Effect Of Glutamine On PCV2 Replication In PK15 Cell And Its Mechanism

PCV2 is currently a ubiquitous pathogeny in pig farms,which can cause porcine circovirus diseases,including postweaning multisystemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),congenital tremor(CT),porcine respiratory disease complex(PRDC),proliferative and necrotizing pneumonia(PNP),reproductive disorder and enteric disease.PMWS is the most important PCVD and becomes an economically important disease in swine-producing areas worldwide.Glutamine is the most abundant free amino acid in the body,acting an important part in providing oxidative fuel,gluconeogenic precursor and lipogenic precursor,regulating gene expression and protein level.Glutamine can affect virus infection and replication.Glutamine is essential for human cytomegalovirus infection,infectious virions are not produced under glutamine-free conditions.Omission of glutamine from culture medium resulted in a marked inhibitory effect on the release of infectious virus and synthesis of envelope protein,while VSV or NDV was not significantly affected by glutamine deprivation.Up to now,there is no reporter about effect of glutamine on PCV2 replication.The present study was conducted to investigate the effect of glutamine on PCV2 replication in PK-15 cells and to explore the mechanism,so as to provide a theoretical reference for application of glutamine on cell culture and PCV2 propagation in vitro.Experiment 1:Effects of glutamine on PCV2 replication in PK15 cellsIn this study we examined the effects of glutamine on PK15 cell proliferation and PCV2 replication.PK15 cells were infected with PCV2 in replete medium for 24 hours,then had the medium changed to various concentrations of glutamine,cells were allowed to grow another 48 hours before determining the cell proliferation by MTT.The results showed that cell proliferation was reduced in PCV2 infected cells,cell viabilities decreased when glutamine concentration was below 4 mM,but there was no improvement when glutamine concentration was above 4 mM.PCV2 replications in replete medium and glutamine-free medium were determined by real-time PCR at different times.The results were shown as follows:the highest PCV2 DNA copies were detected at 72 hours post-infection;at 72 hpi and 96 hpi,PCV2 DNA copies in glutamine-free medium were higher than those in replete medium.PK15 cells were infected with PCV2 in replete medium for 24 hours,then had the medium changed to various concentrations of glutamine,cells were allowed to grow another 48 hours before determining PCV2 DNA copies and relative amount of PCV2 infected cells by real-time PCR and immunofluorescence,respectively.The results showed that PCV2 replication and relative amount of PCV2 infected cells were increased when glutamine concentration was below 4 mM,but there was little effect when glutamine concentration was above 4 mM.These datas suggest that when glutamine concentration was below 4 mM,there were decrease of cell growth and increase of PCV2 infection and replication with lower concentration,when glutamine concentration was above 4 mM,there was no significant difference among different concentrations.Experiment 2:Mechanism of influence of glutamine on PCV2 replicationTo investigate the mechanism of PCV2 replication affected by glutamine,we cultured PK15 cells infected with PCV2 in medium with glutamine at different concentrations below 4 mM,PCV2 replication,antioxidative index and p38 MAPK were detected at 72 hpi.The results were shown as follows:lower glutamine promoted PCV2 replication,reduced GSH level,increased MDA concentration,facilitated p38 phosphorylation,but there were no effects on SOD concentration,p38 mRNA expression and total p3 8 protein level.Then 4 mM G,0 mM Q 0 mM G+20μM BSO,4 mM G+50μM BSO were used in medium after cells were infected,PCV2 replication,relative amount of PCV2 infected cells and p38 MAPK activation were detected at 72 hpi.The results were shown as follows:compared with normal group with 4 mM glutamine,4 mM glutamine added BSO reduced GSH concentration,facilitated p38 phosphorylation and increased PCV2 replication in infected cells.The group with 0 mM glutamine had the same results as groups with 4 mM glutamine added BSO.Previous research demonstrated that the activation of p38 is required for PCV2 replication.We conclude that PCV2 replication was increased by reducing GSH level in cells with lower glutamine,which was associated with changes of p3 8 MAPK activation.