Diagnostic Value Of Yellow Fever Virus Nonstructural Protein 1(YFV-NS1)in The Early Stage Of Infection And Preparation Of Anti-YFV-NS1 Monoclonal Antibody

Yellow fever(YF)is an acute infectious disease relying on mosquito vectors for transmission.The predominant endemic area is in tropical and subtropical regions of Africa and South America.Most infections manifest mild symptoms and self-limiting,However,progressed cases was about one-fifth,with a mortality rate of 20%to 50%.Till now,safe and effective treatment measures are unavailable.Therefore,to prevent and control the epidemic mainly relying on mass vaccination.In recent years,as the coverage of vaccination reducing and population flow for international trade and tourism increasing,yellow fever re-emerged in epidemic areas of the southern United States,East Africa,enen in the previously unaffected areas of the South and Central America.Though there haven’t yet outbreak in Asia,imported cases of yellow fever were found by China Customs in 2016.Additionly,it was reported that YFV could be carried by Asian strains of Aedes aegypti,which was common in the tropical and subtropical regions of south China.Therefore,the risk of yellow fever transmission in China cannot be ignored.Early diagnosis and isolation of infected patients is critical to prevent the yellow fever virus(YFV)spreading.Currently,the laboratory diagnosis of yellow fever virus included viral nucleic acids mostly by RT-PCR,serological detection of IgM-specific antibodies and virus isolation.There were somewhat limitations in these detection methods for early diagnosis of yellow fever virus infection.It was necessary to develop a detection method with good sensitivity and specificity,high throughput,as well as convenient and economical,for screening the yellow fever virus infection,especially in dealing with the emerging infectious disease.Yellow fever virus is member of the Flavivirus genus of the family Flaviviridae.YFV is a small enveloped virus with an 10862 bp single-stranded positivesense RNA genome encoding 3 structural proteins(E,M,C)and 7 non-structural proteins(NS1,NS2A,NS2B,NS3,NS4A,NS4B,NS5).NS1 is an secretory glycoprotein,existing predominantly as a dimer in the membranes of cell infected,and secreting from it as a soluble,detergent-labile hexamer.At present,NS1 protein has been successfully used as a detection target for the early diagnosis of West Nile virus,dengue virus,and Japanese encephalitis virus infection.However,little is known about the dynamics of NS 1 secretion in animal and YFV infected cells,both at home and abroad.In this study,we aim to validate whether NS1 protein could be used as a biomarker for the early diagnosis of YFV infection,to illustrate the dynamics of NS1 secretion,and to consequently prepare anti-YFV NS1 monoclonal antibodies for developing immunoassay method.Briefly,the recombinant plasmid pQE30-YFV NS1 was constructed and successfully expressed NS1 protein in prokaryotic expression system.After infected by YFV 17D,YFV NS1 protein and YFV RNA in both the susceptible cells(C6/36 and Vero)and suckling mice models,were detected by NS1 capture enzyme-linked immunosorbent assay(ELISA)based on anti-YFV NS1 polyclonal antibodies and real-time RT-PCR respectively.As YFV NS1 protein was confirmed to be used as a biomarker for the early diagnosis of YFV infection,the monoclonal antibody against YFV NS1 was successfully prepared by hybridoma cell technology,which was helpful for developing immunoassay method of YFV NS1 detection in the further study.This work included three parts:First,the expression and immunity identification of yellow fever virus NS1 protein.YFV NS1 gene fragment was amplified by RT-PCR using YFV 17D vaccine RNA as a template.The NS 1 amino acid sequences of the YFV wild-type virus strains and YFV 17D vaccine strain were aligned and confirmed to have high homology.The YFV NS1 gene fragment was introduced into the prokaryotic expression vector pQE30,and the recombinant plasmid was verified to be correctly connected and transferred into the competent cell E.coli M15.The YFV NS1 recombinant protein was induced by IPTG induction.The recombinant protein was denaturalized and dissolved,purified and recovered.The concentration of