Preparation And Application Of CCHFV TecVLP

Crimean-congo hemorrhagic fever virus（CCHFV）is highly infectious and lethal.According to the classification of ICTV,CCHFV belongs to the order Bunyavirales,Nairoviridae,and Orthonairovirus^[1].Crimean-congo hemorrhagic fever（CCHF）,an acute infectious disease,is widely prevalent in Africa,Eastern Europe and the Middle East.It is a tick-borne natural epidemic disease^[2].Clinically,the disease is characterized by fever,gastrointestinal bleeding,hematemesis,hypotension and shock,with a high fatality rate of 3-30%^[3].In 1965,this disease was first discovered in Bashu,Xinjiang,China,and is also known as Xinjiang Hemorrhagic Fever（XHF）^[4].In addition,antibodies against the disease were found in Anhui,Qinghai,Sichuan and other regions of China,indicating that the disease may not be limited to Xinjiang in China^[4].At the same time,on a global scale,involved in the disease area,there are many countries belong to our country economic belt"silk road"advocated by the country,along with the global economic integration and increasingly frequent international exchanges,tourism,trade and overseas,our country also always faced the threat of CCHF input sex,so the control task is very difficult.Currently,there is no specific vaccine or drug for the prevention and treatment of CCHFV.In the aspect of vaccine research and development,it mainly includes the development of traditional vaccine--inactivated vaccine and new vaccine^[5].In terms of drug use,the drugs currently used for clinical treatment of CCHF are mainly broad-spectrum drugs,such as ribavirin,etc.,while general supportive therapy cannot specifically and effectively exert the best efficacy.An important reason for this situation is that CCHFV has a high safety level,requiring b SL-4 biosafety conditions to conduct live virus research,but most laboratories around the world do not have high biosafety conditions.Therefore,this paper intends to find a safe and reliable substitute by means of experimental technology,and treat it as a"virus"for vaccine development and drug screening and other related research.The substitution is transcription and entry-competent virus-like particles（tecVLP）.TecVLP consists of virus-like particles（VLP）and virus microgenomes.VLP contains no viral nucleic acid and is formed by self-assembly of viral structural proteins.Microgenomes include microgenomic plasmids,which replace viral genes with reporter genes to construct recombinant plasmids,and helper plasmids,which insert structural protein genes into eukaryotic expression vectors to construct recombinant plasmids.When tne VLP is combined with the microgenome,the tecVLP formed is similar to the morphology and structure of virus particles,and the virus genome is destroyed,so it does not have the ability to self-replicate and has high safety.At the same time,the tecVLP has the ability of single entry and transcription and translation.Therefore,this project intends to obtain CCHFV tecVLP through experimental technology to replace live virus for vaccine evaluation and drug screening.In this study,microgenomic plasmids carrying Nano Luc,helper plasmids expressing L,M and S genes,and PCAGGS-T7were constructed by p CAGGS vector.After plasmid construction and identification,the results of co-transfection of microgenomic plasmid and auxiliary plasmid indicated that the microgenomic system was correct,and cells should be lysed for detection during subsequent identification.After the microgenome system was verified to be correct,tecVLP was initially packaged.On the basis of the four plasmids mentioned above,recombinant plasmids expressing envelope glycoproteins were added for co-transfection.The results showed that intracellular samples should be collected 72h after transfection,and the auxiliary plasmid should be pre-transferred for identification,and detected 48h after infection.On this basis,a large amount of CCHFV tecVLP was packed,concentrated and its titer was determined.In addition,tecVLP particles were observed by electron microscopy.Huh7 cells and C57 mice were infected with packaged CCHFV tecVLP,respectively.The luciferase kit and Q-PCR assay showed that CCHFV tecVLP cells and animal infection models were successfully constructed,and the benign results were obtained by cell imaging.On this basis,we used cell platform and animal platform to evaluate whether four drugs,itraconazole,sorafenib,chlorpromazine and chloroquine,could inhibit tecVLP entry and transcription translation.At the same time,the neutralization efficacy of CCHFV Gc,Gn and NP vaccines was detected by tecVLP.The results showed that,through the above platform,we successfully screened the drugs that could inhibit virus entry and transcriptional translation ability,and evaluated the neutralizing antibody titer and animal protection effect of the vaccine.In this paper,through the expression of CCHFV tecVLP and the establishment of infection platform in cells and animals,it is finally successfully applied to the evaluation and drug screening of CCHFV vaccine.This study will lay a solid foundation for the development of CCHFV vaccine and the extensive development of drug screening,and ultimately serve the prevention,control and treatment of CCHF.