Figure 4.

CX₃CR1 ablation results in delayed viral clearance from peripheral lymphoid tissues. (A) Viral burden in peripheral lymphoid during JE progression. (B) Infectious JEV in sera and viral burden in CNS tissues during JE progression. Viral burden in popliteal LNs, spleen, and brain of CX₃CR1^+/+ and CX₃CR1^−/− mice was assessed by real-time qRT-PCR at the indicated days after infection with JEV via footpad inoculation. Viral RNA load was expressed by viral RNA copy number per microgram of total RNA. The levels of infectious JEV in sera were determined by focus-forming assay. (C) Confocal imaging for detection of JEV Ag⁺CX₃CR1⁺CD11c⁺ DCs in popliteal LNs. Sections of popliteal LNs derived from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice infected with JEV via footpad inoculation were co-stained for JEV Ags [E and NS1 (PE red)] and DC marker CD11c (APC purple) at 2 dpi. The CX₃CR1^gfpCD11c⁺ DCs co-localizing with JEV Ags in lower magnification images (upper pictures) and higher magnification images (lower pictures) are denoted by white arrows. (D) Visualization of JEV in the CNS. Brain sections obtained from JEV-infected CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice were co-stained with JEV Ags [E and NS1 (PE red)] and nuclear stain DAPI (blue) at 5 dpi. Images are representative of sections derived from at least five mice per group. Data show the average ± SEM of levels derived from at least three independent experiments (n = 4–5). ^**p < 0.01 and ^***p < 0.001 for CX₃CR1^+/+ vs. CX₃CR1^−/− mice at the indicated dpi.