Development of a small animal model for deer tick virus pathogenesis mimicking human clinical outcome

Abstract

Powassan virus (POWV) is a tick-borne flavivirus that encompasses two genetic lineages, POWV (Lineage I) and deer tick virus (DTV, Lineage II). In recent years, the incidence of reported POWV disease cases has increased, coupled with an expanded geographic range of the DTV tick vector, Ixodes scapularis. POWV and DTV are serologically indistinguishable, and it is not known whether clinical manifestations, pathology, or disease outcome differ between the two viruses. Six-week-old male and female BALB/c mice were footpad-inoculated with DTV doses ranging from 101 to 105 FFU. Dose-independent mortality, morbidity, and organ viral loads were observed for mice inoculated with sequentially increasing doses of DTV. By study completion, all surviving mice had cleared their viremias but detectable levels of negative-sense DTV RNA were present in the brain, indicating viral persistence of infectious DTV in the central nervous system. For mice that succumbed to disease, neuropathology revealed meningoencephalitis characterized by microscopic lesions with widespread distribution of viral RNA in the brain. These findings, coupled with the rapid onset of neurological signs of disease and high viral titers in nervous tissue, highlight the neurotropism of DTV in this mouse model. Additionally, disease outcome for DTV-infected mice was not affected by sex, as males and females were equally susceptible to disease. This is the first study to comprehensively characterize the clinical disease outcome in a small animal model across a spectrum of POWV/DTV infection doses. Here, we developed a small animal model for DTV pathogenesis that mimics the manifestations of POWV disease in humans. Since it is currently not known whether DTV and POWV differ in their capacity to cause human disease, the animal model detailed in our study could be utilized in future comparative pathogenesis studies, or as a platform for testing the efficacy of vaccines, and anti-virals.

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. Detection of DTV in organs.

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.

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.