Role Of HMGCR And PKC In PCV2 Infection And Preparation Of PCV2 Vaccines

Porcine circovirus 2(PCV2)is the etiological agent of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD),which are present in every major swine-producing country in the world.This virus was first confirmed in 1982 and subsequently identified in pigs in the USA,France,Japan,Korea,China,and other countries.However to date,there is no effective drug for PCVD/PCVAD.ue to the small genomic size and highly limited coding capacity,viral DNA replication requires cellular DNA replication machinery,and the life cycle of PCV2 relies predominantly on host cell factors.Therefore,information about host factors involved in PCV2 infection will provide novel strategies to control the disease.And it is urgent to establish a high permissive cell for the virus production in vitro.The enzyme 3-hydroxy-3-methylglutaryl coenzyme A(HMG-Co A,HMGCR)reductase catalyzes the conversion of HMG-Co A to mevalonate,a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids.It was suggested that HMGCR can affect virus infection.We previously found that HMG-Co A reductase was inactivated by phosphorylation during PCV2 infection.HMG-Co A reductase was negatively associated with PCV2 infection in vitro and in vivo.Protein kinase C(PKC)is a large family of protein kinases,which regulate numerous cellular responses by phosphorylating threonine and serine residues of target proteins.Recent reports demonstrated that PKC participated in virus infection,cellular antiviral responses and virus-induced cellular apoptosis.However,it remainsunclear whether PKC participates in PCV2 infection.Furthermore,whether PKC is involved in HMGCR or JNK1/2 pathway during PCV2 infection needs to be elucidated.In this study,effects of PKC and HMGCR on PCV2 infection were evaluated using real time PCR and western blot.We found that PKC and HMGCR participated in different stages of PCV2 infection.HMGCR works on the early stage of the infection to inhibit the virus infection,while PKC enhances the infection at the late stage.Furthermore,PKC enhances PCV2 replication by activating JNK1/2 and inactivating HMGCR via regulating phosphorylation of these two proteins,while HMGCR can suppress phosphorylation of JNK1/2.HMGCR may interact with Cap protein in the early stage of the infection,PKC interacts with Rep protein in the later period of the infection.The results in the present study will provide new sights in the pathogenesis of PCV2 infection,as well as interactions between host factors during PCV2 infection.Currently,several commercial vaccines are effective to induce protective immunity against PCV2 infection.However,many of these available vaccines are based on inactive viruses that need to be propagated in susceptive cells.Although porcine kidney cell(PK-15)is widely used in PCV2 propagation and vaccine preparation,the virus titers is much lower than expected.The reasons are partly due to that the virus propagates extreme slowly and the poor susceptible of the cell for PCV2 infection.Therefore,it is urgent to establish a high permissive cell for the virus production in vitro.In this study,a PCV2 sensitive cell line PK-15-IL-2,which was constructed previously,was treated with D-glucosamine for a short-term,followed by culturing in the microcarrier culture system.The results showed that PCV2 proliferation can be significantly enhanced in cells stably expressing porcine IL-2gene using microcarrier culture system.Furthermore,efficiency of the inactived virusprepared in this study as well as several commericialized vaccines to produce antibody against PCV2 were compared.The results indicated that polyclonal antiserum prepared from the inactived virus had better immunity in comparison to that of the commericialized vaccines.These results will be helpful for further studies focusing on pathogenesis of PCV2 and serology diagnostic test or vaccine development against PCV2.