Fig 1.
Balb/c mice with MAYV infected below the forelimb.
Animals showing eye irritation and ruffled fur at 8 days post-infection. The irritation in the eye presented unilaterally, with tearing.
Fig 2.
Mayaro virus induces weight loss in 15-day old Balb/c mice.
Mice weight was evaluated throughout the experiment. 15-day old Balb/c mice were infected with 2.57 x 106 PFU in the rear footpad and 1.25 x 107 PFU below the forelimb by subcutaneous injection. A) Infected mice did not present a statistical difference in hind limb pad pathway. B) A statistical difference was observed for the below the forelimb pathway. C) Behavior of the groups throughout the experiment. Control groups received only PBS 1X. The animals were monitored at 24-hour intervals for weight gain or loss, and statistics were ascertained with two-way ANOVA, Tukey’s multiple comparisons test (* P value ≤ 0.05;*** P value <0.0002). N = 12 animals/group.
Fig 3.
Survival of 15-day-old Balb/c mice infected below the forelimb subcutaneously.
(N = 12 animals/group).
Table 1.
Clinical signs observed in Balb / c mice during virus infection Mayaro.
Fig 4.
Evaluation of muscle weakness in 15-day-old Balb/c mice.
Mice were subjected to the Wire-hang physical test at 24-hour intervals. 15-day old Balb/c mice were infected with 2.57 x 106 PFU in the rear footpad and 1.25 x 107 PFU below the forelimb by subcutaneous injection. Control groups received only PBS 1X. (A and B) average time that animals were able to remain attached to the grid, infected groups presented significantly lower means than the control time. (C) Behavior of the groups throughout the experiment. Statistics were performed with two-way ANOVA, Tukey’s multiple comparisons test. (* P value ≤ 0.05;** P value <0.0034; **** P value <0.0001). N = 12 animals/group.
Fig 5.
Mayaro virus-induced myositis in the ipsilateral muscle in mice infected below the right forelimb with 1.25x107 PFU.
The animals were sacrificed on days 3, 7, 10, 15 and 20 PI. for tissue damage analysis. The quadricep muscle was fixed in Karnovsky, embedded in paraffin, and 5μm sections were made and stained with HE. (A) Negative control. (B) MAYV infection at 3 days PI. (C) MAYV infection at 7 days PI (D) MAYV infection at 10 days PI (E) MAYV infection at 15 days PI. Fibers with centralized nuclei (arrowhead). (F) MAYV infection at 20 days PI. (G) Fiber in degeneration, with pale color and swollen appearance. (H) Region of degenerating fiber undergoing phagocytosis. (I) Vasculitis. (J) Histological score. Images A, B, C, D, E, F and I (200X magnification, 40 μm bar); G and H pictures (magnification 400X, bar 40 uM). N = 3 animals/group.
Fig 6.
Myositis Mayaro virus-induced contralateral muscle in mice infected below the right forelimb with 1.25x107 PFU.
The animals were sacrificed on days 3, 7, 10, 15 and 20 PI. for tissue damage analysis. The quadricep muscles were fixed in Karnovsky, embedded in paraffin, and 5μm sections were made and stained with HE. (A) Negative control. (B) MAYV infection at 3 days PI. (C) MAYV infection at 7 days PI. (D) MAYV infection at 10 days PI. (E) MAYV infection at 15 days PI. (F) MAYV infection at 20 days PI. (G) Negative control, fibers in cross-section. (H) Fibers in cross section showing centralized nuclei (arrowhead). (I) Fibers undergoing phagocytosis. (J) Histological score. Images A, B, C, D, E, F (200X magnification), G, H and I images (400X magnification). 40μm bar. N = 3 animals/group.
Fig 7.
Mayaro virus-induced arthritis in mice infected below the right forelimb with 1.25x107 PFU.
Animals were sacrificed at 3, 7, 10, 15 and 20 days p.i. for tissue damage analysis. The ankle joint was fixed in Karnovsky, decalcified in 10% EDTA, embedded in paraffin, and 5μm sections were made and stained with HE. (A) Negative control. (B) Infiltrated in the joint capsule, muscle and synovial membrane with 7 days PI. (C) Infiltrated the ligament, tendon and muscle with 10 days PI. (D) Infiltrated synovial membrane with 15 days PI. (E) MAYV infection with 20 days PI. (F) Tendonitis and myositis with 7 days PI. (G) Periostitis with 10 days PI. (H) Tenosynovitis with 15 days PI. (I) Synovitis with 7 days PI. (J) Histological score. M: muscle; T: tendon; L: ligament; B: bone; P: periosteum; SM: Synovial membrane. Images A, B, C, D, E (40X magnification, 200 μm bar); F and I images (100X magnification), G and H images (200X magnification) 100 μm bar. N = 3 animals/group.
