Identification Of Major Target Cells For SFTSV Infection In Human Peripheral Blood

BackgroundSevere fever with thrombocytopenia syndrome virus(SFTSV)is a novel bunyavirus that was first discovered in China in 2010.Infection of SFTSV causes severe fever with thrombocytopenia syndrome(SFTS),an emerging tick-borne hemorrhagic fever disease.Most patients have a history of tick bites within two weeks before the onset of the disease.The main clinical characteristics include acute fever,malaise,myalgia,gastrointestinal symptoms,leukopenia and thrombocytopenia.SFTSV can affect almost all age groups,especially the elderly population,which is more threatening and has a higher fatality rate.The incidence of SFTS is increasing worldwide,including China,Japan and Korea,and has caused serious public health problems.The key features of SFTSV infection are the persistent replication of the virus,severe reduction of platelets and MODS.Preliminary studies have been conducted to explore the potential pathogenic mechanisms of SFTSV.however,The pathogenic mechanism of SFTSV remains unknown.Up to now,SFTS patients are mainly treated with supportive treatment,and there is no approved specific effective antiviral drugs.The binding of the virus to the surface molecules of the host cell is the first step for virus infection,and the selectivity of virus to infect target cells makes the virus only infect certain tissues or organs.Thus the identification of the target cells for virus has become a key problem.In the process of arbovirus infection,the virus through the tik bites on the skin enter into the blood and fights with the immune system to determine the pathogenesis of the organism,determines the pathogenesis of the body after a struggle with the body’s immune system.Currently,target cells for SFTSV infection and the pathogenesis have been investigated exploratively by in vitro cell lines,autopsy samples from a few deceased patients,and animal models of immunodeficiency;however,little research has been focused on peripheral blood immune cells.Studies have revealed that patients with severe SFTS displayed persistent high serum viral load,and changed composition of white blood cell subsets in peripheral blood,suggesting that peripheral blood cells may serve as target cells for SFTSV infection.Recent studies have shown that SFTSV may be sexually transmitted,and it remains to unkown whether white blood cells can carry SFTSV as a vehicle for transmission of the virus across the testicular barrier.Therefore,determining whether peripheral blood leukocytes can be infected by SFTSV is of importance for the prevention and treatment of SFTS.MethodsWe collected fresh peripheral blood samples from SFTS patients at the 990th PLA Hospital in Xinyang City,Henan Province,the designated hospital for SFTS treatment.We detected SFTSV infection rate in five major immune cells,T cells,B cells,NK cells,monocytes and neutrophiles,through using Flow Cytometry(FCM),and compared the changes in the composition of the five immune cells in pheripheral blood.We also collected demograhic information,clinical and laboratory data and follow-up outcomes of SFTS patients,and then analyzed the relationship between the feature of peripheral blood target cells and disease progression,prognosis of SFTS patients.Based on the identification of target cells for SFTSV infection in peripheral blood of SFTS patients,healthy volunteers were also recruited for in vitro infection of fresh peripheral blood,and flow cytometry was used to detect the SFTSV infection rate.At the same time,the isolated PBMCs and purified primary monocytes of healthy volunteers were used for in vitro infection.Flow cytometry was used to analyze the infection rates of lymphocyte and monocyte populations as well as changes in the composition of monocyte subsets.ResultsBy flow cytometry analysis of T cells,B cells,NK cells,monocyte,and neutrophiles SFTSV infections rate in peripheral blood of 15 SFTS patients,we found that the highest SFTSV infection rate in monocytes(14.86%±9.127.64%),compared to T cells(0.77%±0.74%),B cells(2.50%±1.60%),NK cells(0.68%±0.91%),and neutrophiles(0.00%±0.00%)(P<0.05).Moreover,the percentage of neutrophiles to leukocytes in peripheral blood was higher in SFTS patients compared to healthy controls(P<0.001),while the percentage of T cells,B cells,and monocytes to leukocytes was lower in SFTS patients(P<0.001,P<0.05,and P<0.001;respectively).The infection rate of SFTSV in monocytes was found to be positively correlated to viral load(R~2=0.570,P<0.001)and age in SFTS patients(R~2=0.97,P<0.001).We also collected Peripheral blood mononuclear cells(PBMCs)from 98 SFTS patients and 39 healthy controls to detect changes in the proportion of monocyte subpopulations.We found that the proportion of classical monocytes(CD14~+CD16~-)was decreased(50.05%±2.79%vs.91.74%±3.71%,P<0.001)and the proportion of intermediate monocytes(CD14~+CD16~+)was increased in SFTS patients compared to healthy controls(46.10%±21.06%vs.3.95%±2.08%,P<0.001);the proportion of intermediate monocytes was higher in the deceased patients compared to the survivors(61.01%±20.30%vs.42.17%±19.34%,P<0.001).In addition,the higher the viral load or age of SFTS patients,the higher the proportion of intermediate monocytes in monocytes(both P<0.05).Peripheral blood samples from 13 healthy donors were subjected to in vitro SFTSV infection,and the infection rates of five major immune subpopulations(T cells,B cells,NK cells,neutrophils and monocytes)in peripheral blood were analyzed by flow cytometry.Compared to the infection rates of T cells(0.09%±0.30%),B cells(1.45%±1.18%),NK cells(0.08%±0.27%),and neutrophils(0.00%±0.00%),we found the highest infection rate of SFTSV in monocytes(8.58%±2.93%),which was consistent with the results of in vivo study.Isolated PBMCs(n=13)and purified monocytes(n=8)from healthy donors were used to analyze the changes in the proportion of monocyte subpopulations after SFTSV infection,and we found that the higher proportion of intermediate monocytes was related to the inoculated concentration of SFTSV and the older age in the healthy donor(both P<0.05).Isolated PBMCs(n=8)were also infected with SFTSV,and flow cytometry was used to sort out the classic monocytes and intermediate monocytes from monocytes to detect the intracellular SFTSV titer,and it was found that the SFTSV titer in intermediate monocytes was higher than that in classic monocytes(P<0.05).ConclusionThe major target cells for SFTSV infection in human peripheral blood were monocytes,followed by B cells.SFTSV infection induced the change of the composition of monocyte subsets,with a significantly increased proportion of intermediate monocytes and a high viral titer in intermediate monocytes.The high infection rate of monocytes was positively correlated with the viral load and age of SFTS patients.Through the follow-up of SFTS patients,we demonstrated that the phenotypic shift from classical monocytes to intermediate monocytes was related to fatal SFTS,indicating the important role of monocytes in the progression of SFTS.