Combination of Nonstructural Protein 1-Based Enzyme-Linked Immunosorbent Assays Can Detect and Distinguish Various Dengue Virus and Zika Virus Infections

Abstract

The recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.

Keywords: Zika virus; dengue virus; nonstructural protein 1; serodiagnosis.

FIG 1

Results of DENV1, DENV2, DENV3, DENV4, and DENV1-4 NS1 IgG ELISAs tested with primary DENV infection panels. NS1 IgG ELISAs based on DENV1 (A), DENV2 (C), DENV3 (E), DENV4 (G), and DENV1-4 (H) were tested with convalescent-phase samples of different panels (negative control [NC], primary DENV, pZIKV, pWNV, or sDENV). Results of pDENV1 (B), pDENV2 (D), and pDENV3 (F) panels tested with different NS1 IgG ELISAs. Dotted lines indicate cutoff rODs. Data are the means from two experiments (each in duplicates); horizontal lines are the means from each panel. D1, DENV1; D2, DENV2; D3, DENV3; D4, DENV4; D1-4, DENV1-4.

FIG 2

Results of DENV1-4 NS1 IgG ELISA tested with secondary DENV infection panels. (A) DENV1-4 NS1 IgG ELISA was tested with postconvalescent-phase samples of the sDENV panel. Comparison of DENV1 and DENV1-4 NS1 IgG ELISAs tested with convalescent- or postconvalescent-phase samples of sDENV (B), ZIKVwprDENV (C), and probable ZIKVwprDENV (D) panels. Dotted lines indicate cutoff rOD values. Data are the means from two experiments (each in duplicates); horizontal lines are the means from each panel. D1, DENV1; D2, DENV2; D3, DENV3; D4, DENV4; and D1-4, DENV1-4.

FIG 3

Results of DENV1-4 and ZIKV NS1 IgG ELISAs for different panels over time. DENV1-4 and ZIKV NS1 IgG ELISAs were tested for convalescent- and postconvalescent-phase samples of pZIKV (A), ZIKVwprDENV (B), pDENV1 (C), and pDENV2 (D) panels. Dotted lines indicate cutoff rOD values. Data are the means from two experiments (each in duplicates); horizontal lines are the means from each panel. D1-4, DENV1-4.

FIG 4

Results of DENV1-4 and ZIKV NS1 IgG ELISAs for sDENV and ZIKVwprDENV panels. DENV1-4 NS1 (A, D) and ZIKV NS1 (B, E) IgG ELISAs were tested for convalescent-phase (<3 month PSO [A to C]) and postconvalescent-phase (3 to 12 months PSO [D to F]) samples of sDENV and ZIKVwprDENV panels; the rOD ratio of ZIKV to DENV1-4 NS1 (C, F) is shown. Dotted lines indicate cutoff rOD values; dashed lines are the cutoff rOD ratio (0.24). Data are the means from two experiments (each in duplicates); horizontal lines are the means from each panel. The two-tailed Mann-Whitney test was used to compare two groups. D1-4, DENV1-4.