Clinical Characteristics Of Hemorrhagic Fever With Renal Syndrome And Inhibitory Mechanisms Of Hantavirus On Type Ⅰ Interferon

Hemorrhagic fever with renal syndrome(HFRS)is a natural foci disease caused by Hantavirus infection,with a fatality rate of about 2-10%.Jilin Province is a high-incidence area of Hantavirus infection in China.Lack of specificity in clinical manifestations,the rate of missed diagnosis and misdiagnosis were higher.Relevant clinical and epidemiological studies are insufficient.It is currently agreed that vascular endothelial injury,platelet dysfunction and excessive immune response may be related to the pathogenesis of HFRS.The main target cells of hantavirus are endothelial cells.The innate immune system recognizes pathogen-associated molecular patterns(PAMPs)of foreign pathogenic microorganisms through pattern recognition receptors(PRRs),and induces the production of type I interferon(IFN-Ⅰ)and inflammatory factors.The specific molecular mechanism is still unclear.This study determined the epidemiological and clinical characteristics of HFRS through retrospective analysis of clinical cases of HFRS in Jilin Province,and studied the mechanism of HTNV structural protein inhibiting the antiviral response of host interferon,providing a theoretical basis for the clinical diagnosis of HFRS and the study of the pathogenesis of HTNV.1.Epidemiological and clinical characteristics of HFRS in Jilin Province: Through a single-center retrospective study,the clinical data of 2113 patients with HFRS in the First Hospital of Jilin University from 2012 to 2019 were collected,including onset-admission time interval,hospital stay,clinical manifestations(fever,headache,low back pain,orbital pain,skin flushing and bleeding spots,etc.),five-stage progression(fever,hypotensive shock,oliguria,polyuria and recovery),complications,laboratory tests and renal Ultrasound,hantavirus antibodies,prognosis and other related data,SAS9.3 software was used to statistically analyze the 7-year prevalence of HFRS and the changes in the severity rate.It was found that the prevalence of HFRS in Jilin Province was high in young adults and men,and advanced age was a risk factor for severe disease.The epidemic season shows an obvious bimodal trend,with the main peak from April to June,and the secondary peak from October to December,in which the severe HFRS rate is higher from October to December;in recent years,HFRS endemic areas have spread from mountainous areas to plains,and rural areas to cities.The severe rate is still the highest in mountainous areas,and the epidemic trend is mixed.Through multivariate Logistic regression analysis of clinical symptoms and auxiliary examination and other related data,it was found that the typical HFRS "three reds and three pains" combined with gastrointestinal symptoms are more prominent.It is an important factor in predicting severe HFRS.Leukocytes,platelets,blood urea nitrogen,creatinine,lactate dehydrogenase and α-hydroxybutyrate on the 4th to 7th day after virus infection,the severe group was significantly different from the mild group at each time point from 4 to 5 days Significantly,it was shown that leukocytes,platelets,blood urea nitrogen,creatinine,lactate dehydrogenase and α-hydroxybutyrate can be used as reference indicators for early prediction of disease severity.Abdominal ultrasound showed that severe patients were more prone to pancreatitis,ascites,and gallbladder wall edema,suggesting that they can be used as predictors of severe disease.Except for a slight increase in 2014,the case fatality rate showed a downward trend as a whole,which was lower than 1%,which may be related to the improvement of clinicians and early intervention.2.Identification of clinical inflammatory markers in HFRS: The clinical inflammatory markers(ferritin,PCT and CRP)of 373 HFRS patients from 2013 to 2019 were separately counted,and their relationship with disease severity,co-bacterial infection and prognosis was analyzed;The value of inflammatory markers in HFRS was analyzed using ROC curves for disease severity,co-bacterial infection or prognostic variables and quantified using SPSS 19.0 software to calculate the area under the ROC curve(AUC)and 95%confidence interval(CI).The results showed that the AUC values of PCT and serum ferritin for predicting the severity of HFRS were 0.824(95% CI 0.773-0.875,p<0.001)and 0.732(95%CI 0.678-0.786,p<0.001),respectively,while CRP predicted the severity of HFRS.The AUC value of 0.614(95% CI 0.552-0.676,p=0.001)indicated that PCT and ferritin were more meaningful in predicting the severity of HFRS.The AUC values of PCT and CRP for predicting bacterial infection were 0.527(95% CI 0.464-0.589,p=0.401)and 