Study On Transmission Mechanism Of The Severe Fever With Thrombocytopenia Syndrome Virus And The Role Of The Inflammasomes And Pyroptosis In The Course Infection

Severe fever with thrombocytopenia syndrome virus(SFTSV)is a recently identified phlebovirus in the family Phenuiviridae.SFTSV infection can cause high fever,drastic loss of platelets and white blood cells,and is clinically called severe fever with thrombocytopenia syndrome(SFTS).In severe cases,bleeding and multi-organ failure may occur,resulting in a high fatality rate.Currently,SFTS is considered to be a zoonotic disease.However,its transmission vector and natural hosts are not clear,and its circulation model in nature is also lack of research.In this study,the epidemic characteristics of SFTS in Jiangsu and Anhui provinces were analyzed.Monitoring sites were set up in the epidemic areas of SFTS in Jiangsu Province to explore the circulating pattern of SFTSV transmission among humans,vectors and host animals.Our data showed that SFTS in eastern China is mainly prevalent in the border areas of Anhui and Jiangsu provinces.The circulating SFTSV genotypes in this region are mainly genotypes A and D,and their genomes have undergone gene recombination.For the first time in China's mainland,we have found the presence of gene E in Jiangsu and Anhui provinces.The viral nucleic acid or live virus strains have also been detected or isolated in larvae,nymph and adult H.longicornis collected from the endemic area of SFTS.This project has further worked out an experimental model of H.longicornis for examing the vector efficacy of H.longicornis in the transmission of SFTSV.The results showed that SFTSV was effective in infecting larvae,nymph and adult H.longicornis,and the infection rate was up to 50% after their biting mice during the viremia stage.Temale adult ticks infected with the virus could pass the virus through the eggs to the next generation of young ticks and the minimum infection rate(MIR)was 7.5%.In addition,artificially infected larvae,nymph and adult H.longicornis could transmit the virus to the mice through biting with high transmission efficiencies,and adult ticks could cause successful infection in up to 75% of mice.These above results re-confirmed that SFTSV could be transmitted by H.longicornis and the transmission could be vertical through eggs.We had also detected SFTSV nucleic acid and antibodies in many small and medium-sized wild animals,and isolated the viruses from the spleens or serum of hedgehogs and shrews(sorex araneus Linnaeus),suggesting that small and medium-sized mammals in nature could be the reservoir and amplification hosts of SFTSV.SFTSV antibodies were also detected from spotted doves and wild geese collected from Jiangsu and Anhui provinces.We also preformed an experimental infection with spotted dovesand found that SFTSV genotype A and E could infect the birds.The infection could cause viremia up to seven days post inoculation,which provided aboundant period for tick biting and being infected.The genotype E virus,which could be isolated both in the coastal region of China and in Japan,infected the birds resulting in a higher level of viremia in comparison to the genotype D virus,isolated mainly in early years in China,indicating that migratory birds could be involved in long distance transmission.This may help explain why the SFTSV could be spread so rapidly in the past decade througout China and to the other countries even across the East China SeaSFTSV causes viral hemorrhagic fever(VHF)in severe cases but the underlying pathogenesis of the syndrome has been poorly characterized.In this study we explored the pathogenesis of SFTSV infection and proposed that SFTSV activates inflammasomes and pyroptosis,which may have played a key role in SFTS pathogenicity.Clinically,the serum levels of pro-inflammatory factor interlukin-1?(IL-1?)significantly increased with SFTSV infection in patients.We infected C57/BL6 mice with SFTSV and found that the secretion of IL-1? in serum was also significantly elevated.At the cellular level we infected human peripheral blood monocytes(PBMC)with SFTSV and found that caspase-1 activation occurred and IL-1? secretion significantly increased as well.These results suggested that SFTSV infection may have activated inflammasome and promoted IL-1? maturation and secretion.To elucidate the molecular mechanism about how the inflammasome was activated during SFTSV infection,we used sh RNA,specific for the NLR family receptors NLRP1,NLRP3,NLRC4 and AIM2,respectively,by constructing lentiviral plasmids to knock down the four corresponding receptors in PBMCs.We found that after the knockdown of NLRP3,caspase-1 activation and IL-1? secretion were significantly reduced.We further applied the NLRP3 specific inhibitor,glibenclamide,to block NLRP3 activation in PBMCs and found that the secretion of IL-1? decreased significantly when infected with SFTSV,and the replicaton of the virus increased.We used specific inhibitor Ac-YVAD-cmk to block caspase-1 cleavage in PBMCs and C57/BL6 mice,and found that the secretion of IL-1? decreased while the replication of the virus increased both in PBMCs and mice.Increased cell damage and death occurred when PBMCs were infected with SFTSV,and the pyroptotic executioner GSDMD was activated,indicating the occurrence of pyroptosis.In summary,our data indicated that SFTSV infection in PBMCs and mice activated NLRP3 inflammasomes,promoted IL-1? secretion,and induced pyroptosis.Increased IL-1? secretion and pyroptosis played a key role in host pathogenicity and innateimmune defense.Analysis of clinical data showed that serum IL-1? levels were negatively correlated with the severity of symptoms and serum viral load(P<0.001).Therefore,inflammasome and pyroptosis may have involved in both pathogenicity andresistance to SFTSV infection in patients.This study provides a new perspective to understand the pathogenesis of SFTSV in which inflammasome activation and pyroptosis appeared to play an important role.