The Establishment Of Six Kinds Of The Arboviruses Protein Chip Detection Method

Arboviruses (arthropod-borne viruses) are viruses transmitted by hematophagous arthropodvectors and susceptible vertebrate hosts. Arboviruses cause infections in humans, which are mostlyasymptomatic but can extend from mild fever to severe encephalitis. The epidemiology ofarboviruses typically presents as seasonal and locally distribution. Arboviruses emerge maily insummer and autumn which is positively correlates with the active season of the tick and mosquitovectors, and severely threat human health and public security. As some of the arboviruses cantransmit by aerosols and regarded as important viral bio-warfare agents. Arboviruses include manyviruses within the family Togaviridae, Bunyaviridae and Flaviviridae. Among them, Flavivirusinfection is the most dangerous. Japanese encephalitis virus (JEV), dengue virus (DENV),Crimean-Congo hemorrhagic fever virus (CCHFV)(also known as Xinjiang hemorrhagic fevervirus, XHFV), and tick-borne encephalitis virus (TBEV) are the four principal arboviruses of publichealth importance in mainland China at present. The pathogenesis of these viruses is still unclearand there are no effective vaccines or antivirus medicines so far. The specific early diagnosis of theinfection is critical for the effective prevention and therapy.Because of the similar clinical symptoms and epidemiology, the accurate detection andidentification of pathogens is of critically importance. Most frequently used diagnosis for antibodiespresently are IFA and ELISA which can detect one target in one reaction. The aim of our presentstudy was to establish a protein-chip platform to detect antibodies against JEV, TBEV, DENV,WNV, WEEV and EEEV simutaniously. This will provide a new way for clinical diagnosis ofarboviruses infection. The principal of the platform is to select the specific diagnostic antigens ascapture antigen to spot on chip to react with antiviral antibodies, and then to combine with afluorescence labeled second antibody for analysis and determination of the results. So far, noprotein array for detection of anti-arbovirus antibodies was reported.The main results of our study are summarized as following:1. The expression, purification and identification of arbovirus potential diagnostic antigensWe selected recombine antigens include epitopes to induce neutralizing antibody as ourpotential diagnose antigens for the protein array. This is based on the previous findings that-EDⅢprotein of Flavivirus consists of specific type and subtype epitopes to induce neutralizing antibodywithout epitopes for inducing cross-react antibodies. Structural protein PrM, nonstructural proteinNS1and NS3can also induce neutralizing antibody and have been employed as diagnostic antigens.It is also reported that E1protein of Alphavirus includes at least four non-lapped epitopes to induce neutralizing antibody and have widely been used as diagnostic antigens. Combined with the specialcharacteristics of viral strains epidemic in our country, we used DNAstar and Primer5softwares todesign specific PCR primers and added the suitable enzyme digest cites. Totally,17pair of primerswere designed for amplification of genes of TBEV,JEV,DENV1,DENV2,DENV3,DENV4,YFV,WEE,EEE and WNV. Recombinant expression plasmids were constructed and verified byRT-PCR and sequencing. Seventeen combined antigens were expressed by E.coli and purified. Inaddition, animal immune serum were prepared by inoculate rabbits with inactive whole virus andtheir titers and specificity were confirmed. The specificity of expressed antigens was identified byELISA through react with animal immune serum. Twelve diagnostic antigens were selected withgood specificity. These diagnostic antigens provide good materials for establishment of proteinarray.2. The establishment of protein array and the optimization of reaction parametersSeventeen expressed potential diagnostic antigens were spotted on a three-dimensional chip toconstruct protein-array. Rabbit IgG was used and positive control and PBS contained20%glycerinewas used as negative control. Each antigen was spotted three times and air-dried at roomtemperature. Firstly, rabbit immune serum was used to react with the spotted protein array to selectthe diagnostic antigens for capture antigens. After reaction and analyse, twelve diagnostic antigenswere selected with good specificity, the same as selected with ELISA method. This indicated thatcombined different antigens on one chip did not affect their individual specificity. Then, thereaction parameters were optimized. The results showed that when concentration of antigens werewithin the scale of0.125～0.6mg/ml could obtain good specificity. The detected antibodies rangedfrom1:100to1:1000. The optimized parameters were as following:2hrs of inculbation time,1:1000of the dilution of second antibody,45min of incubating period of second antibody, theconcentration of glycerine was15%of the protein dilution buffer. After the opitimization, theprotein array was verified with the immune serum again. All12antigens showed good specificityon the array.3. The evaluation and validation of the protein arrayThe purpose of this study is to apply the protein array to clinical diagnosis of arbovirusinfected patients, and to provide an etiological evidence for the treatment of arbovirus infecteddiseases. Therefore, after the establishment and optimization of the protein array, we used clinicalpatient serum for the evaluation and validation of the method. Totally,33suspected TBEV infectedsera,8suspected JEV infected sera and22suspected DENV infected sera were collected and another20sera from healthy voluteers were used as negative controls. Firstly, IFA coated withwhole virus was employed to detect the suspected serum. Then identical ELISA and protein arrayused the same antigens were carried out in parrellel for comparison.The results showed among the33suspected TBEV infected serum,23were TBEV IgG positive and23were IgM positive detectedwith IFA, while identical ELISA and protein array all tested16IgG positive and17IgM positive,the correlation of ELISA and protein array was100%. Among the8suspected JEV infected serum,8were JEV IgG positive and6were IgM positive detected with IFA, while the traditional ELISAtested4IgG positive and5IgM positive, and protein array detected5IgG positive and5IgMpositive. Among the22suspected DENV infected serum,13were DENV IgG positive and15wereIgM positive detected with ELISA, and14IgG positive and15IgM positive detected by proteinarray, the sensitivity of protein array was slightly higher than the ELISA. All healthy serum gavenegative results. Protein array has very good correlation with the ELISA after Kappa statistical test.Protein array and the identical ELISA used the recombinant antigens as capture antigen had lowerpositive numbers compared with the IFA used whole virus as capture antigen. In order to increasethe sensitivity of the protein array, we mixed EDⅢ and NS1antigens as capture antigen fordetecting of TBEV antibodies.20TBEV IgM positive sera were identified. Combined antigencould increase the sensitivity of the protein array method.After screening and optimization of the potential antigens and the reaction parameters,relatively good specificity and sensitivity were obtained. The specificity of the protein array fordetecting of clinical serum was100%and the sensitivity was as well as the traditional ELISA.Additional study indicated that by combining the antigens, the sensitivity of the protein array couldbe increased.This study reported a protein array for detection of antibodies against six arboviruses for thefirst time. The protein array can detect six viruses as well as subtype of four DENV subtypes. Itprovides a new method for diagnosis of the six arboviruses infected diseases.