Complete Genome Sequence Analysis And Construction Of A Full-length CDNA Clone Of Cell Fusing Agent Virus

Cell fusing agent virus,(CFAV)is a kind of mosquito-borne viruses.CFAV and dengue virus,an important human pathogen,both belong to flavivirus,flaviviridae.Similar to dengue virus and yellow fever virus,the genome of CFAV is also composed of coding region,5' untranslated region(5?UTR)and 3'untranslated region(3?UTR).The coding region codes 10 kinds of proteins in total,including 3 structural protein(C,pr M/M,E)and 7 non-structural protein(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5).Cell fusing agent virus was named for causing Aedes albopictus cells to fuse.This virus was first found in laboratory-grown Aedes aegypti cells.Later on,researchers also isolated the same virus in natural mosquitoes.Genome evolution analysis indicates,Cell fusing agent virus has some similarities with dengue virus and yellow fever virus.Compared with other flaviviruses,cell fusing agent virus is specific for mosquitoes,so that it cannot infect vertebrate or replicate in other animal cells except for mosquito cells.According to research,there is coinfection phenomenon of cell fusing agent virus and dengue virus in natural mosquitoes.The influence of coinfection on the intracellular replication and epidemiological distribution of dengue virus is not clear yet.So far,research of cell fusing agent virus mainly focuses on isolation and identification of virus and evolution analysis,and little attention is paid to its molecular mechanism or biological significance.This is due to lacking technique for manipulating the virus directly on gene level reverse genetics system.And the first step of constructing reverse genetics system is to construct the full-length clones of c DNA cell fusing agent virus.Reverse genetics system is to assemble active individuals and study structure and function of organism genome by modifying the target gene,such as insert/delete site-directed mutated genes and gene replacement,then constructing modified genomecontaining necessary elements for organism on the basis of getting full-length gene sequence of organism genome.RNA reverse genetics system is salvaging living virus from susceptible cells using the genetic material of virus.The procedure is to insert c DNA copy of the whole virus genome into bacterial plasmid in order to make c DNA itself or RNA transcribed in vitro from c DNA infectious.Reverse genetics system of RNA virus can detect the phenotype of the salvaged modified virus by directional modifying the genome sequence of virus and can study virus gene structure,function and virus-host cell interaction in vivo efficiently.Now reverse genetic technique has been very mature.Many full-length infectious clones of RNA virus have been successfully constructed.Constructing reverse genetics system of cell fusing agent virus successfully enables manipulating the genes directly and plays an important role in understanding and modifying virus.Furthermore,it can serve as a tool for studying the mechanism of virus-specific infection.This study first constructed phylogenetic tree of 23 flaviviruses and clarified the evolutionary relationship between cell fusing agent virus and other flaviviruses.The genetic sequence differences between cell fusing agent virus and Zika virus,kamiti river virus,Chaoyang virus was mainly compared.Meanwhile,homology comparison was performed on its structural protein and non-structural protein.Furthermore,we analyzed the gene sequences of 2 cell fusing agent viruses from Genbank and designed primers and probes for q RT-PCR after getting its conserved sequence.Using the primers and probes we can detect the genome of cell fusing agent virus specifically and efficiently.It does not cross-react with Zika virus or Japanese encephalitis virus and thus provided efficient technique for detecting cell fusing agent virus.Later on,we got full DNA sequences of virus with total genetic synthesis method in vitro and ligated them into complete full-length c DNA of cell fusing agent virus in vitro.This study established the base for constructing reverse genetics system of cell fusing agent virus.This study is composed of the following 3 parts:1.Whole genome sequence analysis of cell fusing agent virus:We downloaded 3 of cell fusing agent virus sequences via Genbank database.Two gene sequences were completely identical.Our study also