Fig. 9: HTNV NP promotes M1 polarization by activating the notch pathway and accelerates disease procession.

a qRT-PCR analysis of Hes1 (-i) or TNFα (-ii), and immunoblot analysis for NICD (-iii). The mBMDM are infected with HTNV (MOI = 1), treated with heat/Co⁶⁰-devitalized HTNV (MOI = 5), transfected with viral RNAs or stimulated with viral proteins (NP, 1 μg/ml; VLP, MOI = 5). qRT-PCR analysis of inflammatory genes in NP-stimulated mBMDM with DPAT pre-treatment (-iv). Mock as control, in the left part of (a)-(i), p = 0.0167 (vs. HTNV) or 0.0008 (vs. NP); in the right part of a-(ii), p < 0.0001 (vs. HTNV, Heated HTNV, Co60-HTNV, NP or VLP). DMSO + NP vs. DAPT + NP, in (a)-(iii), p < 0.0001 (Tnf)/ = 0.002 (Il6)/ = 0.0025(Ccl2)/ < 0.0001 (Nos2). b (-i) Serum NP production in HFRS patients with distinct disease severity detected by ELISA. The sample number of each group is shown in the figure. (-ii) The correlation of HFRS patient serum NP with the M1- or M2-like monocyte percentage (n = 20). The exact P values are shown in the figure. c Flow cytometry analysis of TNFα⁺ or iNOS⁺ mBMDM at 24 h post-NP stimulation (5 μg/ml) (n = 4). The mBMDM are pretreated with DAPT for 12 h and then subjected to NP stimulation. The NP (5 μg/ml) is co-incubated with control antibody 4G2 (the flavivirus group antibody, 50 ng/ml) or anti-NP antibody 1A8 (50 ng/ml) for 2 h at room temperature and then added to stimulate mBMDM. DMSO + NP vs. DAPT + NP, p = 0.0026 (TNFα)/0.0014 (iNOS); NP + 4G2 vs. NP + 1A8, p = 0.0002 (TNFα)/ < 0.0001 (iNOS). d Flow cytometry analysis of CD80⁺ TNFα⁺ or CD14⁺ CX3CR1⁺ mBMDM at 36 h post-HTNV infection or NP stimulation. The truncated NP peptides are constructed and purified with the baculovirus system. e Survival data for 4-day-old neonatal mice challenged with HTNV (i.p., 8 × 10⁵ TCID₅₀/g) and then treated with 4G2 or 1A8 (0.25 μg/g) from 1 dpi to death (every two days). HTNV + 4G2 vs. HTNV + 1A8, p = 0.0061. Data are shown as the mean ± SEM, and are representative of two independent experiments. Analysis is performed mainly with one-way ANOVA (a-(i), Dunnett’s multiple comparisons test), two-sided unpaired Student’s t test (a-(iii), c), or the survival curve comparison (e, log-rank [Mantel–Cox] test). *p < 0.05, **p < 0.01, ***p < 0.001. Molecular weight markers are shown to the left of the blots in kDa, and antibodies used are indicated to the right. Source data are provided as a Source Data file.