Construction Of Reverse Genetics Systems And Research On The Viral Genome Reassortment And Non-Coding Regions Of Emerging Bunyaviruses

Banyangvirus belongs to the order of Bunyavirales,family of Phenuiviridae and genus of Banyangvirus.Up to now,there are three types of banyangviruses released by the International Virus Classification Committee(ICTV),namely severe fever with thrombocytopenia syndrome virus(SFTSV),Heartland virus(HRTV),and Guertu virus(GTV),all of which are emerging bunyaviruses.Similar to other bunyaviruses,banyangvirus mainly consists of three negative strand RNA fragments,namely L segment,M segment and S segment.The viral genome segments are composed of open reading frame(ORF)and 5' and 3' untranslated region(UTR).Heartland virus is an emerging bunyavirus isolated and confirmed in the United States between 2009 and 2010.It is currently prevalent in the Midwest and Southern states of the United States.Heartland virus is mainly transmitted by tick bites.The clinical symptoms caused by HRTV are very similar to those caused by severe fever with thrombocytopenia syndrome bunyavirus,including fever,fatigue,decreased appetite,headache,nausea,diarrhea,muscle or joint pain,and the number of white blood cells and platelets reduced.At present,there is no effective vaccine or therapeutic drug for HRTV,and the reverse genetics system of HRTV has not been successfully constructed.In this study described here,we have successfully constructed HRTV L,M and S minigenome systems,combinatorial minigenome systems and combinatorial rescue systems based on T7 promoter and L protein and NP of SFTSV.Then with the reverse genetics systems,we have successfully rescued wild-type HRTV and the reporter virus without expressing NSs.The successfully constructed and applied HRTV reverse genetics system will provide a powerful tool for the molecular biology research on HRTV and the development of related vaccines and the screening of inhibitors or therapeutic drugs.Severe Fever with thrombocytopenia syndrome virus(SFTSV)is also an emerging bunyavirus isolated and confirmed in China in 2009.It is currently prevalent in some countries in East Asia,including China,Japan,South Korea and Vietnam.The spread of SFTSV is mainly through tick bites,and the lethal rate caused by SFTSV can reach to 6%-30%,and there are no effective preventive vaccines or therapeutic drugs currently.Although HRTV and SFTSV have a natural barrier of gene reassortmentt or recombination in geographical location,the viral genes of HRTV and SFTSV have high homology.With respect to the foregoing,exploring whether HRTV and SFTSV have the potential of gene reassortment can not only deepen our understanding of the evolutionary process of banyangviruses but also reminds us to pay enough attention to the threat caused by the potential gene reassortment or recombination risk of the emerging bunyaviruses.For segmented bunyavirus,gene reassortment and recombination within and between species are common in nature.In this study,the minigenme system,infectious virus-like particles(i VLP)system and viral superinfection method were used respectively to explore the substitutability of different components of the banyangviruses during the formation of virus RNP and the process of virus packaging,meanwhile we also revealed the different strength of each genome segment viral promoter activity of banyangviruses with these systems constructed.Minigenome system based on the mouse pol I promoter was first constructed for SFTSV and then we found that the L protein and NP of SFTSV can recognize the UTRs of HRTV L,M,and S to form combinatorial RNPs.These heterologous RNPs finally can be packaged by HRTV Gn / Gc into infectious virus-like particles that can infect fresh HEK-293 T cells.At last,we used HRTV and SFTSV L,M and S minigenome assays combined with viral superinfection to further investigate whether SFTSV and HRTV have the potential for gene reassortment in the context of viral infection.The results in our study show that the transcripts of all the minigenome plasmids above can be recognized by homologous or heterologous viral components to form RNPs and i VLP.In summary,this study shows that SFTSV and HRTV have the potential risk of gene reassortment.The UTR of each genomic segment of bunyavirus contains important elements including the viral promoter,polymerase binding site,transcription,replication,and viral packaging and regulatory sequences.But for banyangvirus UTRs,the elements that have an influence on the viral promoter activity and even the gene reassortment potential between viruses have not been studied.In this study,we used reverse genetics systems including minigenome and i VLP systems together with sequence and structure analysis software to analyze the UTRs of different banyangviruses including SFTSV,HRTV,and GTV.The results show that L and M UTRs of banyangviruses contain a conserved region(5 '13 nt,3' 12 nt)with a non-basepairing protruding nucleotide,namely 10 / 11 C;However,a conserved region does not exist for banyangvirus S segments.After experiments such as site-directed mutation,replacement and deletion of nucleotides,our results show that: 1)In the minigenome systems,for L and M of different banyangviruses,in addition to the conserved sequence,the nonbasepairing protruding nucleotide 10 / 11 C also has an significant effect on viral segment promoter activity;2)For S segments containing non-basepairing protruding nucleotide,when this nucleotide is deleted,the viral segment promoter activity is significantly reduced.As for the S segment in which there is no non-basepairing protruding nucleotide,when the protruding nucleotide is introduced at a specific position,the viral segment promoter activity increases significantly.