Metabolomics Profiling Of Classical Swine Fever Virus-infected Cells And Glucose Promotes The Propagation Of Classical Swine Fever Virus

Classical swine fever virus(CSFV),a member of the Pestivirus genus of the Flaviviridae family,is an small enveloped,non-segmented single stranded RNA virus,the genome of which is about 12.3 Kb and carries one large ORF encoding a polyprotein,the latter can be processed by viral and host protease to 4 structural and 8 nonstructural proteins.Classical swine fever caused by CSFV is a severely swine infectious disease,which is characterized by extensive hemorrhage,high fever,and immunosuppression.The outbreak of classical swine fever has caused huge economic losses,it is listed as an animal infectious disease that must be declared by the world organization for animal health(OIE),and ruled as class one animal disease by Chinese government.So it is necessary to clarify the interaction between host and CSFV during viral infection,in order to develop vaccines or targeted drugs to effectively control CSFV infection.Many viral infections hijacked the host aerobic glycolysis,that is the Warburg effect,they also can exploit host fatty acid synthesis and glutamine metabolism.Alteration of the carbon source is suitable with the demand for energy in the process of viral replication and the assembly of viral particles,these changes can not only provide specific biosynthetic precursors for the proliferation of virus particles,but also can increase the survival of host cells.The virus is not only common to the change of metabolism in infected cells,but also has a unique metabolic change due to the difference of virus species.So it is necessary to identify how the virus changes cell metabolism and these metabolic changes occurred in which stage of the virus life cycle,which improves better understanding about the virus requirements in replication,and this wil also provide new insights into exploring potential targets for inhibition of virus proliferation in cell and then developing effective antiviral drugs.In this study,ultraperformance liquid chromatography-quadrupole time of flight/mass spectrometry(UPLC-Q-TOF/MS)and Gas Chromatography-Mass Spectrometer(GC-MS)were used to determine the differential metabolites in infected swine testicular cells(ST)occurred at 12 h,24 h and 48 h post infection,and subsequently analyzed with principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA).The differential metabolites screened by UPLC-Q-TOF/MS under both positive and negative ions were aligned with progenesis QI software database to qualitative the obtained metabolites,and the differential,metabolites screened by GC-MS were aligned with NIST database and Feihn database.The integrated experimental data of UPLC-Q-TOF/MS and GC-MS show that 214 differential metabolites were found in CSFV infected-ST cells at three different time points,including 102 lipids(75 out of 102 are phospholipids),36 amino acids,33 carbohydrates,24 nucleotides,and 19 coenzyme and hormones substances.For metabolic pathway analysis,the differential metabolites were analyzed with KEGG and several metabolic pathways were significantly altered during CSF infection,including phospholipid metabolism,biosynthesis of amino acids,aminoacyl-tRNA Pyrimidine metabolism,carbon metabolism and pantothenate and CoA biosynthesis,planine,aspartate and glutamate metabolism,andphenylalanine,tyrosine and tryptophan biosynthesis.The above metabonomic results confirmed that CSFV infection notably altered the host cell metabolism.Metabolomics profiling of CSFV-infected cells showed that a number of metabolites in the key metabolic pathway were significantly altered,but it is not clear which altered metabolites are more meaningful to CSFV replication.In this case,the effects of glucose and glutamine metabolism on the proliferation of CSFV were studied.First,ST cells infected with CSFV cell-adapted field strain GD53/2011 were cultured with DMEM deprived of glucose or glutamine,and the results showed that glucose deprivation significantly inhibited the titer of CSFV compared to the mock group,but glutamine deprivation has few effect on the CSFV propagation.Inhibition of CSFV replication by glucose starvation was also confirmed by the decreased Npro expression levels in infected cells.Further,different concentrations of glucose were added to the DMEM deprived of glucose,which were used to culture ST cells infected with CSFV,and the viral titer was measured at 24 h post infection.As a result,the infectious titer of CSFV increased with the glucose concentration and reached peak at 1 g/L glucose,indicating that glucose is critical for the optimal replication of CSFV.In addition,glucose analog 2-deoxy glucose(2-DG)was added to normal DMEM at 0 mM,25 mM and 50 mM respectively,then used to culture CSFV-infected cells,the results showed that the proliferation of virus infection was significantly inhibited in 2-DG treated cells,the inhibition effect depends on the concentration of 2-DG.In addition,the effects of glucose on theviral stages of CSFV life cycle were also analyzed,results of the infectious titer and genome copy numbers in the intracellular and extracellular samples indicated that the number of CSFV genome copies and the infectious titer in intracellular and extracellular samples were significantly lower than that of the samples in the complete medium.Meanwhile,the infectious titer in intracellular samples under glucose starvation is identical with that in the extracellular samples,although the genome copy numbers in intracellular was 10 times higher than in extracellular,indicating that glucose is a essential material in CSFV replication and mature rather than in release.Further,we found that inhibition of lactic acid production had no effect on the generation of infectious virus particles.Finally,more glucose was observed to be taken into the CSFV-infected cells in comparison with the control group.In this study,the metabolomics profiling of CSFV-infected ST cells were screened by UPLC-MS and GC-MS,many metabolites and associated with metabolic pathways were altered during CSFV infection,including sugars,lipids,amino acids and nucleotides,the types and the variation of individual metabolites were different at different time points,which is corresponds to the life cycle of CSFV.In addition,we also studied the role of glucose and glutamine in the viral replication,assembly and release of CSFV,and glucose was found to be critical for the optimal replication of CSFV,but glutamine is not necessary.Taken together,thus,this study will be helpful to reveal the molecular mechanisms of replication and pathogenesis of CSFV.