Figure 1.
CML1 is pathogenic for athymic nude mice.
Body weight evolution (A, E), survival curve (B), viral titers (C, D, G, H) and disease indices (F) recorded in nu/nu mice infected i.n. (A–D) or i.c. by light scarification on the back (E–H). (A–B, E–F) Ten mice per group (uninfected group and CML1-infected group) were followed for 80 days for their body weight and disease progression. Survival curves (B) were built based on i.n. infected animals that were euthanized due to (i) failure in gaining weight and (ii) severe disease when compared to uninfected controls (see materials and methods section). Disease indices (F) were attributed as stated in materials and methods. (C–D, G–H) Viral loads (n = 4 mice per time point) of serum, liver or back skin samples of CML1-infected mice were determined by qPCR (see materials and methods) and plotted on a logarithmic scale as viral DNA copies per 50 µl serum or per mg tissue. Results are presented as means±standard error of the mean (SEM). ***p<0.001: statistic differences in body weight evolution (unpaired t test) and in survival (Log-rank test). Results are representative of three independent experiments.
Figure 2.
Symptoms observed at day 30 post-infection in nu/nu mice infected i.n. with CML1.
Note the vesicles localized on the leg (A and B), the swelling of the joint of the leg (B and C), and the presence of lesions along the tail (D and E). Similar symptoms are seen with i.c. infection.
Figure 3.
Histological examinations of lungs (A), proximal leg (B) and tail (C) of nu/nu mice infected i.n. with CML1.
Hematoxylin and eosin-stained tissues are shown at day 45 post-infection for uninfected nu/nu mice (upper panels) and animals infected with CML1 by i.n. instillation (lower panels). (A) In CML1 infected animals, in comparison with uninfected animals, the lung tissue is characterized by open alveolar spaces, bronchioli without changes, and some dilated vessels in the interalveolar septa. (B) The disease induced in CML1 infected animals is also characterized by a massive edema of the muscular compartment of the proximal leg. (C) Histopathological changes in the tail upon infection are indentified by a diffuse infiltrate of neutrophils that extends from the deep dermis up to the underlying bone. Magnifications are indicated on each panel, and the 10× or 20× images represent the boxed region of the corresponding 2.5× image.
Figure 4.
Histology of back skin (scarified-zone), proximal leg and tail in nu/nu infected by scarification with CML1.
(A) Hematoxylin and eosin-stained skin tissues are shown for uninfected and CML1-scarified nu/nu mice at 15, 30, 45 and 75 dpi. Inflammation is centered around hair follicles, as well as in the deep muscle, and the hyperplastic epithelium invaginates to form a crater. (B) Histological examinations of proximal leg and tail of CML1-scarified nu/nu mice are shown at 45 and 75 days after virus infection. Note the epithelial hyperplasia and ulceration in the skin of the leg and of the tail, and the massive edema that extends between individual muscle fibers of the leg at 75 dpi. Magnifications are indicated on each panel, and the 10× images represent the boxed region of the corresponding 2.5× image.
Figure 5.
Identification of changes in immune cell populations from spleen of nu/nu mice exposed to CML1.
Mice (n = 4 per group) were challenged i.n. (A–D) or i.c. (E–H) with PBS (uninfected) or with CML1 at a dose of 2.0×106 PFU. At 25 and 45 dpi for the i.n. model or at 30 and 75 dpi for the i.c. model, spleen were harvested and CD11b+F4/80+ macrophages, CD11b+Gr1+ neutrophils, DX5+ NK cells and B220+CD19+ mature B cells were identified by FACS. Results are presented as percent gated cells. Bars represent mean±SD, standard deviation (n = 4). ***p<0.001; **p<0.01 and *p<0.05: CML1 differs significantly from uninfected group as determined by unpaired t test; representative of two independent experiments.
Figure 6.
DLN responses to CML1 in nu/nu mice following i.n. or i.c. infection.
Mice received PBS (uninfected) or 2.0×106 PFU of CML1. At 25 and 45 dpi for the i.n. model (A–B) or at 30 and 75 dpi for the i.c. model (C–D), DLNs (pool of inguinal, axillary, mesenteric and lumbar lymph nodes) were collected and the CD11c+CD8α+ lymphoid and CD11c+CD11b+ myeloid DCs were identified. The magnitude of DC responses is depicted as percent gated cells plotted on box- and whiskers-graphs. Bars represent Min to Max whiskers (n = 4 individual mice for each group). Results were submitted to unpaired t test but the differences between the groups were not significant.
Figure 7.
Cytokine production in the sera of nu/nu after exposure to CML1.
Sera from mice inoculated i.n. (A) or i.c. (B), isolated at two time points after exposure to PBS or to CML1, were used to measure cytokine levels by ELISA. Data are the average±SEM (n = 4 mice for each group). ***p<0.001; **p<0.01 and *p<0.05, CML1 versus the corresponding values of the uninfected group by unpaired t test. Boxed regions represent a zoom-in of the corresponding field, i.e., IL-1β and IL-6.
Figure 8.
HPMPC protects against lethal CML1 i.n. challenge, whereas HPMPDAP showed an intermediate protection.
Nu/nu mice were inoculated i.n. with PBS (uninfected group) or with CML1 at a dose of 2.0×106 PFU (CML1, HPMPC and HPMPDAP groups). HPMPC and HPMPDAP groups were treated intraperitoneally for 3 days, starting the day of infection, with 50 mg/kg of HPMPC or HPMPDAP, respectively; uninfected and CML1 groups received a similar treatment regimen but with PBS. Animals were monitored for 70 days for body weight (A). Survival curves (B) were built based on i.n. infected animals treated or not that were euthanized due to (i) failure in gaining weight and (ii) severe illness in comparison with uninfected controls (see materials and methods). Viral loads in the serum and various organs (C) and in skin swabs of tail and leg (D) were determined by qPCR at days 25 and 45 post-infection. Data are mean±SEM. Statistical analyses were performed as described in materials and methods. Results are representative of two independent experiments.
Figure 9.
Effects of topical HPMPC and HPMPDAP against CML1 propagation in cutaneously infected nu/nu mice.
Animals were scarified with PBS (uninfected group) or with CML1 at a dose of 2.0×106 PFU (CML1, HPMPC and HPMPDAP groups). Topical application of 1% HPMPC- and HPMPDAP-cream started the day of infection for 5 days, once per day. Uninfected and CML1 groups were treated similarly with a placebo-cream. Animals were monitored for 75 days for body weight (A, n = 5), lesions development (B–C), and viral loads (D). B; Disease indices or scores were given as stated in materials and methods. Data are mean±SEM (n = 5). C; photos were taken at 4, 18, 30, 46 and 75 dpi, and show the evolution of the primary lesions and their spread to the tail. D; the disease index of the sacrificed mice (n = 4 per group) is shown. Viral loads in various tissues and swabs were determined by qPCR at days 30 and 75 post-infection. Data are mean±SEM. For statistical analyses, see materials and methods. Results are representative of two independent experiments.