The Role Of ORF6 On The Pathogenicity Of Porcine Circovirus Type 2

The earliest report on the infection and epidemic of PCV2 in China was in 2001,and it has been widespread in the pig industry in China,which has a serious impact on the development of pig industry in China.PCV2 is widely epidemic around the world.five of the 11 proteins predicted by PCV2 have been identified as ORF1-5.ORF6 was identified by us in previous study.The ORF6 gene?nucleotide sequence 1611-1522 bp,total 90 bp?is overlapped with ORF2,encodes 30 amino acids with 2.8 kDa.This subject mainly analyzed and studied the biological role of PCV2 ORF6 in PCV2 pathogenicity.The role of ORF6 in the Replication of PCV2,the influence of ORF1⁴,the apoptosis induced by ORF6,and the pathogenicity experiments in animals were investigated to reveal the effect of ORF6 gene on PCV2 Replication and its pathogenicity.The main research contents and results are as follows:1.Virus Replication and the effect of ORF6 on ORF1-4 mRNAPreviously,the infectious clones of both PCV2 wild-type and ORF6-deleted mutant had been successfully constructed,and the recombinant wild-type PCV2 virus W-2PCV2and the ORF6–deleted mutant virus M-2PCV2 had been obtained.The infected doses of M-2PCV2 and W-2PCV2 were adjusted to the same MOI,and the viral Replication was detected by fluorescence quantitative PCR.One step multiplication curve of virus revealed that M-2PCV2 had a lower titer than W-2PCV2.This indicated that ORF6affects the proliferation of PCV2 in vitro.The effect of ORF6 on the transcription of ORFs1-4 mRNA was further investigated.PK-15 cells were infected with M-2PCV2 and W-2PCV2.The infected cells were collected at different times after infection and total cellular RNA was extracted.Using specific ORF1-4 primers,it was observed by real-time fluorescence quantitative PCR that there was no significant difference to mRNA copies of ORF1,ORF2,ORF3 and ORF4between M-2PCV2 and W-2PCV2.It was indicated that the PCV2 ORF6 gene had no significant effect on the transcription levels of ORF1,ORF2,ORF3 and ORF4.2.ORF6-mediated apoptosisPK15 cells without PCV contamination were transfected with ORF6-expression plasmid,cell samples were collected at different times after transfection.The activation of caspase 3,8 and 9 of the apoptosis-related caspase family was detected.As a result,ORF6expression could not activate caspase 3,8 and 9.At the same time,Annexin V-PI double staining was used to detect apoptotic cells.It was observed that ORF6 expression could not induce apoptosis.The PCV2 recombinant wild type virus W-2PCV2 and ORF6–deleted mutant virus M-2PCV2 were respectively infected in PK15 cells.The activity of Caspase 3,8 and 9 was detected.It was observed that the ORF6 deletion mutant upregulated the activity of caspase 3 and down-regulated the activity of caspase 8.3.Animal pathogenicity experimentThe 10^4.0 TCID₅₀ of W-2PCV2 and M-2PCV2 recombinant viruses were challenged to Balb/c mice,and the same dose of PCV2 wild-typeand DMEM blankwere used simultaneously as control.The clinical symptoms of infected mice were observed daily after infection.The sera of the challenged mice were collected at 0,7,14,28,and 42 days after challenge infection to detect the expression of IFN-?,IL-4,RANTES,IL-8 and IL-10,respectively.At the same time,3 mice of different infected groups were randomly killed.Lung,spleen,liver and inguinal lymph node of the challenged mice were collected to detectviral loads.In additional,tissues sections were made,and the histopathological changes were analyzed.It would clarify that the role of ORF6 gene in the pathogenicity of PCV2 infection in vivo.Results:No significant changes in the clinical symptoms of the mice were observed in the experimental and control groups.The sera from the challenged mice were collected at 7,14,28,and 42 days after challenge.Cytokine levels in serum at different time points were detected and analyzed by fluorescent quatitative method.The cytokine levels of IFN-?,IL-4,RANTES,IL-8 and IL-10 induced by W-2PCV2 were significantly higher than that induced by M-2PCV2 at different time points.It indicated that ORF6 can significantly increase expression levels of IFN-?,IL-4,RANTES,IL-8 and IL-10.Different serum and tissue samples of the challenged mice were collected at different time points,and the viral load was detected by real-time PCR.As a result,the viral load of sera and liver,spleen,lung and inguinal lymph nodes of M-2PCV2 mice was higher than that of the sera and tissues of w-2PCV2.This indicates that the ORF6 gene can reduce the viral load in infected mice.In addition,inguinal lymph nodes,spleen,liver,and lung samples of experimental and control mice were collected at different time points,tissue sections were pRepared,and pathological changes were observed by HE staining.It is observed that the mice of M-2PCV2,W-2PCV2,and PCV2 groups showed reduced lymphocytes in spleen and inguinal lymph nodes,the germinal center expanded with proliferation of histiocytes and lymphoblastocytes.Degeneration of Hepatic cells in liver with scattered hepatocyte necrosis was also observed.The most obviously pathological change was observed in mice infected with M-2PCV2.The lymphocytes of spleen and inguinal lymph nodes were disappeared heavied.Meanwhile,a large number of neutrophils and monocytes were infiltrated in the lungs of the mice in the M-2PCV2group,and the alveolar septum was thickened,the haemorrhage was more severe,and the red cells were infiltrarted heavylied in the tissue.In mice,the ORF6 deletion strain M-2PCV2 showed stronger pathogenicity,indicating that ORF6 is closely related to the virulence and pathogenicity of PCV2.Its involvement in the regulation of virulence in this animal pig remains to be further studied.