Figure 7.

CCR2-Dependent Migration and Infection of Myeloid Cells Is Required for Mosquito Bite Enhancement of Virus Infection

(A) Mice were infected intradermally with SFV4(Xho)-EGFP (green) in back dorsal skin and at 6 hpi sections stained for lyve1 (red) and DAPI (blue). Arrows indicate double-positive cells in lower magnification image.

(B) Skin SFV4(Xho)-EGFP-infected cells were identified by both their expression of EGFP and staining against EGFP and back-gated onto a CD11b/Lyve1 plot, represented as green dots. Luciferase-expressing SFV4 was used as a control.

(C) Quantification of CD11b⁺Ly6C^hi myeloid cell numbers in bite sites at 4 and 16 hr. Live cells were first gated for CD45^hi cells and to remove Ly6G^hi neutrophils (n = 4).

(D) CD11b and Gr-1 staining for all cells (left) and back gating of virus-infected cells at 16 hpi (green dots, right plot).

(E) Skin sections were stained for the bone-marrow-derived myeloid marker ER-HR3 (red) in SFV4(Xho)-EGFP-infected skin at 16 hpi.

(F) Percentage CD45⁺Lyve1⁺ macrophages numbers of all live cells at 24 hpi in the skin (n = 4).

(G and H) SFV4-infected bite sites (n = 4) were digested at 16 hpi to release cells and CD11b⁺ cells sorted on columns to generate a CD11b⁻ fraction and a CD11b⁺ enriched fraction (G). Infectious virus released by each cell fraction after 6 hr in culture was quantified by plaque assay (H).

(I) Gene transcripts for bite-associated innate immune genes and viral RNA at 4 hpi (n ≥ 7).

(J) Level of neutrophils at 4 hpi and monocytes at 18 hpi in the skin were determined by flow cytometry (n ≥ 4).

(K and L) Ccr2^−/− mice are protected from bite-enhanced arbovirus infection at 24 hpi. Wild-type and Ccr2^−/− mice were infected with either 10⁴ SFV4 (K) or 10⁴ PFU of the genetically unrelated arbovirus BUNV (L) in the presence or absence of a mosquito bite.

(K) qPCR analysis of SFV RNA at 24 hr in the skin and viraemia at 24 hpi (n = 6).

(L) qPCR analysis of BUNV M-segment RNA at 24 hr in the inoculation site (skin), remote tissue (spleen), and viraemia at 24 hpi (n = 6).

All column plots show the median value ± interquartile range. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ns = not significant. Results shown are representative of either two or three experiments. See also Figure S7.