Regulation Of Host MicroRNA-98 On Replication And Expression Of Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2)infection is prevalent widely all over the world,it is the main pathogens of weaned piglet multi-system failure syndrome(PMWS).PCV2 was reported to cause diseases such as porcine dermatitis and nephrotic syndrome(PDNS),porcine respiratory disease syndrome(PRDC)and reproductive disorders,and these diseases are also referred as PCV2 systemic diseases(PCV2-SD).The main symptoms of PCV2 are manifested mainly as weanling piglet multiple system failure syndrome and other secondary symptoms,such as dyspnea,weight loss,anemia,diarrhea and jaundice;microscopic lesions include lymphadenopathy,nephritis,pancreatitis,hepatitis and granulation Interstitial pneumonia.The pathogenesis of PCV2 is mainly related to the immunosuppression of the host that caused by the virus.PCV2 infects the monocyte/macrophage cell line and grows itself in the macrophages and dendritic cells.Blood circulation causes the virus spread to the whole body,leading to the disease.During PCV2 infection,the DNA replication of host cell was also involved in the regulation progress of viral DNA replication,the virus multiplied with cell division and proliferation,and thus the life cycle of the virus regulated by the host cytokine.At the same time,the host cells are also affected by the virus,resulting in a variety of regulatory signals changed,including cell apoptosis,autophagy start and cytokine changes.Changes in cytokines affect the antiviral response of the host.The expression of cytokine’ mRNA was changed after PCV2-SD infection,IL-10 was overexpressed in the thymus,and IFN-y was overexpressed in tonsil.In other lymphoid tissues,the expression of IFN-γ and IL-10 increased,and the expression of IL-12p40,IL-4 and IL-2 mRNA decreased.So far,little is known about the mechanism of these biological processes,such as the regulation of IL-10 expression,and there has been a mechanism of feedback between IL-10 and multiple miRNA.miRNA is a large number of endogenous small RNAs derived from nonprotein-encoding genes,which are about 17-25 bp in length.miRNA play its important role by inhibiting mRNA translation or inducing gene degenerative or regulatory protein.One miRNA can modulate multiple gene expression,and each mammal produces more than 1000 miRNA,and these miRNA silenced about 30%Protein-encoded gene of the mammal by binding to target mRNA complementary sites,especially untranslated regions(UTRs).It has been shown that host miRNA can create a virus-friendly condition that causes viral immunity escape or inhibit viral infection by binding to the virus or the host’s own target mRNA.At present,there are few reports about PCV2-related miRNA.The role of PCV2 replication-related miRNA in viral replication and pathogenesis has should be further studied.It is important to elucidate the interaction between PCV2 and host,and to provide new targets for the development of new anti-PCV2 drugs and strategy.In order to explore the PCV2 replication-related miRNA,one PCV2 strain was isolated and named as 2015JS.The infection model was established by PK15 cell line(PK15 cell,suitable for PCV2 replication expression),and miRNA related to PCV2 replication were screened by high throughput sequencing.And then through study the molecular mechanism between miRNA and the replication and expression of PCV2 to clarify the role of miRNA in the PCV2-host interaction process.The main contents of this study include the following three aspects:1.Isolation,identification and genome sequence of porcine circovirus 2(PCV2)2015JS strainWe selected PCV2 positive samples as raw materials for isolating strains,which detected by PCR at clinical.The isolates were identified by PCR and indirect immunofluorescence assay(IFA),and the TCID50 of the isolate was been determined.after sequencing the DNA sequence,homology comparison and phylogenetic analysis were carried out to determine the isolation,we sure we successfully isolated a PCV2 strain,the total genome length 1766 bp,named as 2015JS strain.And it has the highest homology(99.8%)with Uruginean strain Uy99(GenBank accession number:KP867050),all belong to type PCV2d.The isolation of the isolate provided a reference material for the epidemiological study,genetic variation analysis and prevention,control of related diseases of PCV2 in Jiangsu Province.The isolate also provided a basis for PCV2 vaccine strains.The virus used in the subsequent trials is 2015JS.2.Screening of the miRNA associated with PCV2 replication and their impaction on the replication and expression of PCV2Inorder to analysis PCV2 replication related miRNA,we use PCV2 infect PK15 cell line to establish cell infection model,extracted the total RNA of PK15 cells to study the differential expression of miRNA by high-throughput sequencing.MiRNA-98 increases gradually along with the increasing replication and expression of PCV2,which was selected as a candidate miRNA that may be associated with PCV2 replication.MiRNA-98(miRNA-98 mimics)was transfected into PCV2-infected cells,the copy number of PCV2 ORF2 was detected by fluorescence quantitative PCR(RT-PCR),indirect immunofluorescence(IFA)was used to observe the condition of diseased cells.The expression level of Cap protein(PCV2 major immunogenic protein)was determined by Western blotting.The results showed that miRNA-98 could promote PCV2 replication and expression.Inhibiting endogenous miRNA-98 expression could reduce the replication and expression of PCV2.Therefore,miRNA-98 was selected as the study object to study its molecular mechanism on PCV2 replication and expression.3.Molecular mechanism of miRNA-98 regulating PCV2 replication and expressionBased on miRNA-98 sequences,we attempt to analyze whether miRNA-98 can directly target the PCV2 genome(the main reading frame ORF 1/2/3)by bioinformatics.It was found that there were possible target sites for miRNA-98 on the PCV2 genome.Continuously using bioinformatics to analyze whether miRNA-98 can affect viral replication and expression by targeting host cell factors,we found that miRNA-98 possible targets GANAB,ALG11 and MAN2A2,which exist on the host cell genome.The target site and its target mutation site sequence were constructed into the double fluorescent reporter(pgl3 vector)and co-transfected with miRNA-98 mimics.The results of double luciferase reporter assay showed that miRNA-98 did not affect the activity of reporter gene pGL3-ORF1/2/3 and pGL3-MAN2A2,that is,miRNA-98 could not directly target PCV2 transcripts;However,miRNA-98 can reduce the activity of the reporter gene GANAB and ALG11,and does not affect the activity of the target mutation site,indicating that miRNA-98 can directly target ALG11 and GANAB.Further studied the effects of miRNA-98 on the expression of GANAB and ALG11 mRNA,protein and cell cycle.It was found that overexpression of miRNA-98 promoted the expression of GANAB and ALG11 mRNA and protein.Cell cycle detection showed that the cell numbers of G1 phase in the test group compared with the positive control group were decreased,while the number of S phase and G2 phase increased relatively;inhibition of endogenous miRNA-98 inhibited the expression of ALG11 and GANAB mRNA and protein,and the results of cell cycle analysis are contrary to the results of overexpression of miRNA-98.It is suggested that miRNA-98 is involved in the regulation of cell cycle and apoptosis.It is possible to hinder the synthesis of N-glycans on the surface of the host cell membrane by targeting GANAB,which makes the virus enter the cell more convenient;and in the other side,the cell cycle is prolonged by GANAB and ALG11 thus promoting the replication and expression of the virus.