0.588(95% CI0.525-0.652,p=0.005),respectively.The results suggest that CRP is a better potential inflammatory marker than PCT for predicting co-bacterial infection in HFRS patients.The AUC values of serum ferritin,PCT and CRP for predicting mortality were 0.853(95% CI0.774-0.933,p<0.001),0.737(95% CI 0.587-0.887,p=0.002)and 0.703(95% CI 0.577-0.828,p=0.008),indicating that serum ferritin is the best inflammatory marker for predicting mortality in HFRS.Therefore,serum ferritin and PCT can be used as predictors for evaluating the severity and mortality of HFRS,and CRP is more meaningful in evaluating HFRS complicated with bacterial infection.3.Transcriptome sequencing analysis of HNTV structural proteins NP and Gc transfected cells: "cytokine storm" may be a key factor in the pathogenesis of hantavirus.This study intends to construct a GFP-tagged NP and Gc eukaryotic expression vector in vitro,and transfected it into human renal epithelial cells(HEK293T),using the GFP blank plasmid as a control,sorting GFP-positive cells for transcriptome analysis,and using bioinformatics to analyze the changes in the cell transcriptome caused by the overexpression of related proteins,to explore the mechanism of Hantavirus inflammatory storm.Transcriptome analysis of 9samples in NP group,Gc group and control GFP group,a total of 70.24 Gb of data was obtained,each sample reached more than 6.58 Gb,and the percentage of Q30 bases was more than 93.93%.This analysis detected a total of 35,304 expressed genes,including 34,838 known genes and 466 new genes;a total of 176,196 expressed transcripts,including 155,323 known transcripts and 20,873 new transcripts.Transcriptome differential gene analysis showed that there were 3329 differentially expressed genes(DEGs)in the transfected Gc protein group,of which 1567 DEGs were up-regulated and 1762 DEGs were down-regulated;there were 3595 differentially expressed genes(DEGs)in the transfected NP protein group,of which 1744 DEGs were up-regulated and 1851 DEGs were down-regulated.Compared with the GFP control group,the NP and Gc groups have many co-regulated genes,such as upregulated neurodevelopment-related genes Nefm and Ina,melanoma-associated antigen(MAGE)family genes Magea1,Magea2,Magea3,Magea6 and Magea2b;and the downregulated interferon signaling pathway-related genes Ifit2,Ifit3,Ddx58,Ifnb1,and Bst2,we speculate that hantavirus NP and Gc proteins inhibit interferon signaling pathway gene expression,making the virus easier to replicate in host cells and spread,which in turn stimulates immune cells to secrete more inflammatory factors,eventually leading to a "cytokine storm".4.Interferon inhibition mechanism of HNTV structural proteins NP and Gc: The results of transcriptome sequencing suggest that hantavirus NP and Gc proteins can inhibit the expression of genes related to the interferon signaling pathway.We verified the differentially expressed genes by quantitative PCR(q PCR)experiments.The results showed that Gc and NP could significantly inhibit the expression of Ifnb1,Ifit1 and Tfnsf4.In order to determine whether HTNV Gc and NP inhibit the antiviral response of interferon by targeting RLRs(including RIG-I and MDA5)signaling pathway,we used fluorescence quantitative PCR,western blotting,co-immunoprecipitation and other techniques to study the effect of HTNV Gc and NP on the interference The regulation of key target molecules(RIG-I and MDA5,TBK1/IRF3 phosphorylation,MAVS,TRIM25)of the IFN signaling pathway,the results show that HTNV NP and Gc can inhibit the production of interferon induced by RIG-I and MDA5,but not MAVS.The induced interferon production indicated that the target molecules of viral proteins were located upstream of MAVS.Finally,we found that HTNV Gc and NP inhibited the RLRs signaling pathway-induced type I interferon production by targeting the E3 ubiquitin ligase TRIM25.In conclusion,analysis of 2113 clinical cases of HFRS in Jilin Province found that advanced age,overlapping multiple stages,leukocytes,platelets,blood urea nitrogen,creatinine,lactate dehydrogenase and α-hydroxybutyrate can be used as reference indicators for early prediction of disease severity.Abdominal ultrasound showed pancreatitis,ascites and gallbladder wall edema,which can be used as risk factors for predicting severe HFRS;serum ferritin and PCT can be used as predictors for evaluating the severity and mortality of HFRS,and CRP in evaluating HFRS complicated with bacterial infection more meaningful.HTNV Gc and NP inhibit RLRs signaling-induced type I interferon production by targeting the E3 ubiquitin ligase TRIM25.The research results provide the basis for the clinical diagnosis,auxiliary examination,prognosis evaluation of HFRS and the pathogenic mechanism of HTNV.