Establishment Of Chikungunya Virus Nucleic Acid Detection Method And Identification Of Monoclonal Antibodies Against Structural Protein E2

Chikungunya virus(Chikungunya virus,CHIKV)isanenveloped,single-stranded positive sense RNA virus.It is a member of the Alphavirus genus of the Togaviridae family.The mosquito is the main vector of CHIKV transmission.CHIKV infection is mainly responsible for fever,headache,rash,systemic arthritis and other symptoms,with a few cases presenting meningeal encephalitis,myocarditis,liver function damage,skin and mucous membrane hemorrhage.With global warming and increasing communication among countries,CHIKV is spreading worldwide.Since 2008 when the first imported case was discovered in China,a few imported cases have been reported every year in China.Given that the widely distribution of mosquito species capable of transmitting CHIKV in China,local transmission of CHIKV might take place in China.Currently,there are no effective therapeutics against CHIKV infection.Therefore,the early detection of CHIKV was particularly important.This study mainly focused on the establishment of the Chikungunya virus diagnostic test methods,including(1)Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of CHIKV and Zika virus(ZIKV);(2)Preparation and identification of monoclonal antibodies against CHIKV E2 protein.(1)Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of CHIKV and ZIKV.The symptoms of CHIKV and ZIKV are very similar in the early stage of infections,such as fever,headache,full Body muscle pain.It is difficult to determine which virus has infected a patient through solely relying on clinical symptoms.In such a case,to establish a highly sensitive nucleic acid diagnostic method is most necessary,for example a real-time PCR method.In order to simultaneously detect CHIKV and ZIKV,we established a one-step multiplex real-time RT-PCR assay.This assay is highly sensitive and specific for detecting either CHIKV or ZIKV,and could be used for clinical diagnostics,which is important for making the appropriate treatment therapy for patients.(1)We designed and synthesized the primers and TaqMan probes of CHIKV and ZIKV.The primers and probes are located in the regions of CHIKV nsP1 and ZIKV NS5,respectively.The amplification efficiency of the reaction system was calculated by establishing a standard curve.The sensitivity and specificity of this assay were evaluated.The results showed that(a)the amplification efficiencies of this assay for CHIKV and ZIKV were 97.99%and 99.98%,respectively.(b)the correlation coefficients R~2 were 0.9977 and 0.9994,respectively;(c)the detection limits were 1PFU and 0.5 PFU,respectively;(d)there was no cross-reaction with other species of alphavirus and flavivirus,such as Sindbis virus and Semliki forest encephalitis virus in the Alphavirus genus,as well as Japanese encephalitis virus,West Nile virus in the Flavivirus genus.Furthermore,this method was assessed using clinical samples.It indicated that the results using it were consistent with those detected in hospital.Therefore,this newly developed assay could be used to rapidly detect CHIKV and ZIKV with high sensitivity and specificity,providing an effective detection method for both viruses.(2)Preparation and identification of monoclonal antibodies against structural protein E2(CHIKV-E2)of CHIKV.Firstly,the polyclonal antibodies were obtained by immunizing mice with CHIKV-E2 protein as an immunogen that was obtained through prokaryotic expression and purification.The efficiency of the serum antibodies was verified by indirect immunofluorescence assay(IFA)and Western blotting assay.Secondly,the expression of E2 protein in eukaryotic cells infected with CHIKV can be detected by the E2 antibodies.The results of ELISA assay indicated that the mice serum antibodies with dilutions from 1:300,000 to 1:1,000,000 still had neutralizing activities after four rounds of immunizations.Thirdly,hybridoma cells were obtained by fusing spleen cells from an immunized mouse with activated myeloma cells(SP2/0).Using CHIKV-E2 expressing cell lysates coated plates,15strains were initially obtained by ELISA screening.Furthermore,nine positive clones were identified by IFA that could react with virus-infected cells.Cells of these IFA-positive clones were subjected to four rounds of subcloning,and then nine monoclonal cell lines were obtained.Finally,high-titer and highly specific ascites antibodies were successfully prepared,and the purified ascites antibody was characterized by tests of neutralizing activity,specificity,and epitope regions.The results showed that five purified antibodies had the activity of neutralizing the virus,and 9 antibodies can be used in IFA test.In addition to a 6E2 fluorescence detected,the other 8 can be used for detection,Western blot indicates that the above 9 cells can be used for detection,and we also conducted a neutralization assay,9 epitopes identified.The subclones belong to three different domain regions of E2 protein.Specific detection revealed that two of the 9 antibodies(4C4,7B7)not only detected CHIKV but also cross-reacted with its subgenus Semliki forest encephalitis virus(SFV).While the remaining seven strains can only specifically detect CHIKV,these monoclonal antibodies may become potential diagnostic and potential antiviral agents,and also provide a good basis for studying the functions of structural protein E2during virus neutralization.