Metabolomics Study Of Amantadine Interfere C6Cells Infected With Borna Disease Virus

BACKGROUNDBorna disease virus was first discovered in southern Germany in the18th century, until it was separated from most warm-blooded animalsincluding humans in many countries,people realized BDV had a more vastinfected area. BDV is a non-segmental single-stranded, negative-strandRNA virus and the typical representative of a single strand ofnegative-strand viruses heads Borna virus family. The virus is highlyneurotropic, and can long-term sojourn in the nervous system.In recentyears, BDV antigen-specific antibody and RNA have been frequentlydetected from peripheral blood mononuclear cells and brain tissues ofneuropsychiatric patients,these studies show that the virus is closely relatedto the human mental illness, Liv Bode and other studies have found thatamantadine has anti-viral effect of BDV, the symptoms of patient withbipolar disorder eased after taking the drug, but the drug mechanisms thatinhibit viral infection are not yet in-depth research. OBJECTIVETo obtain the broad spectrum metabolites of control group, BDVinfected group and drug intervention group through application of a1HNMR-based metabonomic method. Compare the differential metabolitesin the three groups. Explore the pathophysiological mechanisms thatamantadine inhibit BDV infection.METHODSBDV virus liquid was extracted in a certain multiplicity of infectionfrom long-term BDV-infected cells, and C6cells were infected with thevirus solution.Experimental groups were divided into control group, BDVinfected group and amantadine intervention group,in three replicates. Thenintracellular metabolites was extracted using methanol and chloroform,1Hnuclear magnetic resonance Mass was used to characterize metabolicprofiles of the three groups. Q-test and application T-test were used todistinguish metabolites, finally partial least squares discriminant analysiswas used for the three groups of cells metabolite pattern analysis.RESULTSVirus titers measured that BDV virus liquid can infect cellssuccessfully, RT-PCR technology and WB experiments were conducted toverify the successful transcription and translation and protein expression ofBDV-infected cells from the gene level and protein level. 1H NMR analyzed quantitatively27metabolites, wherein BDVinfection elevated levels of20species metabolites,include: isoleucine,valine, lactate, alanine, acetate,-naphthaleneacetic acid, pyruvic acid,glutamine, succinate, glutamine, creatine, choline, inositol, glycine,tyrosine, β-glucose, phenylalanine, methyl formate, AMP, NAD+; reducedwith7species: leucine2-hydroxy isobutyric acid, aspartic acid,pyroglutamic acid, lactose, histidine, ADP.21kinds of metabolite were differentiaed, comprising: isoleucine,valine, lactate, alanine, pyruvate, glutamate, succinate, glutamine, creatine,inositol, glycine, tyrosine acid, β-glucose, phenylalanine, methyl formate,AMP, leucine2-hydroxy isobutyric acid, aspartic acid, lactose, histidine.Metabolites that drug intervention group are not differentiaed fromcontrol group, but are different with the viral infection metabolites include16kinds, respectively: isoleucine, valine, lactate, alanine, pyruvic acid,glutamic acid, succinic acid, glutamine, creatine, glycine, glucose, tyrosine,phenylalanine, acid, lactose, histidine.CONCLUSIONThe results showed that the BDV-infection leads to celluar metabolismdisorders, mainly for changing the amino acid and energy metabolites.BDV possible affect host cells by interfering the energy metabolismpathways. In-depth analysis of BDV-infected group and the interventiongroup shows that can clearly distinguish drug intervention group betweenBDV-infected group,the energy metabolites elevated by BDV-infection significantly reduced in drug intervention group. Thereby,it can be inferredthat BDV infection enhances the activity of the citric acid cycle pathwayscontrolling cellular energy metabolism pathway for its proliferation, reducedrug-related energy metabolism after amantadine substances, but the resultreversed after amnatadine intervention through blocking virus replication,changes in these metabolites reveals that BDV affects the host normalmetabolic activity of cells, to privide evidence for elucidating thebiochemical mechanism of amantadine anti-BDV.