The Infection And Replication Mechanism Research Of Tick-borne Banyangvirus

Tick-borne viruses are a large group of viruses with rich genetic diversity,including a variety of viral pathogens with infectious pathogenicity.In 2009,severe fever with thrombocytopenia syndrome virus(SFTSV)infection was found and reported in the central and eastern part of China.By 2016,SFTSV has infected more than 7000 cases in China,with an average fatality rate of 5.3%.Shortly thereafter,two fatal cases of human infection by Heartland virus(HRTV)were reported in the United States,and the virus was isolated from the patient's serum.So far,more than 30 people have been infected and 4 have died in the United States.In 2014,a new virus,Guertu virus(GTV),was isolated from Xinjiang,China.Although there are no reports of human infection,serological investigations and pathogenicity of animal infection suggest that GTV is a potential pathogen.SFTSV,GTV and HRTV all use ticks as vectors and are closely related.They belong to the genus Banyangvirus of the Phenuiviridae family.In this study,Banyangvirus was taken as the research object,and the mechanism of infection and replication of Banyangvirus was preliminarily explored through reverse genetic system.The relativity among SFTSV,GTV and HRTV was analyzed from the perspective of viral characteristics and protein function.The efficacy of antiviral drugs targeting at nucleoprotein(NP)was tested to further analyze the replication and infection of Banyangvirus.The pathogenic case of SFTSV was investigated and analyzed to reveal the possible infection risk of SFTSV.This study established reverse genetic system of Banyangvirus,and laid an important technical means for further research on the replication and transcription mechanism of virus infection based on this system.In chapter 1,the basic characteristics of SFTSV,GTV and HRTV viruses are briefly introduced,including its feature in infection replication and molecular biology.Subsequently,this chapter introduces the research progress of reverse genetics of Bunyaviridae and discusses the development progress of antiviral drugs of Bunyaviridae.At the end of this chapter,we summarized the content and significance of this thesis.In chapter 2,we established a novel reverse genetic system of GTV.This study is based on the the constructed three segments containing T7 RNA polymerase and RNA polymerase I,and constructed expression plasmids stably expressing GTV NP and RdRp protein.The recombinant plasmids were co-transfected into target cells.After 4-5 days,the supernatants of cultured cells were transferred into Vero cells,the expression in GTV L,M and S genes was detected by Nested PCR,and immunofluorescence assay showed that GTV NP protein could be detected.These results indicated that the recombinant virus was rescued successfully.This means the reverse genetic operation system of GTV has been successfully established.The establishment of reverse genetics system can provide a platform for the research of viral genome function,molecular pathogenesis,new vaccines and new RNA viral vectors.In chapter 3,we summarized the activities of GTV,SFTSV,and HRTV minigenome in the presence of NP and RdRp proteins from cognate or heterologous viruses.These viruses have a tripartite negative-sense RNA genome,if two or more closely related viruses co-infect the same host or vector cells,any RNA fragment of these parental viruses may be incorporated into the progeny virions to produce reassortant viruses.However,little is known about the reassortant ability of tick-borne viruses.We used the minigenome systems of these three viruses to assess the abilities of viral NP and RdRp proteins to recognize,transcribe,and replicate minigenome of heterologous virus.The results showed that several combinations of NP and RdRp proteins of heterologous viruses resulted in the minigenome activity,which proved that the untranslated regions(UTRs)of viral genome were functional promoters for the transcription and replication of NP and RdRp proteins of related viruses.These results show that co-infection of these viruses may lead to viable reassortant progeny.Therefore,this study is aid in elucidate the role of genome reassortant in the evolution of these emerging pathogens in experimental conditions.In chapter 4,we aimed at designing small molecular inhibitors for NP protein space structure,and screened out drugs with antiviral activity.NP is the most abundant viral protein in virions and infected cells.It encapsidates the viral genomic RNA(vRNA),resulting in the formation of ribonucleoprotein complexes(RNPs).In addition to its critical role in protecting the vRNA,NP also plays an active role in RNA transcription and replication,as well as in virion assembly.SFTSV NP shows a unique bilobed architecture.the hexameric ring of NP is assembled by monomer.Oligomerization is mediated by amino and carboxy terminal arms.The RNA binding site is located at the lobe intersection.So,according to the structure of NP,we designed some Antivirus drugs.AMG837 can recognize each other with carboxyl arm,and competitively inhibit oligomerization of NP,while CY1 can recognize RNA binding sites and competitively inhibit the binding of NP protein to viral vRNA,thus inhibiting the transcription and replication of viruses.This chapter lays a foundation for screening potential drugs with effective antiviral activity.In chapter 5,we reported the first laboratory-confirmed SFTS case in 2017.A resident of Hainan International Tourism Island(HNITI)was bitten by ticks when traveling in XJUAR and had illness onset after returning to HNITI.RT-PCR detected SFTSV RNA in the patient's serum samples.Antibodies against SFTSV were detected from the patient and the neutralization from serum samples was evaluated.Moreover,a new SFTSV strain was isolated from the serum sample collected from the patient during acute phase of disease.In addition,SFTSV was detected positive in ticks collected from XJUAR in 2017,which suggested that SFTSV was more widely distributed than we recognized.Therefore,this study identified the first SFTS case from XJUAR where confirmed cases have never been reported and demonstrated the substantial risk from SFTSV infection via tick bite there.It is also the first importing SFTS case in HNITI,which showed the significant role of human transport in disease spread and indicated that the recently authorized international tourism island may face more challenges for controlling other importing cases from different areas and countries.Chapter 6 We overviewed this thesis and made a further discussion.In this study,the reverse genetics system of GTV and SFTSV was constructed to verify the minigenome activity of the virus in the presence of NP and RdRp proteins of homologous or heterologous viruses.Potential antiviral drugs targeting the structure of NP proteins were screened to provide a platform for future research on the genomic function,infection and replication mechanism of the virus and vaccine preparation.