Abstract
The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel “pan-Flavi” suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96–100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.
Highlights
Zika virus (ZIKV) infections emerged during the last decade in several parts of the world, most dramatically during 2016, and are affecting millions [1]
Among the 173 blood donor sera, previous TBE vaccination was inferred as the presence of only a singular (MFI > 500) TBEV whole virus (WV) IgG signal (n = 42), previous YFV vaccination as the presence of only a strong YFV nonstructural protein 1 (NS1) IgG (MFI > 500, n = 4)
Several factors contributed to give a high analytical specificity of pan-Flavi” suspension multiplex immunoassay (PFSMIA); a low background for most sera, a high precision and simultaneous measurement of all flavivirus antibodies in the same reaction
Summary
Zika virus (ZIKV) infections emerged during the last decade in several parts of the world, most dramatically during 2016, and are affecting millions [1]. We developed a data reduction procedure that used the average degree of cross-reactivity and completeness (coincidence criterion) of homologous antibody responses, similar to the principle of an immunoblot confirmation test for HIV. It could partially compensate for heterologous, cross-reactive, signals. The evaluation was focused on differentiating ZVD from DF All of these known flavivirus infections were correctly classified in spite of cross-reactions to heterologous flavivirus(es). We demonstrated that taking antigen coincidence, average cross-reactivity, isotype, serial sampling, and avidity into account considerably increases the discrimination of ZVD from DF, and most likely, the specificity of flavivirus serology as a whole
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