Fig 1.
General conditions and motor function assessment of peripheral nerves in JEVs-infected mice.
(A) Experimental timeline of murine model. Mice were intraperitoneally inoculated with MEM (mock control) or 1 × 105 PFU JEV NX1889, GZ56, P3, and XZ0934 strains. In each group, half of the mice were euthanized at 10-12 dpi to perform sciatic nerve electromyography, HE staining, immunohistochemistry, and qRT-PCR. The remaining cohort was monitored daily for general conditions, neurological function and survival status over a 14-day observation period. (B) Representational pictures of JEVs-infected mice during peak illness. (C-F) Daily monitoring of the body weight change, VPS scores, HWT scores, and survival curves of JEVs-infected mice or mock control over a 14-day period. VPS and HWT scores were used to assess the limb strength of mice; n = 6. (G) Representative electrophysiological recordings of the sciatic nerve compound muscle action potentials (CMAPs) of JEVs-infected mice during peak illness. (H-J) Statistical analysis of the bilateral sciatic nerve CMAP in amplitude, end latency and NCV.
Fig 2.
Neuropathological alterations in the sciatic nerve, DRG, and brain of JEVs-infected mice during peak illness.
(A-C) Representative images of HE staining of the (A) sciatic nerve, (B) DRG and (C) brain. ↓ infiltration and aggregation of inflammatory cells,▼perivascular cuffing accompanied by congestion, ∇lymphocytic infiltration in the meninges, ★neuronal damage, 〇gliosis. (D, E) Representative images and statistical analysis of MBP in the sciatic nerve. n = 3. Scale bar for (A, B) 50 μm, (C) 100 μm (up) and 50 μm (down), and (D) 20 μm.
Fig 3.
Assessment of neuroinflammatory responses of JEVs-infected mice during peak illness.
(A-D) Representative images of immunohistochemical staining for CD45 and quantification of CD45 + immune cells in the (A, C) sciatic nerve and (B, D) DRG. The quantitative analysis of CD45 staining was performed by counting CD45-positive cells. CD45-positive cells were stained brown. DRG neurons, which are larger in volume than CD45-positive cells, exhibited nonspecific staining, with their cytoplasm stained in a light brown color. n = 5. (E, F) The mRNA expression of pro-inflammatory factors in the brain and sciatic nerve. n = 3. Scale bar is 50 μm.
Fig 4.
Viral loads of tissues and organs in JEVs-infected mice.
(A-C) JEV RNA load of the (A) serum, (B) brain, and (C) sciatic nerve in JEVs-infected mice during peak illness. JEV RNA load was detected by qRT-PCR. (D, E) Representative images and statistical analysis of JEV-NS1 protein in the sciatic nerve of JEVs-infected mice during peak illness. (F) CT value of tissues and organs in JEV NX1889-infected mice, including tissues of the CNS, i.e., brains and spinal cords, tissues of the PNS, i.e. DRGs and sciatic nerves, and peripheral organs or tissues. (G) Visualization of SCs, co-stained with JEV NS1 protein in the sciatic nerve of NX1889-infected mice. (H) Visualization of neurons, co-stained with JEV NS1 protein in the DRG of NX1889-infected mice. SCs labeled by s100β and DRG neurons labeled by Tuj1. n = 3. Scale bar is 20 μm.
Fig 5.
Replication of JEVs in RSC96 cells.
(A) Growth curves of strain NX1889 in RSC96 cells. RSC96 cells were infected with JEV NX1889 at MOIs of 0.01, 0.1, and 1. Viral titers were quantified by plaque assay on BHK-21 cells at 12, 24, 48, 72, and 96 hpi. (B) Comparison of growth curves of four JEV strains (MOI = 0.1) in RSC96 cells assessed by plaque assay. (C) Comparison of growth curves of four JEV strains (MOI = 0.1) in RSC96 cells detected by qRT-PCR. (D, E) Representative immunofluorescent staining images and statistical analysis of JEV NS1 protein expression in RSC96 cells at 48 hpi (MOI = 1). n = 3. Scale bar, 20 μm.
Fig 6.
Impact of JEVs (P3, GZ56, and NX1889) infection on proliferation and cell cycle of RSC96 cells.
(A) Effects of JEVs infection on the proliferation of RSC96 cells at different infection times and MOIs. Cell viability (proliferation) was quantified using Cell Counting Kit-8 (CCK-8) assay. Data normalized to uninfected controls; n = 5. (B, C) Impact of NX1889 infection on the cell cycle of RSC96 cells at different infection times. RSC96 cells infected with NX1889 (MOI = 1) were harvested at 24, 30, and 36 hpi, and detected by flow cytometry; n = 3. (D, E) Impact of JEVs infection (MOI = 1) on the cell cycle of RSC96 cells at 36 hpi; n = 3.
Fig 7.
Impact of strain P3, GZ56, and NX1889 infection on apoptosis in RSC96 cells.
(A) Morphology of JEVs-infected RSC96 cells at different time (MOI = 3). (B, D) Representative images and statistical analysis of apoptosis in NX1889-infected RSC96 cells at different times detected by flow cytometry (MOI = 3). (C, E) Representative images and statistical analysis of apoptosis in JEVs-infected RSC96 cells at 96 hpi detected by flow cytometry (MOI = 3). (F, G) Representative images and statistical analysis of cl-Casp3 protein of JEVs-infected RSC96 cells at 96 hpi (MOI = 3). (H) Co-localization of cl-Casp3 with JEV NS1 in NX1889-infected RSC96 cells at 96 hpi (MOI = 3). (I) Relative mRNA expression of pro-inflammatory factors in JEVs-infected RSC96 cells at 12 hpi. (MOI = 3). n = 3. The Scale bar is 50 μm for (A) and (F), and 20 μm for (H).
Fig 8.
Correlation analysis among limb motor deficits, viral load, and inflammatory response of the sciatic nerves in NX1889-infected mice.
Sciatic nerves were obtained between 8 to 12 dpi, and JEV RNA load and relative mRNA expression of pro-inflammatory factors were determined by qRT-PCR. (A) Correlation analysis between VPS score and JEV RNA load of the sciatic nerves; n = 30. (B-D) Correlation analysis between VPS score and the relative mRNA expression of (B) CCL2, (C) IFN-γ and (D) TNF-α in the sciatic nerves; n = 24. (E-G) Correlation analysis between JEV RNA load and the relative mRNA expression of (E) CCL2, (F) IFN-γ, and (G) TNF-α in the sciatic nerves; n = 24.