Enzyme-linked immunosorbent assays using virus-like particles containing mutations of conserved residues on envelope protein can distinguish three flavivirus infections

Figures & data

Figure 1. Results of ZIKV and DENV1 VLP IgG ELISA. Convalescent- and post-convalescent-phase serum or plasma samples from different panels were tested with ZIKV (A,C,E) and DENV1 (B,D,F) VLP IgG ELISA. (A,B) WT-VLP, (C,D) FL/BCL- or FL-VLP, (E,F) rOD ratio of mutant to WT VLP. Data are the means of two experiments (each in duplicate). Dashed lines indicate cutoff rOD and dotted lines cutoff rOD ratio in panels E and F.

Figure 2. Results of ZIKV and DENV1 VLP IgM ELISA. Convalescent-phase samples from different panels were tested with ZIKV (A,C,E) and DENV1 (B,D,F) VLP IgM ELISA. (A,B) WT-VLP, (C,D) FL/BCL- or FL-VLP, (E,F) rOD ratio of mutant to WT VLP. Data are the means of two experiments (each in duplicate). Dashed lines indicate cutoff rOD and dotted lines cutoff rOD ratio in panels E and F.

Figure 3. Results of WNV VLP IgG and IgM ELISA. Convalescent- and post-convalescent-phase samples from different panels were tested with WNV VLP IgG ELISA (A,C,E), and convalescent-phase samples with WNV VLP IgM ELISA (B,D,F). (A,B) WT-VLP, (C,D) FL-VLP, and (E,F) rOD ratio of mutant to WT VLP. Data are the means of two experiments (each in duplicate). Dashed lines indicate cutoff rOD and dotted lines cutoff rOD ratio in panels E and F.

Table 1. Sensitivity and specificity of different VLP-IgG ELISA.

ELISA^a		% Sensitivity (95% CI)^b	% Specificity (95% CI)^b,c
ZIKV WT-VLP	Overall	100 (100–100)	52.9 (44.6–57.2)
	Subgroup	pZIKV:100, ZIKVwprDENV:100	naïve:98.5, pDENV1:0, sDENV:0, pWNV:22.2
ZIKV FL/BCL-VLP	Overall	100 (100–100)	83.3 (77.1–86.5)
	Subgroup	pZIKV:100, ZIKVwprDENV:100	naïve:100, pDENV1:93.8, sDENV:20.0, pWNV:83.3
rOD ratio ≥0.57 (ZIKV FL/BCL to WT VLP)	Overall	93.9 (85.8–98.1)	92.2 (85.6–95.5)
	Subgroup	pZIKV:100, ZIKVwprDENV:86.7	naïve:NA, pDENV1:93.8, sDENV:85.0, pWNV:96.4
DENV1 WT-VLP	Overall	100 (100–100)	70.0 (61.8–74.2)
	Subgroup	pDENV1:100, sDENV:100, ZIKVwprDENV:100	naïve:98.5, pZIKV:0, pWNV:52.8
DENV1 FL-VLP	Overall	100 (100–100)	93.3 (88.9–95.6)
	Subgroup	pDENV1:100, sDENV:100, ZIKVwprDENV:86.7	naïve:100, pZIKV:94.4, pWNV:80.1
rOD ratio ≥0.66 (DENV1 FL to WT VLP)	Overall	88.2 (79.4–92.8)	91.4 (82.2–96.2)
	Subgroup	pDENV1:100, sDENV:100, ZIKVwprDENV:60.0	naïve:NA, pZIKV:100, pWNV:82.4
WNV WT-VLP	Overall	100 (100–100)	56.3 (47.9–60.6)
	Subgroup	pWNV:100	naïve:100, ZIKVwprDENV:0, pZIKV:50.0, pDENV1:0, sDENV:5.0
WNV FL-VLP	Overall	100 (100–100)	83.0 (76.6–86.2)
	Subgroup	pWNV:100	naïve:100, ZIKVwprDENV:46.7, pZIKV:100, pDENV1:93.8, sDENV:30.0
rOD ratio ≥0.50 (WNV FL to WT VLP)	Overall	100 (100–100)	66.1 (54.0–72.3)
	Subgroup	pWNV:100	naïve:NA, ZIKVwprDENV:53.3, pZIKV:100, pDENV1:93.8, sDENV:36.8

^aBCL: BC loop; CI, confidence interval; ELISA: enzyme-linked immunosorbent assays; FL: fusion loop; NA: not applicable; pDENV1: primary dengue virus type 1 infection; pWNV: primary West Nile virus infection; pZIKV: primary Zika virus infection; rOD: relative optical density; sDENV: secondary dengue virus infection; Sens: sensitivity; Spec: specificity; VLP: virus-like particles; WT: wild type; ZIKVwprDENV: Zika virus infection with previous dengue virus infection.