Fig 8.
Mayaro virus-induced myositis in the ipsilateral muscle in mice infected via the rear left footpad with 2.57 x 106 PFU.
Animals were sacrificed on days 3, 7, 10, 15 and 20 PI. for tissue damage analysis. The quadricep muscle was fixed in Karnovsky, embedded in paraffin, and 5μm sections were made and stained with HE. (A) Negative control. (B) MAYV infection at 3 days PI. (C) MAYV infection at 7 days PI. (D) MAYV infection at 10 days PI. (E) MAYV infection at 15 days PI. (F) MAYV infection at 20 days PI, with centralized nuclei (arrow). (G) Histological score. Images (200X magnification, bar 40 μm) N = 3 animals/group.
Fig 9.
Mayaro virus-induced myositis in the contralateral muscle in mice infected via the rear left footpad with 2.57 x 106 PFU.
The animals were sacrificed on days 3, 7, 10, 15 and 20 PI. for tissue damage analysis. The quadricep muscles was fixed in Karnovsky, embedded in paraffin, and 5μm sections were made and stained with HE. (A) Negative control. (B) MAYV infection at 3 days PI. (C) MAYV infection at 7 days PI. (D) MAYV infection at 10 days PI. (E) MAYV infection at 15 days PI. (F) MAYV infection at 20 days PI. (G) Histological score. Images (200X magnification, 40 μm bar). N = 3 animals/group.
Fig 10.
Cytokine profile of 15-day old Balb/c mice infected below the forelimb with 1.25x107 PFU.
Serum was collected 3, 10, 15 and 30 days post-infection for analysis of cytokine levels. At day 3 PI levels of MCP-1 (A), TNF (B), INF-γ (C) and IL-6 (D) were high, and TNF (B) and INF-γ levels up to the 10th day. IL-10 (E) and IL-4 (F) levels increased at day 10 PI. Statistics were performed with one-way ANOVA, Bonferroni’s multiple comparisons test. Gray bars: Infected. Black bars: negative control. (* P value ≤ 0.05; ** P value ≤ 0.01; *** P value ≤ 0.001; **** P value ≤ 0.0001). N = 3 animals/group.
Fig 11.
Cytokine profile of 15-day old Balb/c mice infected via the rear footpad with 2.57 x 106 PFU.
Serum was collected 3, 10, 15 and 30 days post-infection for analysis of soluble factor levels. The levels of MCP-1 (A), TNF (B), INF-γ (C) and IL-6 (D) were elevated on day 3 PI. At day 10, TNF (B) and INF-γ (C) remained high and the level of IL-10 (E) was elevated. At day 30 PI TNF (B) and IL-4 (F) were present. Statistics were performed with one-way ANOVA, Bonferroni’s multiple comparisons test. Gray bars: Infected. Black bars: negative control. (* P value ≤ 0.05; ** P value ≤ 0.01; **** P value ≤ 0.0001). N = 3 animals/group.
Fig 12.
Detection of MCP-1 in muscle tissue.
In the group infected below the forelimb (1.25x107 PFU) MCP-1 was elevated only on day 3PI (A). While in the group infected via the rear footpad (2.57x106 PFU), MCP-1 was detected on days 7 and 10PI (B). Gray bars: Infected. Black bars: negative control. Statistics were performed with one-way ANOVA, Bonferroni’s multiple comparisons test. (**** P value ≤ 0.0001). N = 3 animals/group.
Fig 13.
MAYV load on organ and tissues.
15-day old Balb/c mice were infected below the forelimb with 1.25x107 PFU. At 24, 48, 72, 96, 120 and 144 hpi, liver (A), spleen (B), heart (C), serum (D), right and left ankles E and F), right and left quadricep muscles (G and H) and eyes (I) were harvested and homogenized, and the amount of infectious virus present was quantified by plaque assay on Vero cells. N = 3 animals/group.
Fig 14.
MAYV load on organ and tissues.
15-day old Balb/c mice infected in the rear footpad with 2.57 x 106 PFU. At 24, 48, 72, 96, 120 and 144 hpi, liver (A), spleen (B), heart (C), serum (D), right and left ankles E and F), right and left quadricep muscles (G and H) and eyes (I) were harvested and homogenized, and the amount of infectious virus present was quantified by plaque assay on Vero cells. N = 3 animals/group.
Fig 15.
Evaluation of the pain threshold by the von Frey method in animals infected with the Mayaro virus.
Statistics were performed with two-way ANOVA, Tukey’s multiple comparisons test (*** P value ≤ 0.001; **** P value ≤ 0.0001). N = 8 animals/group.