingframe 5(ORF5) were constructed respectively using enzyme digestion. By using the methodsof polymerase chain reaction(PCR) detection, PCR-restriction fragment length polymorphismanalysis (PCR-RFLP) and observation under transmission electron microscope, thetransmissibility of these gene deletion mutational strains to IBRS-2 cells was identified, andanimal inoculation experiment was carried out to research the pathogenicity andimmunogenicity of these mutational strains to piglets. The objective of this study is toinvestigate the function of genes mentioned above and explore their influences on replicationand pathogenicity of PCV2, and to provide some methods and theoretic bases for constructionof candidate strains for a new type of genetically engineering vaccine.A pair of specific primers AF-P1 and AF-P2 were designed, which introduce the SacⅡenzyme digestion site convenient for gene performance later. Using this pair of primers, thecomplete genome of PCV2 was amplified directly from the pathological sample derived froma pig farm in Chengdu area, which was detected firstly by multiplex PCR method to be PCV2positive, and this complete genome of PCV2 was named PCV-CD strain. The virus genomewas cloned to pMD18-T Simple vector to construct recombinant plasmid pMD18-TSimple-PCV2-CD, which was identified by enzyme digestion and sequence determination.Analysis on the nucleotide sequence showed that, compared with the other twelve PCV2strains published in Genbank, the homology of PCV2-CD is high up to 95.0%～100%.The recombinant plasmid pMD18-T Simple-PCV2-CD was digested by restriction enzyme EcoRⅤand BmgBⅠtogether to delete 187bp in ORF1 gene, the target linearDNA fragment with 4272bp was recollected and connected circularly to construct therecombinant plasmid pMD18-T Simple-PCV2-CD-A. The recombinant plasmid pMD18-TSimple-PCV2-CD was digested by restriction enzyme NcoⅠand BstBⅠrespectively toobtain two target linear DNA fragments with 4459bp, which were blunted andconnected circularly to construct the recombinant plasmids pMD18-T Simple-PCV2-CD-Cand pMD18-T Simple-PCV2-CD-E with insertion in ORF3 and ORF5 genes respectively. Thenewly constructed recombinant plasmids with gene deletion were identified to be correct byenzyme digestion and sequence determination. The recombinant plasmids pMD18-TSimple-PCV2-CD-A, pMD 18-T Simple-PCV2-CD-C and pMD 18-TSimple-PCV2-CD-E were digested by SacⅡto obtain linear genomes of PCV2-CDwith gene deletion. The target DNA fragments with 1580bp, 1771bp and 1769bpwere recollected respectively and connected circularly in vitro, and then three mutationalstrains of PCV2 with gene deletion in ORF1, ORF3 and ORF5 genes were constructedrespectively, which were named mPCV2-A, mPCV2-C and mPCV2-E. Three mutationalstrains were transfected into IBRS-2 cells(PCV1 and PCV2 negative) mediated by liposomeinfection protocol. By using the methods of PCR and PCR-RFLP, in cells after transfectionand cultivation for 4 blind passages, mPCV2-A, mPCV2-C and mPCV2-E were detectedspecificly respectively and distinguished with parent strain PCV2-CD. Observation undertransmission electron microscope showed that, the virus particles of three mutational strainsof PCV2 were observed respectively in the nucleus and kytoplasm of IBRS-2 cells whichwere cultivated for 6 blind passages. The identification results showed that, the gene deletionmutational strains of PCV2, mPCV2-A, mPCV2-C and mPCV2-E have transmissibility,which can replicate and proliferate in IBRS-2 cells. The results also showed that, ORF3 andORF5 genes are not the indispensable genes for replication of PCV2, and the deletion of187bp in ORF1 gene doesn't influence the replication ability of PCV2 obviously.Twenty four health piglets(PCV1 and PCV2 negative) aged 28 to30 day-old weredivided to five groups at random, in which A, B and C were experimental groups for 5piglets per group, D was control group of PCV2-CD for 5 piglets, and E was negativecontrol group for 4 piglets. Every piglet in group A, B, C and D was injectedintramuscularly with 2ml of cell cultures of mPCV2-A, mPCV2-C, mPCV2-E and PCV2-CDrespectively, and every piglet in group E was injected intramuscularly with 2ml ofphysiologic saline. One of piglets in group A, B, C and D was slaughtered on 10d postinoculation (DPI) respectively, the organs and tissues were collected for detection of virus' replication and proliferation in vivo and observation on histopathology. The precava bloodsamples were taken on 0, 7, 14 and 21 DPI for dynamic determination of the T lymphocytesubgroup and the antibody of PCV2, the data were analyzed with DPS v6.55 statisticalanalysis software. By using the established methods of PCR and PCR-RFLP, the DNA ofgene deletion mutational strains in the inguinal lymph nodes of slaughtered piglets on 10 DPIwas found respectively, which proved the gene deletion mutational strains mPCV2-A,mPCV2-C and mPCV2-E could replicate and proliferate in the piglet body, and proved thegene deletion didn't influence replication and proliferation of the viruses in vivo. Observation onhistopathology showed that, in three experimental groups(mPCV2-A, mPCV2-C andPCV2-E), the decrease of secondary lymph nodule amount and or enlargement of germinal centerwere observed in lymphoid nodes in slaughtered piglets, the particle and or water vacuoledisvolution were observed in liver cells, and the alveolar wall epithelium swelling and lymphocyteInfiltration were observed in lung tissue. Similar pathological changes of PCV2-CD controlgroup were more severe than that of experimental groups, there were no pathological changesin all other tissues and organs in all experimental groups and PCV2-CD control group,including spleen, kidney and heart etc., the gene deletion didn't influence the corrodingapproaches and resulted in decrease of pathogenicity of the mutational viruses. By using flowcytometry (FACS), the dynamic determination of CD3~+, CD4~+ and CD8~+ cells' percentcontent of T lymphocyte subgroup in peripheral blood of the animals showed that, the genedeletion didn't influence the cellular immune function of the viruses, and mPCV2-A, mPCV2-Cand mPCV2-E showed the tendency to induce cellullar immunologic response in piglets.Using enzyme linked immunosorbent assay (ELISA), the dynamic changes of the antibodyagainst PCV2 in peripheral blood of the animals were determined. The results showed that,gene deletion didn't influence the humoral immune function of mutational strains mPCV2-A,mPCV2-C and mPCV2-E, which induced the humoral immunity in piglets obviously. Theresults showed that, mPCV2-A, mPCV2-C and mPCV2-E could be the candidate strains for genedeletion vaccine of PCV2.