PMC

Quantitation of the influx of immune cells into the airways of infected wild-type (WT) BL/6 or CCL3^−/− mice. Total numbers of immune cells from wild-type BL/6 (white bars) or CCL3^−/− mice (gray bars) infected intranasally with RacL11 (striped bars) or mock infected (open bars). At different days p.i., inflammatory cells were harvested by BAL, washed, and counted (A). Absolute numbers of neutrophils (B), macrophages (C), and lymphocytes (D) in BAL cells are shown. Data are presented as the mean ± standard deviation, and asterisks indicate statistically significant differences (P < 0.05).

Histological analysis of lung sections. (A) A total lung inflammation score was determined for two mice in each group (four lung sections per mouse) and graded on a scale of 0+ (normal) to 5+ (severe). Lung sections of wild-type infected (white bars) or CCL3^−/− inoculated mice (light gray bars) were analyzed on days 1, 2, and 4 p.i. (B) H&E-stained images showing histological features in the lungs of wild-type (WT) and CCL3^−/− infected mice at days 1, 2, and 4 p.i. Bars, 100 μm. IP, interstitial pneumonia (m, mild); PE, perivascular edema; V, vasculitis; B, bronchiolitis.

Quantitation of the influx of immune cells into the airways of wild-type BL/6 mice after reinfection. Total numbers of immune cells from wild-type BL/6 mice that were infected with 1 × 10⁴ PFU of vL11ΔgGR (black bars) or vL11ΔgG (gray bars) or mock infected (white bars) and then reinfected with virulent strain RacL11 4 weeks later. At different days after reinfection, inflammatory cells were harvested by BAL, washed, and counted (A). Absolute numbers of neutrophils (B), macrophages (C), and lymphocytes (D) in BAL cells are shown. Data are presented as the mean ± standard deviation, and asterisks indicate statistically significant differences (P < 0.05).

Infection of wild-type BL/6 or CCL3^−/− mice with EHV-1. (A) Development of mean body weights after infection. CCL3^−/− (closed symbols) or wild-type (WT) BL/6 (open symbols) mice (groups of 10) were treated intranasally with MEM (circles) or with 10⁴ PFU of RacL11 (squares). Mean body weights were determined on the day of infection (day 0) up to day 14 p.i. Mean body weights and standard deviations are shown. Asterisks indicate statistically significant differences (P < 0.05) between inoculated wild-type and CCL3^−/− mice. (B) Virus titers in lungs. Viral titers were determined in two mice of each group on days 2 and 4 p.i. Mean titers in lungs and standard deviations are shown. The limit of detection was 1 × 10¹ PFU/organ, and <1 indicates that no virus was recovered from the lungs. Asterisks indicate statistically significant differences (P < 0.05) between wild-type (white bars) and CCL3^−/− (black bars) mice.

(A) Presence of gG-specific antibodies in EHV-1-hyperimmune serum. The inhibition of binding to coated gG of polyclonal rabbit anti-gG antibodies in EHV-1-hyperimmune horse serum with an SN titer of 192 (○) or preimmune serum (□) was determined by an inhibition ELISA as described in Materials and Methods. (B) Antibodies against gG interfere with the proper function of gG as a vCKBP in vitro. Chemotaxis assays with murine macrophages were performed with 10 ng/ml murine CCL3 preincubated with 0.3 μg/ml recombinant gG in the presence or absence of 20% EHV-1-hyperimmune horse serum or preimmune serum (pre serum). Data are expressed as the mean ± standard deviation of at least three independent experiments. Asterisks indicate no statistically significant differences in CCL-3-induced chemotaxis (P > 0.1).

(A) gG of EHV-1 inhibits CCL3-induced chemotaxis of murine immune cells in vitro. Chemotaxis assays with murine neutrophils and macrophages were performed with 10 ng/ml murine CCL3 preincubated with (gray bars) or without (white bars) 0.3 μg/ml recombinant gG. Data are expressed as the mean ± standard deviation of at least three independent experiments. Asterisks indicate statistically significant differences (*, P < 0.01; **, P < 0.05). (B) In vivo relevance of gG to pathogenesis in a wild-type BL/6 murine infection model. Wild-type BL/6 mice (groups of 10) were infected intranasally with 1 × 10⁴ PFU of vL11ΔgGR (white bars) or vL11ΔgG (black bars). Mean body weights and titers in lungs at day 2 p.i., together with their standard deviations, are shown. Asterisks indicate statistically significant differences (P < 0.05) between vL11ΔgGR- and vL11ΔgG-inoculated BL/6 mice. (C) Histological analysis of lung sections. A total lung inflammation score was determined for two mice (four lung sections per mouse) in each group and graded on a scale of 0+ (normal) to 5+ (severe). Lung sections of mice infected with vL11ΔgGR (white bars) or vL11ΔgG (light gray bars) were analyzed on days 1, 2, and 4 p.i.

Infection and reinfection of wild-type BL/6 mice with EHV-1. (A) Development of mean body weights. Six-week-old BL/6 mice were infected with 1 × 10⁴ PFU of vL11ΔgG (▵) or vL11ΔgGR (⋄). One group was initially mock infected (×). Four weeks later, mice were reinfected with RacL11 at a dose of 1 × 10⁵ PFU. Mock-infected animals of the same age were used as negative controls (○). Mean body weights were determined on the day of reinfection (day 0) up to day 5 after reinfection. Mean body weights and standard deviations are shown. Asterisks indicate statistically significant differences between the different groups of mice. (B) Virus titers in lungs. Viral titers were determined in two mice of each group on days 2 and 4 after reinfection. Titers in lungs and standard deviations are shown. The limit of detection was 1 × 10¹ PFU/organ, and <1 indicates that no virus could be recovered from the lungs. Asterisks indicate statistically significant differences (P < 0.05) between primary (white bars) and vL11ΔgGR (black bars)- or vL11ΔgG (gray bars)-inoculated mice after reinfection with RacL11. (C) Histological analysis of lung sections. A total lung inflammation score was determined for two mice (four lung sections per mouse) in each group and graded on a scale of 0+ (normal) to 5+ (severe). Lung sections of naive mice (white bars) or mice that had previously been infected with vL11ΔgGR (dark gray bars) or vL11ΔgG (light gray bars) were analyzed on days 1, 2, and 4 p.i.