Fig 1.
Comparative analysis of the fusion loop region in JEV and ZIKV E proteins.
(A) Sequence alignment of the FL region core region, showing complete conservation between JEV and ZIKV. Colors indicate amino acid types. (B) Tertiary structure superposition of the FL region, with a Cα RMSD of 0.247 Å, indicating high structural similarity. Orange/red: ZIKV protein/FP loop; purple/blue: JEV protein/FP loop. (C) Surface electrostatic potential of the FL region. ZIKV exhibits a positively charged surface (blue), while JEV is neutral (white). Color scale: blue, positive; white, neutral; red, negative. (D) Linear B-cell epitope prediction. ZIKV FL region forms a strong epitope of seven residues (98–104), whereas JEV comprises five residues (98–102), indicating stronger predicted antigenicity in ZIKV(* indicates > 0.01512).
Fig 2.
Cross-reactivity and concentration-dependent functional effects of anti-JEV fusion loop antibodies in vitro.
The BALB/c mouse image (https://scidraw.io/drawing/799) and syringe image (https://scidraw.io/drawing/385) were obtained from SciDraw and are licensed under the Creative Commons Attribution 4.0 International (CCBY 4.0) license. (A) Schematic of the mouse immunization and experimental workflow. (B) ELISA titers of mouse sera against the JEV FL peptide, showing successful immunization. (C) Comparison of cross-reactivity (area under the curve [AUC]) between five JEV FL-positive and five negative sera with ZIKV virions (p < 0.0001). The AUC was calculated using GraphPad Prism to quantify the overall binding response across the dilution series; all measurements were performed at OD450. (D) Correlation between anti-JEV FL antibody titers and ZIKV cross-reactivity.(E) Antibody-dependent enhancement (ADE) in K562 cells at different serum dilutions, measured by relative ZIKV RNA levels. (F) Plaque reduction neutralization percentage in Vero cells at different serum dilutions. (G) Representative plaque assays of ZIKV in Vero cells treated with undiluted JEV FL-negative (top) or positive (bottom) sera. All experiments were independently repeated three times.
Table 1.
Median survival time and life extension rate in each group of mice.
Fig 3.
Concentration-dependent protective and ADE effects of anti-JEV FL antibodies in vivo.
The C57BL/6 mouse image was obtained from Wikimedia Commons (https://commons.wikimedia.org/wiki/File:201707_mouse_3D_black.svg) and is licensed under CCBY 4.0. The syringe image was sourced from SciDraw (https://scidraw.io/drawing/385) and is also licensed under CCBY 4.0. The mouse symptom photograph was taken by the authors. (A) Schematic of the passive immunization and ZIKV challenge experimental design.(B) Clinical symptoms on day 8 post-infection in each group.(C) Kaplan–Meier survival curves of mice.(D) Body weight changes over the course of infection.(E) Clinical disease scores of infected mice.(F) Viral loads in blood at different time points post-infection, measured by qRT-PCR. Statistical significance at dpi 5 compared with virus-only controls was determined by t-test.(G) Serum TNF-α concentrations in each group.(H) Serum IL-6 concentrations in each group. Data were analyzed using two independent-sample t-tests;ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001. (* indicates statistical significance compared with the PBS control. The color of * corresponds to the color of its respective treatment group in the figure).
Fig 4.
Histopathological alterations and semi-quantitative lesion scoring of major organs in mice.
(A) Representative H&E-stained sections of brain, liver, and kidney tissues (the black box indicates the magnified field of view).Brain (hippocampal region): black arrows indicate perivascular edema; yellow arrows indicate pyramidal neuron nuclear shrinkage; red arrows indicate focal hemorrhage in the DG region; green arrows indicate nuclear shrinkage of granular cells in the DG region.Liver: black arrows indicate hepatocellular steatosis; green arrows indicate vascular congestion.Kidney: black arrows indicate tubular epithelial swelling; yellow arrows indicate vacuolar degeneration; red/white arrows indicate glomerular lesions; green arrows indicate tubular dilation; brown arrows indicate interstitial congestion; blue arrows indicate inflammatory (lymphocytic) infiltration. (B) Semi-quantitative composite pathological score of brain lesions. (C) Semi-quantitative composite pathological score of liver lesions. (D) Semi-quantitative composite pathological score of kidney lesions.Pathological grading was performed according to the INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) four-tier scoring system. Data were analyzed using two independent-sample t-tests; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.