Fig 11. Acute phase protein MBL binds to RRV but does not affect viral infectivity.

(A) Serum from RRVD patients (n = 21) or healthy controls (n = 10) were analyzed by ELISA for MBL levels. Data are presented as mean ± SEM. ***P < 0.001, Mann-Whitney U test. (B) 21-day-old C57BL/6 WT mice (n = 4–5 per group) were subcutaneously injected with 10⁴ PFU of RRV or PBS (mock). Mice were sacrificed at 2, 5, 10 and 15 dpi, and serum was collected for analysis of MBL-C expression by ELISA. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, two-way ANOVA, Bonferroni post-test. (C) Increasing concentrations of mouse recombinant MBL-C were added to RRV-coated plates for 2 hours at 37°C, followed by binding to biotin-conjugated anti-MBL-C antibody for additional 2 hours at 37°C. Optical density at 450 nm was read using Horseradish Peroxidase Substrate kit. (D) Dose-dependent infection of C2C12 cells was performed at MOI 0.1, 1, 2.5, 5 and 10 for 24 h, using EFGP-RRV. The percentage of infected cells (EGFP⁺) was assessed using flow cytometry analysis. (E) C2C12 cells were infected with EGFP-RRV (10⁴ PFU RRV) and pre-bound MBL-C-RRV or PTX3-RRV complex (1 μg/ml of mouse recombinant proteins + 10⁴ PFU RRV) for 6, 12 and 24 hours. The percentage of infected cells (EGFP⁺) was assessed using flow cytometry analysis. Horizontal dotted line represents the mean percentage of EGFP⁺ cells detected in mock control. *P < 0.05, ***P < 0.001, one-way ANOVA, Bonferroni’s post-test.