Article Figures & Data
Figures
- FIG. 1.
(A) Infection of mixed neural cell cultures with either wt JHMV or srr7. Mixed neural cell cultures were infected with either wt JHMV or srr7 and fixed with 4% paraformaldehyde at 8 h, 12 h, or 24 h after infection. Viral antigens in cells were detected by staining with anti-MHV MAbs and anti-mouse IgG. (B) Immunofluorescent double staining of srr7-infected cells with anti-MHV MAbs and microglial cell/macrophage-specific GS-lectin. Mixed neural cell cultures were infected with srr7 and incubated at 37°C for 12 h. Viral antigens, as well as microglial cells, were stained with anti-MHV MAbs (Alexafluor 488, green) and GS-lectin (Texas Red, red), respectively, and the images were merged.
- FIG. 2.
Blockade of wt and srr7 infection in mixed neural cell cultures by anti-MHVR MAb CC1. Mixed neural cell cultures were treated with 10-fold-stepwise-diluted CC1 at 37°C for 1 h and then infected with either wt virus or srr7. After infection, cells were cultured in the presence of CC1. Viral antigen was visualized by staining with anti-MHV MAbs at 12 and 24 h after wt and srr7 infection, respectively, and antigen-positive cells were counted. The blockade of viral infection by CC1 was calculated as the percentage of viral antigen-positive cells in the presence of CC1 compared with cell numbers obtained in the absence of CC1.
- FIG. 3.
Examination of MHVR-bearing cells by flow cytometric analysis. Neural cells separated from cerebrums of 1- to 3-day-old mice were labeled with either GS-lectin (PE) (A) or anti-MHVR MAb CC1 (Alexafluor 488) (B). Cells were also analyzed for coexpression of GS-lectin and MHVR (D) by double staining as described in Materials and Methods. (C) Background control.
- FIG. 4.
(A and B) Detection of MHVR and JHMV binding to cells by flow cytometric analysis. Cells isolated from neonate mouse brain were mixed with wt JHMV and stained with anti-MHV MAb-Alexafluor 488. Those cells were also examined for binding by GS-lectin PE and analyzed by flow cytometry (B). (A) Background control. (C to E) Inhibition of wt JHMV attachment on neural cells by CC1. Cells isolated from neonatal mice were first treated with CC1 at 4°C for 30 min and then infected with wt JHMV. JHMV attached to cells was detected with anti-MHV MAb, biotinylated anti-mouse IgG1, and avidin-PE (E). As a control, cells single stained with CC1 (C) or those with attached virions (D) were also analyzed by flow cytometry.
- FIG. 5.
Infection of microglial cell-enriched cultures by wt JHMV or srr7. Microglial cell-enriched cultures were prepared as described in Materials and Methods. Those cells were infected with either wt JHMV or srr7, and viral antigens were examined with anti-MHV MAb (Texas Red) as well as for the presence of GS-lectin (Alexafluor 488) on the cultured cells at 12 h after infection. The images of GS-lectin-positive and MHV-positive cells were merged.
- FIG. 6.
(A) Infection of astrocyte-enriched cultures by wt and srr7. Astrocyte cultures were prepared as described in Materials and Methods. Cells were infected with either wt JHMV or srr7, and viral antigens were monitored with anti-MHV MAb (Alexafluor 488) at 12 h after infection. (B) Infection of srr7 in astrocyte-rich cultures. srr7-infected cells in astrocyte-rich cultures were also examined for GS-lectin positivity (using Texas Red) as well as with anti-MHV MAb. These two images were merged.
- FIG. 7.
(A) Effect of CC1 on wt virus spread from initially infected cells. Mixed neural cell cultures were infected with wt virus. After 1 h of adsorption, cells were washed with HBSS and cultured in the presence or absence of CC1. Cells were fixed at 24 h after infection and stained with anti-MHV MAbs. (B) Effect of CC1 on the spread of wt virus and srr7 from initially infected cells in microglial cell-enriched cultures, which were infected with either the wt or srr7 and incubated at 37°C for 1 h. After washing, cells were cultured with medium in the presence or absence of CC1. Cells were stained with anti-MHV MAbs at 24 h after infection.