soluble YFV NS1 protein was 0.3 mg/mL after dialysis..The antigenicity of the protein was identified by indirect ELISA and Western blotting.The results showed that the YFV NS1 recombinant protein reacted significantly with ascites in mice immunized with yellow fever virus,suggesting that although the spatial conformation of the NS1 recombinant protein is different from native NS 1 protein.However,recombinant NS 1 proteins still retain some activities and functions of natural NS1 proteins.In addition,the recombinant YFV NS1 protein cross-reacts with several other flaviviruses(1-4 Dengue virus,Japanese encephalitis virus,West Nile virus,Zika virus)immune ascites and immune sera,suggesting that YFV NS1 protein is structurally similar and conserved.Second,the secretion law of yellow fever virus NS 1 protein.Ten mice were immunized with the renatured YFV NS1 recombinant protein and anti-YFV NS1 polyclonal serum was collected.Identifying the titer and immunity of mice serum.The results showed that the polyclonal antibodies in the serum of the Balb/c mice immunized with YFV NS1 not only reacted with YFV NS1 recombinant protein,but also reacted with NS1 protein in YFV infected C6/36 cell ultrasound supernatant.Ten mice polyclonal sera were randomly divided into 2 groups.After purification,anti-YFV NS1 polyclonal antibody was prepared,one group was used as capture antibody,and the other group was labeled with horseradish peroxidase(HRP)as detection antibody.Multiple anti-sandwich ELISA method.The detection limit of YFV NS1 recombinant protein was approximately 200 pg/mL,and the minimum detection limit of YFV infected C6/36 cell supernatant was approximately 400 pfu/mL.YFV virus replication and NS1 protein secretion in infected C6/36,Vero and suckling mice was detected by this ELISA method and real-time fluorescence quantitative RT-PCR.The results showed that the YFV NS1 protein was detected in the C6/36 and Vero cells culture supernatant 36 hours after infection of the virus,and the YFV nucleic acid was detected 48 hours after the virus infection of the C6/36 and Vero cells;after 2 days of virus infection,YFV NS1 protein can be detected in some mice blood,no YFV nucleic acid was detected;after 3 or 4 days of infection,2 mice had no YFV nucleic acid detected in the blood,but serum could detect low concentration of YFV NS1 protein.It was demonstrated that NS1 protein was secreted from the supernatant of YFV infected cells and animal serum,and the detection of NS1 was slightly earlier than that of YFV.The YFV NS1 protein has potential value for the early diagnosis of YFV infection.Third,the preparation and identification of yellow fever virus NS1 protein monoclonal antibody.The mice were immunized five times with YFV NS1 recombinant protein and the sera titer of the immunized mice was determined by indirect ELISA.When the titer reached 1:500000,the mouse spleen cells were electrofused with mouse myeloma cells SP2/0 to prepare hybridoma cells.The positive clones were screened by indirect ELISA and then sub-cloned by limited dilution method.Finally,12 cell lines secreting anti-YFV NS1 monoclonal antibody were screened out.The subclasses of 12 monoclonal antibodies were identified.The results showed that 8 monoclonal antibodies were IgGl,2 monoclonal antibodies were IgG3,and 2 monoclonal antibodies were IgG2b.In addition,the specificity of the 12 monoclonal antibodies was identified by indirect immunofluorescence and western blotting.The results showed that the 8 monoclonal antibodies were specific monoclonal antibodies against the yellow fever virus NS1 and the 2 monoclonal antibodies were specific for flavivirus NS1.The remaining 2 monoclonal antibodies cross-react with the NS1 of flavivirus.In conclusion,this study primarily explored the secretion of NS1 protein in supernatants of YFV infected cells and animal serum,suggesting that NS1 protein has potential value for the early diagnosis of YFV infection.In addition,YFV NS1 recombinant protein with good antigenicity was successfully obtained,and 8 monoclonal antibodies specific for YFV NS1 protein were prepared,which providing experimental materials for the establishment of YFV NS1 protein immunological assays,researching YFV NS1 protein structure and function.