Epidemiological Study Of Severe Fever Thrombocytopenia Syndrome Virus And Its Interaction With Autophagy

Background and Purpose:Severe fever with thrombocytopenia syndrome(SFTS)is an emerging infectious disease that was first detected in China in 2009 and subsequently reported in Korea and Japan.Symptoms of severe SFTS patients often develop rapidly.The case fatality of this disease is very high(5-50%)and till now there is no effective vaccine or treatment for SFTS.SFTS is prevalent in East Asian countries in areas where the climate is warm and humid and where bushes are abundant.Epidemiological studies have shown that SFTS mainly occurs in farmers with old age.The most important transmission pathway is being bitten by ticks,especially the Haemaphysalis longicornis.The pathogenic agent of the disease is SFTSV[Severe fever with thrombocytopenia syndrome phlebovirus,SFTSV).In addition to infecting humans,SFTSV can also infect a wide range of livestock(including goats,cattle,dogs,pigs)and wild animals(including Rattus norvegicus,Mus musculus,Apodemus agrarius and Acarus communis),which may be potential amplification animal hosts of SFTSV in nature.However,compared to domestic animals,the wild animals have a relatively high viral load and a long duration of viraemia,so it is speculated that they may be more effective animal hosts for SFTSV.However,there have been few studies on the infection of SFTSV in wildlife.The clinical manifestations of SFTS are mainly fever,thrombocytopenia,leukopenia,elevation of serum alanine aminotransferase(ALT)and other enzymes,accompanied with elevation level of a variety of serum cytokines including IFN-a,TNF-a and IL-10 etc.,and decreased T cells.These data all have a correlation with the prognosis of patients.SFTSV is a member of genus Phleboviridae,which belongs to the family Bunyaviradae.SFTSV is a segmented,negative-stranded RNA virus.The SFTSV RNA consists of three segments,S,M,and L,and they encode NSs(non-structure proteins),NP(nucleic protein),envelope glycoprotein(Gn/Gc)and RNA-dependent RNA polymerase,respectively.At present,researches on the pathogenic mechanism of SFTSV mainly focus on the study of the interaction between NSs protein and interferon-sinaling pathway;and the study on the molecular structures of NP and Gn/Gc protein.The mechanism of the interaction between SFTSV and autophagy has not been studied yet.Autophagy is a conserved cellular metabolic pathway in eukaryotic cells.It captures part of the cytoplasmic components or senescent organelles into autophagic bodies and transmits them to lysosomes,which eventually hydrolyze them into small fragments,which are recycled by cells.By this way autophagy plays an important role in maintaining cell homeostasis.Autophagy also plays a role in physiological processes such as cell starvation,morphogenesis,and cell differentiation,but also participates in cell pathological processes including viral infections,tumors,and neurodegenerative diseases.A number of studies have shown that autophagy plays an important role during the infection processes of various viruses.Not only can it play a role in the degradation of virus particles,but it can also help cells activate innate immunity and adaptive immunity.Moreover,autophagy plays an important role in affecting on virus replication,assembly,and release.However,the mechanism of interaction between autophagy and SFTSV has not been reported yet.In summary,the purpose of this study is as follows:1.To investigate the seroprevalence of SFTSV in hedgehogs in Shandong Province;2.To elucidate the effect of autophagy on SFTSV replication during SFTSV infection;3.To explain the influence of SFTSV infection on autophagy and the mechanism of interaction between SFTSV and autophagy;Methods:1.Exploration of epidemiological characteristics of SFTSV in hedgehogs:(1)Collection of serum samples:host animals were captured in Huangdao district,Qingdao city and Changqing district,Jinan city in 2014 and 2016,respectively.Heart blood samples were collected from the host animals after anesthesia,and stored at-80?.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect total SFTSV antibodies in serum samples;(3)Extraction of viral RNA from serum was performed,then reverse transcriptase polymerase chain reaction(RT-PCR)and nested PCR was used for detection of SFTSV M fragment.2.Mechanism of interaction between autophagy and SFTSV during infection:(1)Vero cells were infected with SFTSV or mock infected,then cells were harvested at 4 h,8 h,16 h,and 24 h respectively after infection.Western blot was used to detect expression of LC3 and NSs protein,and gray values of bands were calculated by ImageJ software.(2)Vero cells were infected with SFTSV for 12 hours.Then autophagy inhibitors pepstatin A and E64d were added to the culture system and 12 hours later cells were collected for protein analysis.The expression of LC3 protein was detected by western blot,and gray values were calculated by ImageJ software.(3)Vero cells were infected with SFTSV.Then cells were harvested at 6h,12h and 24 h after infection;immunofluorescence staining was performed after fixation,confocal microscopy was used to detect intracellular LC3 protein expression after virus infection.The LC3 expression was also detected at the same time in cells after being starved for 24h;(4)Vero cells were transfected with small interfering RNA(Si RNA)of LC3B gene for 24 h.Then cells were infected with SFTSV,and cell supernatant and cell pellet were collected at 24 h,48 h,and 72 h after infection,respectively.Western blot and real-time PCR were performed to detect LC3B protein expression and LC3B mRNA level in the cells respectively.At the same time,viral RNA was extracted from the culture supernatant;the SFTSV M fragment was amplified by real-time PCR to see whether decreased expression of LC3B protein had an influence on SFTSV replication;(5)Vero cells were infected with SFTSV for 24 hours.Cells were harvested,fixed and then immunofluorescence stained Confocal microscopy was used to observe NSs protein expression in the infected cells.