New Methods For Serological Detection Of Antibodies Agains Zika Virus And MERS-CoV

Background and objectivesZika virus(ZIKV)belongs to flavivirus genus within the family of Flaviviridae.It was first isolated from the sera of febrile rhesus monkeys in Africa in 1947.There have been several outbreaks in the world.In 2016,the imported cases of ZIKV infection were confirmed in China.MERS-CoV was first discovered in Saudi Arabia in 2012.It is also called "SARS-CoV like virus" because the clinical symptoms are similar to SARS-CoV infection.It is mainly prevalent in the Middle East although Eurasia and North America also have MERS cases,which have direct or indirect relationship with those in the Middle East.The mortality rate is as high as 35%.A case of MERS imported from Korea was diagnosed for the first time in China in 2015.With further expansion of globalization,these two emerging infectious diseases may cause outbreaks in China,which will result in great economic and disease burden and become major public health problem in China.Therefore,the establishment of rapid serological detection assays is urgently needed and has great significance in preventing the epidemics of these diseases.Luciferase immunosorbent assay(LISA)is a high-throughput and ultra-sensitive method for detection of antibodies based on a novel luciferase,Nano-luciferase.It is simple,rapid,sensitive and does not require species specific secondary antibody.The assay has already been applied to the detection of anti-HIV antibodies and achieved great success.The purpose of this research is to establish a new method for detecting anti-ZIKV and anti-MRS-CoV antibodies based on LISA platform.MethodsWe chose several target antigens of ZIKV and MERS-CoV,including ZIKV-NS1,ZIKV-NS2B,MERS-S1,MERS-S2,MERS-RBD,MERS-NTD and MERS-NP.The expression plasmids which contain target antigens and luciferase genes were constructed and transfected into 293T cells to express fusion protein.Cell lysates containing fusion antigens were collected to establish LISA methods.Protein G was immobilized on the surface of solid phase carrier as capture molecules and incubated with samples to be tested.Protein G captures total IgG antibodies in the samples.The target antigen was then bound to the specific antibody and attached to the solid phase carrier after adding the above cell lysates.After washing away the excess lysates,the luciferase substrate was added.The presence and concentration of specific antibodies in the samples were determined by detecting the fluorescence light units.Human and animal serum samples were collected from different sources,The detection results were compared with those obtained by the commercial kits to evaluate the sensitivity and specificity of LISA.ResultsIn the present study,the C-NS1 protein was identified as the best antigen for detection of antibody against ZIKV,and the corresponding antibody detection method,C-NS1-LISA,was established.A good correlation between LISA and ELISA was also obtained with Pearson’s correlation coefficients of>0.8.The LISA is at least 4 folds more sensitive than the commercial ELISA.However,there were cross-reactivity with other viruses of Flavivirndae and Togaviridae family.The false positive rates were as follows:DENY 8.5%(4/47),JEV 0%(0/6),YFV 20%(2/10),HCV 0%(0/10),CHIKV 33%(3/9).Furthermore,we found that the combination of ZIKV NSI and NS2B-based LISA could dramatically increase the specificity with the false positivity as follows:DENV 2%(1/47),JEV 0%(0/6),YFV 0%(0/10),HCV 0%(0/10),CHIKV 11%(1/9).MERS-RBD,MERS-S1 and MERS-NP were used as target antigens for the detection of anti-MERS-CoV antibodies.RBD was the best antigen.The RBD-based LISA was at least 16 folds more sensitive than the commercial ELISA for MERS－CoV.In conclusion,our results indicate that the two antibody methods for ZIKV and MERS-CoV can be used to detect antibodies from human and animal samples.