^bFor simplicity, the 95% CIs in the subgroup are not shown.

^cCompared to that of WT-VLP, the improved specificity by FL-VLP, FL/BCL-VLP or rOD ratio is bolded.

Table 2. Sensitivity and specificity of different VLP-IgM ELISA.

ELISA^a		% Sensitivity (95% CI)^b	% Specificity (95% CI)^b,c
ZIKV WT-VLP	Overall	87.5 (77.3–92.7)	96.4 (93.3–98.0)
	Subgroup	pZIKV:100, ZIKVwprDENV:75.0	naïve:100, pDENV1:83.3, sDENV:91.7, pWNV:97.2
ZIKV FL/BCL-VLP	Overall	90.0 (80.7–94.7)	99.3 (97.9–100)
	Subgroup	pZIKV:100, ZIKVwprDENV:80.0	naïve:100, pDENV1:100, sDENV:95.8, pWNV:100
rOD ratio ≥0.51 (ZIKV FL/BCL to WT VLP)	Overall	100 (100–100)	80.0 (44.9–97.9)
	Subgroup	pZIKV:100, ZIKVwprDENV:100	naïve:NA, pDENV1:100, sDENV:50.0, pWNV:100
DENV1 WT-VLP	Overall	80.6 (67.6–87.2)	92.3 (87.9–94.5)
	Subgroup	pDENV1:100, sDENV:70.8	naïve:100, ZIKVwprDENV:75.0 pZIKV:85.0, pWNV:91.7
DENV1 FL-VLP	Overall	75.0 (60.9–82.2)	95.8 (92.5–97.5)
	Subgroup	pDENV1:91.7, sDENV:66.7	naïve:100, ZIKVwprDENV:75.0 pZIKV:95.0, pWNV:100
rOD ratio ≥0.39 (DENV1 FL to WT VLP)	Overall	96.6 (89.9–99.9)	27.3 (1.0–40.7)
	Subgroup	pDENV1:100, sDENV:94.1	naïve:NA, ZIKVwprDENV:0 pZIKV:0, pWNV:100
WNV WT-VLP	Overall	94.4 (87.0–98.3)	91.4 (87.0–98.3)
	Subgroup	pWNV:94.4	naïve:98.4, ZIKVwprDENV:85.0 pZIKV:90.0, pDENV1:58.3, sDENV:95.8
WNV FL-VLP	Overall	100 (100–100)	93.7 (89.7–95.7)
	Subgroup	pWNV:100	naïve:98.5, ZIKVwprDENV:80.0 pZIKV:100, pDENV1:75.0, sDENV:95.8
rOD ratio ≥0.79 (WNV FL to WT VLP)	Overall	100 (100–100)	63.6 (35.2–78.1)
	Subgroup	pWNV:100	naïve:NA, ZIKVwprDENV:33.0, pZIKV:100, pDENV1:60.0, sDENV:100

^aBCL: BC loop; CI, confidence interval; ELISA: enzyme-linked immunosorbent assays; FL: fusion loop; NA: not applicable; pDENV1: primary dengue virus type 1 infection; pWNV: primary West Nile virus infection; pZIKV: primary Zika virus infection; rOD: relative optical density; sDENV: secondary dengue virus infection; Sens: sensitivity; Spec: specificity; VLP: virus-like particles; WT: wild type; ZIKVwprDENV: Zika virus infection with previous dengue virus infection.

^bFor simplicity, the 95% CIs in the subgroup are not shown.

^cCompared to that of WT-VLP, the improved specificity by FL-VLP or FL/BCL-VLP is bolded.

Figure 4. Proposed algorithm of combined VLP IgG ELISA to distinguish three flavivirus infections. Based on the positivity to FL or FL/BCL mutant VLP of three flaviviruses (ZIKV, DENV and WNV), the samples that were negative to all three or positive to one of the mutant VLP could be flavivirus naïve or primary DENV, pZIKV or pWNV infection. For samples that were positive to two or more mutant VLP, the rOD ratio of ZIKV mutant to WT VLP will be calculated to distinguish sDENV and ZIKVwprDENV infections.