Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions

Abstract

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.

Keywords: Yellow fever virus; antibody; flavivirus; premembrane protein; serosurveillance.

Figure 1.

Antibody response to six flavivirus antigens following YF-17D vaccination or DENV, ZIKV or WNV infection. Lysates derived from mock-, DENV1-, DENV2-, DENV4-, WNV-, ZIKV-, and YFV (17D strain)-infected Vero cells were subjected to SDS-12% polyacrylamide gel electrophoresis under non-reducing condition and Western blot analysis probed with different serum/plasma samples or anti-E mouse mAb FL0232 (C). Results of YF-17D vaccinees (A) and NHPs receiving YF-17D vaccine (D), DENV-naïve participant (E), and participants with pDENV (F), WNV (G), sDENV (H), pZIKV (I), and DENV + ZIKV (D + ZK) (J) infections. Lysates derived from 293 T cells transfected with YF-17D prM/E plasmid were subjected to Western blot analysis and probed with a DENV- and YFV-immune serum, short (left) and long (right) exposure (B). The sampling time post-symptom onset, vaccination or TMA test was indicated after sample ID. pDENV, primary DENV infection; sDENV, secondary DENV infection; pZIKV, primary ZIKV infection. The positions of E, NS1 and prM protein bands are indicated. The size of molecular weight markers is shown in kDa. Mo: mock, D1: DENV1, D2: DENV2, D4: DENV4, WN: WNV, ZK: ZIKV, and YF: YF-17D.

Figure 2.

Antibody response to six flavivirus antigens in samples from a fever surveillance programme in the Philippines. (A-E) Results of participants with previous DENV infection (A), previous DENV and YFV infections/vaccination (D + YF) (B), pZIKV infection (C), previous DENV and ZIKV infections (D + ZK) (D), and seronegative to DENV, ZIKV, YFV and WNV (Neg) (E). The positions of E, NS1 and prM protein bands are indicated. The size of molecular weight markers is shown in kDa. Mo: mock, D1: DENV1, D2: DENV2, D4: DENV4, WN: WNV, ZK: ZIKV, and YF: YF-17D. (F,G) The pattern of E and prM proteins recognized and the number/percentage of positive and total samples based on Western blot analysis (F) and a graphic summary (G). (H,I) Results of DENV FL-VLP IgG ELISA (H) and comparison with that of anti-DENV prM reactivity in Western blot analysis (I). rOD: the relative OD. The two-tailed Mann-Whitney test was performed in panel H.

Figure 3.

Antibody response to six flavivirus antigens in samples from suspected ZIKV cased during the ZIKV outbreak in Brazil. (A-D) Results of participants with previous DENV infection (A), previous DENV and ZIKV infections (D + ZK) (B) previous DENV and YFV infections/vaccination (D + YF) (C), and previous DENV, ZIKV and YFV infections/vaccination (D + ZK + YF) (D). The positions of E, NS1 and prM protein bands are indicated. The size of molecular weight markers is shown in kDa. Mo: mock, D1: DENV1, D2: DENV2, D4: DENV4, WN: WNV, ZK: ZIKV, and YF: YF-17D. (E,F) The pattern of E and prM proteins recognized and the number/percentage of positive and total samples based on Western blot analysis (E) and a graphic summary (F).