Abstract
Diagnosing Zika virus (ZIKV) infections has been challenging due to the cross-reactivity of induced antibodies with other flavivirus. The concomitant occurrence of ZIKV and Dengue virus (DENV) in endemic regions requires diagnostic tools with the ability to distinguish these two viral infections. Recent studies demonstrated that immunoassays using the C-terminal fragment of ZIKV NS1 antigen (ΔNS1) can be used to discriminate ZIKV from DENV infections. In order to be used in serological tests, the expression/solubility of ΔNS1 and growth of recombinant E. coli strain were optimized by Response Surface Methodology. Temperature, time and IPTG concentration were evaluated. According to the model, the best condition determined in small scale cultures was 21 °C for 20 h with 0.7 mM of IPTG, which predicted 7.5 g/L of biomass and 962 mg/L of ΔNS1. These conditions were validated and used in a 6-L batch in the bioreactor, which produced 6.4 g/L of biomass and 500 mg/L of ΔNS1 in 12 h of induction. The serological ELISA test performed with purified ΔNS1 showed low cross-reactivity with antibodies from DENV-infected human subjects. Denaturation of ΔNS1 decreased the detection of anti-ZIKV antibodies, thus indicating the contribution of conformational epitopes and confirming the importance of properly folded ΔNS1 for the specificity of the serological analyses. Obtaining high yields of soluble ΔNS1 supports the viability of an effective serologic diagnostic test capable of differentiating ZIKV from other flavivirus infections.
Highlights
Zika virus (ZIKV) is a flavivirus transmitted primarily by Aedes aegypti mosquitoes
Since solubility was an important aspect, we sought to compare the solubility of ΔNS1 produced by E. coli Arctic Express (DE3) and BL21 (DE3) strains using LB, TB and 2xHKSII
The initial results with ΔNS1 indicated that it would be a promising candidate for the differential serological diagnosis of ZIKV infections in the presence of Dengue virus (DENV) and CHIKV
Summary
Zika virus (ZIKV) is a flavivirus transmitted primarily by Aedes aegypti mosquitoes. Many cases of microcephaly and other congenital malformations were reported following ZIKV infections during pregnancy (Franca et al 2016). DENV, the laboratory diagnostic was mostly based on the detection of antibodies against the non-structural protein 1, NS1 (Kikuti et al 2018; Stettler et al 2016). Previous work indicated that serological assays based on NS1 can be used to discriminate ZIKV and DENV infections (Balmaseda et al 2017; Bosch et al 2017; Stettler et al 2016). ZIKV and DENV proteins share high sequence identity resulting in the cross-reactivity of antibodies generated after the infection (Balmaseda et al 2017; Fernanda Estofolete et al 2016; Granger et al 2017; Priyamvada et al 2017). In spite of significant progress made in the last years in our understanding of ZIKV, the improvement of diagnostic assays is still needed (Kikuti et al 2018; Theel and Hata 2018)
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