Construction And Characterization Of A Fluorescent Severe Fever With Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome(SFTS)is an emerging infectious disease caused by a novel bunyavirus(SFTSV).Currently,SFTS is mainly prevalent in China,Japan,South Korea,and other Asian countries.The main symptoms of the disease are fever and thrombocytopenia.In severe cases,patients could appear multiple organ failure and death.So far there have been no specific vaccines or drugs.Reverse genetics is an important tool in virological research and development of antiviral drugs and vaccine.In this thesis,we aim to use reverse genetics to construct a fluorescent SFTSV that can replicate efficiently in cell lines and without the non-structural protein NSs,and characterize the virus in vitro and in vivo.SFTSV-HBMC5,which replicates efficiently in cell lines,was chosen as the parental wild-type virus for reverse genetics.The sequences of the untranslated regions(UTR)of the L,M,and S fragments were obtained by 5’ and 3’ rapid amplification of cDNA ends(RACE),and used for constructing mini-replicon plasmids.In addition,helper plasmids expressing nucleocapsid protein(NP)and RNA dependent RNA polymerase(RdRp)were also constructed.The rescue plasmids containing full-length cDNA fragments of L and M were constructed at the same time.For the rescue plasmid of S fragment,NSs coding sequence was replaced by eGFP.The infectious virus SFTSV-delNSs-eGFP was successfully rescued by co-transfection of rescue plasmid and helper plasmids into BSR-T7/5 cells.The characteristics of SFTSV-delNSs-eGFP and wild-type viruses were analyzed in terms of plaque formation,one-step growth curve,and viral protein expression.The results showed that SFTSV-delNSs-eGFP has similar replication ability to that of the parental virus in Vero cells.Efficacy evaluation on antiviral drugs of Fapilavir(T-705)and chloroquine showed that SFTSV-delNSs-eGFP could be used for drug screening in vitro,and due to its green fluorescence,it was more suitable for high throughput screening of antiviral drugs.The infecting characteristics of SFTSV-delNSs-eGFP and the wild-type virus in interferon receptor deficient(IFNAR-/-)C57BL/6J mice were also analyzed.Unlike the fatal infection of the wild-type virus,no significant pathological changes were observed in major organs in mice.And no amplification of SFTSV-delNSs-eGFP was observed in our mouse model.The results prove that NSs protein is an important virulence factor of SFTSV,and its absence results impaired pathogenicity in IFNAR-/mice,laying a foundation for further application of the virus and the research on the function of NSs.In conclusion,we successfully constructed a SFTSV-delNSs-eGFP recombinant virus,which replicates efficiently in cell lines,but loses pathogenicity in the interferon receptor deficient mice.This virus provides an important tool for high-throughput screening of antiviral drugs,in-depth study on the function of NSs,and development of attenuated vaccines.