picked whole gene sequences of other 21 flaviviruses and constructed phylogenetic tree with cell fusing agent virus.We identified the position of cell fusing agent virus in phylogenetic tree by contrastive analysis and then compared the C,pr M/M,E,NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5 protein sequences and amino acid sequences of cell fusing agent virus and Zika virus,kamiti river virus,Chaoyang virus.We found that although cell fusing agent virus was close to Chaoyang virus in evolutionary relationship,its genetic homology with Zika virus was 41.2%,and its genetic homology with Chaoyang virus was 40.7%.The amino acid sequence homology between cell fusing agent virus and Zika virus was25.4 % while its amino acid sequence homology with Chaoyang virus was 25.5 %.After predicting the secondary structure of non-coding region of cell fusing agent virus,we found the length of 5'-UTR of the non-coding region of Galveston cell fusing agent virus was 103 nt.Its 5'-UTR contained cap structure and 2 conservative stem loop structures(SL): SLA and SLB.the length of 3'-UTR of cell fusing agent virus was553 nt.It contained multiple conservative liner sequences and secondary structures,which were divided into A1,A2 and A3 structural domains.We predicted the function of secondary structure of cell fusing agent virus by comparing with the structures of5'-UTR and 3'-UTR of type 2 dengue virus.2.Constructing detecting method of cell fusing agent virusCell fusing agent virus was first found in laboratory-grown Aedes aegypti cells by Stollar and Thomas.Further analysis indicated that the infection of cell fusing agent virus to Aedes albopictus cells can induce cell fusing phenomenon and that the fusing capacity induced by cell fusing agent virus only exists in Aedes albopictus cells.We analyzed the gene sequences of 2 cell fusing agent viruses from NCBI,got theconservative sequences from the NS2 A gene sequence and designed primers and probes for q RT-PCR.We used standard plasmids containing cell fusing agent virus NS2 A gene sequence to detect the sensitivity of the PCR primers and probes we designed and the result was positive.The lower limit of detection was 150 copies/ml.We used type 2dengue virus,Zika virus and Japanese encephalitis virus to detect the specificity of the PCR primers and probes we designed,the results were all negative.The results above indicated that the detecting method we designed had a good sensitivity and specificity.Based on that,this study analyzed whether the Aag2 and C6/36 cells of our laboratory contain endogenous cell fusing agent virus.We detected the RNA of the Aag2 and C6/36 cells of our laboratory using the RT-PCR primers we designed and the result indicated that no endogenous cell fusing agent virus exists in the Aag2 and C6/36 cells of our laboratory.3.Construction of full-length c DNA clones of cell fusing agent virus genomeAccording to the whole genomic sequence of Galveston cell fusing agent virus,we got 3 DNA fragments covering the whole gene of virus by gene synthesis in vitro and named them A,B and C.The length of A,B and C were1-3414 nt,3400-6596 nt and6591-11395 nt.Each fragment was cloned in p BR002 vector and was named as p BR002-A,p BR002-B and p BR002-C separately.First,we used restriction enzyme Xba I and Age I to digest p BR002-B and p BR002-C fragments,then ligated B and C?using T4 DNA ligase and select the p BR002-BC? clones containing BC? fragment.We ligated A? fragment,which was the PCR products of p BR002-A using Not IF and Bst171 IR primers,with p ACNR vector and got p ACNR-A plasmid.We digested p ACNR-A and p BR002-BC?with Xba I and Bst171 I and got CFAV-p ACNR plasmid,which contained full-length c DNA clone of cell fusing agent virus,after ligating the digested BC fragment and p ACNR-A.After identified by enzyme digestion Sna I and sequencing analysis,the full-length c DNA clone of cell fusing agent virus was obtained.We provided basic data for research of cell fusing agent virus via constructing phylogenetic tree of cell fusing agent virus and analyzing the whole genome.Based on the sequence analysis,we designed specific q RT-PCR primers and probes for cell fusing agent virus and evaluated the sensitivity and specificity of the primers.We obtained full-length c DNA clone of cell fusing agent virus,and thus established a solid foundation for following study of virus replication and molecular mechanism.