Metabolomic Epidemiological Study On Severe Fever With Thrombocytopenia Syndrome

BackgroundSevere fever with thrombocytopenia syndrome(SFTS)is an emerging tick-borne infectious disease that was first discovered in mainland China in 2009.The pathogen was certified as severe fever with thrombocytopenia syndrome virus(SFTSV),a novel bunyavirus.Until 2017,there were reported SFTS cases in 23 provinces in China with an average mortality rate of 5.3%.World Health Organization(WHO)has listed it among the top 10 diseases of the annual disease priority blueprint for many years.However,the understanding of the pathogenic mechanism of this virus and virus-host interaction still remains obscure.One of the two important characteristics and lethal factors of SFTS is that the hemorrhagic tendency caused by the severe reduction of platelets and consistent virus replication induced by immunosuppression.ObjectivesThis study aims to use metabolomics analysis methods to find potential metabolic abnormalities from the perspective of the metabolic perturbation of SFTSV on host patients,and then using case-control study,clinical randomized controlled trials and in vitro cell model to test the relevant metabolic abnormalities caused by the virus and its biological effects,in order to explore the pathogenic mechanism of the virus,and to screen possible biomarkers and therapeutic targets for clinical application.Methods and techniquesIn the first part of the study,serum metabonomics analysis was performed on two independent gourps of population using a targeted quantitative metabolomics technique based on LC-MS/MS.The second part adopts a case-control study design,and uses an automated amino acid profile analysis,flow cytometry,enzyme-linked immunosorbent assay,Primeflow RNA detection and other technical means to perform functional verification and correlation analysis.The third part uses clinical randomized controlled trials to verify the related biological effects of arginine intervention and its clinical efficacy.The last two sections retrospectively analyzed the clinical features associated with endothelial cell function in SFTS cases and relied on the in vitro cell infection model,using laser confocal microscopy,RT-qPCR,Transwell cell culture chamber and Luminex technology to study another suject that arginine metabolism may target: vascular endothelial dysfunction.Results1.SFTS metabolome association study The relative abundance of 63 and 103 metabolites was detected in the first and second groups,respectively.Principal component analysis(PCA)and Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)were used to find that the major differences in metabolites,which can be used to distinguish each group of population.Based on the statistical analysis results,28 and 41 differential metabolites were screened out in the first group and the second group,respectively,and there were 54 differential metabolites in total.Several significant metabolic pathways were found through metabolic pathway analysis.Among them,the arginine and valine metabolic pathways also have the highest pathway impact and with relatively hig significant statistical difference.In addition,the concentration of arginine-related metabolites is closely related to clinical condition.2.Case-control comparison and analysis of biological effects ofabnormal arginine metabolism in SFTS.In the case-control comparison of the validation population in the second part,arginine and citrulline were significantly reduced in the acute phase of infection,and ornithine was significantly increased.The global arginine bioavailability(GABR)decreased significantly and was more pronounced in deaths.At the same time,the proportion of myeloid-derived suppressor cells their expression of intracellular arginase increased significantly.On the other hand,the CD3ζ chain of T cell receptors regulated by arginine was significantly downregulated.The intraplatelet product of arginine,platelet nitric oxide,was significantly down-regulated.These factors have a significant correlation with arginine levels and are closely related to the clinical phenotypes such as platelet count and viral load.Therefore,it is speculated that the decrease of arginine may directly participate in the regulation of immunosuppression and thrombocytopenia,and cause corresponding clinical complications.3.Randomized controlled trials of arginine in the treatment of SFTS The results showed that in the arginine-administered group,platelet nitric oxide increased significantly,T lymphocyte CD3ζ chain was up-regulated,and virus clearance and platelet recovery were accelerated.4.Retrospective analysis and case-control study of endothelial cellinjury in patients with fever and thrombocytopenia syndrome The results showed that the clinical manifestations associated with vascular endothelial cell damage,such as hemorrhage and leakage,occurred during the infection of SFTS patients,and changes in the adhesion factor concentration in the serum of SFTS patients also suggested the presence of inflammatory response and dysfunction of vascular endothelial cells due to viral infection.5.In vitro experimental study on endothelial cell injury of severe feverwith thrombocytopenia syndrome In the in vitro model of endothelial cell infection,we observed that viral infection can directly cause vascular endothelial cell morphology alterations,enhanced cell surface activation molecule expression,increased cytokine secretion,decreased cell viability,decreased cell transmembrane electric resistance,and increased macromolecule diffusion rate,and altered intercellular junction and cytoskeleton protein expression and distribution.ConclusionAbnormal arginine metabolism is an important pathogenic feature in severe fever with thrombocytopenia syndrome.Arginine has several key biological effects such as immune regulation,platelet inhibition and vascular endothelial stability adjustment,which are associated with the disease condition of SFTS.Arginine and its related metabolites have the potential as biomarkers for prognosis and adjunctive therapeutics.Significance and InnovationSevere fever with thrombocytopenia syndrome is a emerging fatal infectious disease that is frequently reported in several provinces across China and is being reported around the world.The distribution of ticks has determined the susceptible population of SFTS to be mostly rural population in remote areas.The continuous epidemic of SFTS in the past decade has brought a heavy burden on the medical system of our nation.However,there is no effective treatment and vaccine for this disease,and there is no clear understanding of its pathogenesis.In this study,we systematically analyzed this novel infectious disease from the view of metabolic for the first time.We found and verified the important characteristics and related effects of arginine metabolism abnormality in SFTS.We brought a new perspective for the prognosis,the pathogenesis and clinical treatment for this disease,and broaden the understanding and application of arginine in acute viral infectious diseases.