(6)Vero cells were infected with SFTSV for 24 hours,and the cells were harvested and fixed for immunofluorescence staining.Confocal microscopy was used to observe the co-localization between NSs protein and LC3 protein.(7)The eukaryotic expression vector LC3B-GFP was transfected into Vero cells,and cells were infected with SFTSV 24 hours after transfection.Cells were harvested 24 hours after infection and fixed by immunofluorescence staining.Confocal microscopy was used to observe co-localization between NSs protein and LC3B-GFP.(8)Eukaryotic expression vector NSs-GFP was constructed,which was then transfected into Vero cells for 24h.Cells were fixed for immunofluorescence staining after transfection for24h.Confocal microscopy was used to observe whether exogenously expressed NSs could sequester LC3 protein;(9)Vero cells were infected with SFTSV for 24 hours,then cells were harvested and fixed for immunofluorescence staining.Confocal microscopy was used to observe co-localization between NSs protein and p62,Lamp1,Lamp2b,Rab5 and Rablla protein respectively;(10)The eukaryotic expression vector NSs-GFP was transfected into Vero cells.After 24 hours,cells were harvested and fixed for immunofluorescence staining.Co-localization between NSs-GFP protein and other proteins were observed by confocal microscopy.Results:2.Exploration of seroprevalence of SFTSV in hedgehogs:(1)In 2014,we captured 8 and 2 hedgehogs in July and October respectively in Huangdao district,Qingdao city,Shandong Province.In July 2016,we captured 4 hedgehogs in the Changqing district,Jinan city,Shandong Province,while none was captured in October.(2)Among the total 14 serum samples of hedgehogs that were captured,9 samples were positive of antibody against SFTSV,and the positive rate of serum antibody was 68.4%;(3)The SFTSV M fragment was not detected in all 14 serum samples of hedgehogs.2.Interaction between autophagy and SFTSV infection:(1)At 4h,8h,16h and 24h after infection,the expression of LC3-? in SFTSV-infected group was all significantly higher than that in mock infected group.The expression of NSs protein in infected group was gradually increased with the prolonging of infection time(within 24 hours).(2)No significant increase of LC3-? was observed in infected cells with autophagy inhibitors compared to those infected cells without autophagy inhibitors.(3)After infection of Vero cells with SFTSV,the LC3-high expressed structures were observed in infected cells.Observed under the microscope,it is a round solid structure,we call it LC3 positive bodies.Different from typical autophagosomes,the LC3 positive bodies were larger.And each infected cell had only 1-3 these structures.As infection time prolonged,the proportion of cells containing LC3 positive bodies was gradually increased(within 24 hours);(4)We successfully used LC3B SiRNA to interfere both LC3B protein expression and mRNA level in Vero cells.In Vero cells with decreased LC3B protein expression,the replication ability of SFTSV was significantly enhanced(5)After SFTSV infection of Vero cells,NSs protein was highly expressed in round solid structures in infected cells.We call this kind of structures the NSs positive bodies.The proportion of cells with NSs positive bodies gradually increased with the prolonged infection time(within 24h),and NSs protein in the bodies increased gradually.At 24 hours after infection,1-3 NSs positive bodies were seen in almost every infected cell(6)In SFTSV-infected cells,LC3 positive bodies completely overlapped with NSs-positive bodies,ie,LC3 protein and NSs protein completely co-localized;(7)In infected cells,NSs protein can also co-localize with exogenously expressed LC3B-GFP,further demonstrating that NSs protein can co-localize with LC3 protein in SFTSV-infected cells;(8)When NSs-GFP was exogenously expressed in non-infected cells,NSs-GFP could not colocalize with intracellular LC3 protein,indicating that NSs protein alone could not sequester LC3 protein;(9)In SFTSV-infected cells,NSs protein could co-localize with p62 and Lamp2b protein,but not with Lamp1,Rab5 and Rablla;(10)When NSs-GFP was exogenously expressed in non-infected cells,NSs-GFP could not colocalize with intracellular p62 or Lamp2b protein,indicating that NSs protein alone could not sequester p62 or Lamp2b protein.Conclusions:1 Hedgehogs captured in the wild has a high positive rate of SFTSV antibody in serum,which is significantly higher than that in other kinds of wild animals.Hedgehog may therefore be one of the main hosts of SFTSV;2 SFTSV infection in Vero cells can induce increased LC3-II protein expression,while autophagic flow may not be enhanced.3 LC3B protein could inhibit the replication of SFTSV,and this may be one of the mechanisms by which autophagy inhibit SFTSV replication.4 In SFTSV-infected cells,NSs protein can co-localize with LC3,p62 and Lamp2b,which may be the main mechanism of how virus escape from autophagy;5 Individual NSs-GFP proteins cannot colocalize with them,indicating that the co-localizations of NSs protein with these three proteins in SFTSV infected cells require participation of other viral proteins.