Study On Propagation Of PVC2in Vitro And The Role Of HMGCR In PCV2Replication

Porcine circovirus type2(PCV2), the primary causative agent of porcinecircovirus-associated disease (PCVAD), is an emerging swine pathogen causingimmense economic losses in the global swine industry. Until present, there has beenno satisfying treatment targeting this virus. PCV2is an extremely slow-growing virus,and PCV2infection and replication in cell culture yield very low viral titers. PCV2has a small genome size and highly limited coding capacity, which restrict theresearch and development of targeted antiviral drugs. It is necessary to investigate thepropagation of PCV2and the interaction between virus and host for us to study onvirus pathogenesis, strategy for blocking viral replication and identifying potentialdrug targets.A PCV2strain, designated CC1, was isolated from pigs with postweaningmultisystemic wasting syndrome (PMWS). The virus genome was sequenced toensure its genetic background and genotype.Due to its small genome size and highly limited coding capacity, the life cycle ofPCV2relies predominantly on host cell factors. The effects of different methods forPCV2replication were compared. PK-15cells were treated with D-glucosamine,methyl-beta-cyclodextrin (MβCD), concanavalin A (ConA), NH4Cl, Clostridiumdifficile toxin B (CdTB), IL-2or IFN-γ before or after PCV2inoculation. It can beconcluded that treatment with IL-2, ConA, D-glucosamine and MβCD consistentlyincreased PCV2production. Furthermore, treatment with a combination of ConA,MβCD and D-glucosamine increased PCV2yield significantly in PK-15cells.3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) isthe rate-limiting enzyme in the synthesis of cholesterol and other isoprenoids. It wassuggested that HMGCR could affect virus infection. However, to date, the possibilitythat the activity of HMGCR may interfere with the replication of PCV2has never been reported. In the present study, the treatment with MβCD to deplete cholesterolcould increase PCV2infection in PK-15cells during consecutive passages of PCV2consistently. Therefore, we hypothesized that there might be a direct or indirectrelationship between HMGCR and PCV2infection.We examined the role of HMGCR during PCV2infection. The resultsdemonstrated that the level of phosphorylated HMGCR, an inactivated form ofHMGCR, was increased in PCV2-infected cells. To determine the relevance ofHMGCR during PCV2infection, different lines of cells derived from PK-15cells inwhich HMGCR was upregulated by overexpression (PK-HMG), silenced by shortinterfering RNA (siRNA)(PK-shHMG) or inactivated by its dominant-negative form(PK-S869A mutant) were generated and infected with PCV2to determine the PCV2replication and yield. The results showed that overexpression of the HMGCR genesignificantly inhibited PCV2replication and yield, whereas PCV2replication andyield were significantly increased in the HMGCR-downregulated PK-shHMG cells.Taken together, PCV2replication and yield were negatively correlated with HMGCRlevels.In the present study, we observed that there was a robust apoptotic response at48hpi in PK-S869A mutant cells. To determine whether HMGCR is associated withapoptosis in PCV2-infected cells, apoptosis and caspase-3activity were analysed. Theresult demonstrated that the apoptotic response at48hpi in PK-S869A mutant cellswas significantly higher than that observed in PK-15cells. And the caspase-3activityresults showed the same tendency. Moreover, caspase-3activity was reduced whenthe PK-S869A mutant cells were treated with the inhibitor Z-VAD-FMK. PCV2replication was dose-dependently increased in PK-S869A mutant cells treated withincreasing amounts of Z-VAD-FMK. Therefore, it is reasonable to conclude thatHMGCR is negatively associated with PCV2-induced apoptosis.Previously, we have found the role of HMGCR in PCV2replication in vitro.Therefore, we hypothesized that inhibition of HMGCR by statin may affect PCV2replication in vivo. We chose BALB/c mice as a experimental model for PCV2infection. In the present study, the BALB/c mice were infected with PCV2andadministered with atovastatin before virus infection. The results in this studydemonstrated that atovastatin significantly stimulated PCV2replication in vivo,indicating that inhibition of HMGCR enhances PCV2replication in vivo. PCV2 antigens in lymph node were detected by IHC. The results demonstrated that PCV2antigens were mainly immunolocalized to the cytoplasm and plasma membrane ofcells in lymph node obtained from inoculated mice.