Fig 1.
Mortality for DTV-infected mice.
A) Kaplan-Meier survival curves for each dose cohort (n = 8 mice per dose), including mock-infected controls (n = 8). The log-rank (Mantel-Cox) test showed statistical significance for all infection doses compared to the control group. (* p < 0.05; ** p < 0.01; *** p < 0.001). The log-rank test indicated no statistically significant differences in survival between the infection doses. B) Mortality rates were recorded for each dose cohort (n = 8). C–G) Kaplan-Meier survival curves for male (n = 4) versus female (n = 4) mice inoculated with C) 101 FFU DTV, D) 102 FFU DTV, E) 103 FFU DTV, F) 104 FFU DTV, or G) 105 FFU DTV. The median survival time (MST) in days is shown for each cohort of mice. “NA” indicates that a median survival time is undefined because greater than 50% of subjects were alive at the end of the study. All mock-infected control mice survived until the end of the study and are not graphed.
Fig 2.
Weight loss for DTV-infected mice.
Mice were weighed daily, and weights are expressed as percentage of initial body weight prior to infection (study day 0). A) Weight change curves for each infection dose cohort (n = 8). B–G) Weight was monitored daily for male (n = 4) and female (n = 4) mice inoculated with B) 101 FFU DTV, C) 102 FFU DTV, D) 103 FFU DTV, E) 104 FFU DTV, F) 105 FFU DTV, or G) media. Error bars represent standard error of the mean (SEM).
Fig 3.
Clinical disease score for DTV-infected mice.
Clinical signs of disease were assessed daily, and each mouse was assigned a cumulative clinical score based on the scoring system shown in S1 Table. A) Mean clinical disease scores for each infection dose cohort (n = 8) are plotted. B–F) Daily mean clinical disease scores are plotted for male (n = 4) and female (n = 4) mice inoculated with B) 101 FFU DTV, C) 102 FFU DTV, D) 103 FFU DTV, E) 104 FFU DTV, or F) 105 FFU DTV. Error bars represent SEM.
Fig 4.
A–E) Organs were harvested at the time of euthanasia and viral loads were analyzed via q-RT-PCR. Viral load data are expressed as FFU equivalents per microgram of RNA after normalization to a standard curve. DTV titers are plotted for each mouse tissue, and horizontal bars indicate mean values for the group. Solid symbols represent mice that succumbed to disease, while open symbols represent mice that survived until the end of the study. Statistical significance between male and female titers was determined by an unpaired two-tailed t-test. * = p < 0.05. F) Male and female mice were retro-orbitally bled on alternating days and blood was analyzed for viral load via q-RT-PCR. Each symbol represents the mean viremia (n = 4), and error bars are SEM. P-values were calculated by Welch’s ANOVA. Asterisk indicates the pair that shows significant difference with Dunnett’s T3 post-hoc test.
Table 1.
Distribution and severity of histologic lesions and detection of viral RNA in the brain of DTV-infected mice.
Sagittal brain sections were examined in the rostro-caudal direction. Each mouse was represented by an H&E-stained section as well as a section stained for viral RNA (teal signal) by RNA in situ hybridization. Viral RNA staining score was graded as — (absence of staining), + (very few to low), ++ (moderate), +++ (numerous). Microscopic lesions, including microgliosis (MG), perivascular cuffing (PC), and/or neuronal necrosis (NN), were graded as – (absence of lesions), + (minimal), ++ (mild), +++ (marked). The presence of meningitis, encephalitis, or meningoencephalitis was coded in the last column of the table as M, E, or ME, respectively. NP = region not present on the slide. OB = olfactory bulb, CTX = cerebral cortex, HPF = hippocampal formation, TH = thalamus, HY = hypothalamus, MB = midbrain, P = pons, MY = medulla, CB = cerebellum.
Fig 5.
Hematoxylin and eosin-stained sections of brain from a mouse inoculated with 103 FFU DTV.
This tissue was harvested from a moribund mouse at 7 dpi and H&E stained. A) Whole brain sagittal cross-section. B) Isocortex C) Thalamus (arrows indicate eosinophilic degenerating neurons characterized by nuclear pyknosis) D) Hypothalamus E) Cerebellum (arrow points to basophilic and shrunken neurons, whereas the arrowhead points to adjacent more normal neurons). G = granule cell layer, P = Purkinje cell layer, M = molecular cell layer.
Fig 6.
Distribution of positive-sense viral RNA in the brain of a mouse inoculated with 103 FFU DTV by RNA in situ hybridization.
This tissue was harvested from a moribund mouse at 7 dpi and stained for positive-sense POWV RNA (blue-green stain) and counter-stained with Hematoxylin (purple). A) Whole brain sagittal cross-section. B) Olfactory bulb C) Isocortex D) Hippocampus E) Cerebellum (arrows point to positively-stained Purkinje cells). G = granule cell layer.