Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures
<p>Lethal dose (LD) range survival data for determination of LD<sub>50</sub> and LD<sub>90</sub>. The mean time-to-death ranged from 8.0 to 10.84 days post-infection (PI) with 700 and 2.44 PFU ECTV-Mos, respectively.</p> "> Figure 2
<p>Kaplan–Meier Plot for ECTV-Mos and sham animals. Two groups were assessed for data (<span class="html-italic">n</span> = 8 per group). All animals infected with ECTV-Mos succumbed to disease or were euthanized by 10 days PI. All mice inoculated with Dulbecco’s phosphate-buffered saline (DPBS) survived until the end of study.</p> "> Figure 3
<p>Change from baseline weight in BALB/c mice post-infection. Significant weight loss in mice infected with ECTV-Mos was observed at eight days PI. Sham-inoculated, Group 35 mice lost a statistically significant amount of weight on day 2 and day 4 PI compared to baseline weights; however, these mice gained weight back by day 6.</p> "> Figure 4
<p>Temperature (°F) with 95% confidence intervals for ectromelia virus (ECTV) and sham mice. Statistically significant changes compared to baseline occurred at random intervals following infection, however were not significant between groups until nine days PI. On study day 9, a dramatic drop in mean temperature was observed in the ECTV-Mos infected group.</p> "> Figure 5
<p>Quantifiable qPCR titers in the blood of ECTV-Mos infected mice were first measured at 4.5 days PI with titers ranging from 2.04 × 10<sup>3</sup> to 3.39 × 10<sup>4</sup> gene copies/mL. Viremia was measured in most animals starting at 5.5 days PI and viral genomic material in the blood increased through 7.5 and eight days PI.</p> "> Figure 6
<p>Animal qPCR titers in tissue. (<b>A</b>) qPCR titers in the spleen following ECTV-Mos infection. Quantifiable viral titers were first measured at 3.5 days and increased as time progressed, with the highest titers present at seven to eight days following challenge; (<b>B</b>) qPCR titers in the liver following ECTV-Mos infection. Viral titers were first measured at 3.5 days PI and slightly increased over time, with the highest titers observed between 7.5 and 8 days following challenge.</p> "> Figure 6 Cont.
<p>Animal qPCR titers in tissue. (<b>A</b>) qPCR titers in the spleen following ECTV-Mos infection. Quantifiable viral titers were first measured at 3.5 days and increased as time progressed, with the highest titers present at seven to eight days following challenge; (<b>B</b>) qPCR titers in the liver following ECTV-Mos infection. Viral titers were first measured at 3.5 days PI and slightly increased over time, with the highest titers observed between 7.5 and 8 days following challenge.</p> "> Figure 7
<p>Viremia as measured by plaque assay. The first positive viremia titers via plaque assay occurred at 4.5 days following ECTV-Mos infection. Titers varied from 10<sup>3</sup> (4.5 days PI) to 10<sup>6</sup> PFU/mL (7.5 days PI).</p> "> Figure 8
<p>Viral load in tissues as measured by the plaque assay. (<b>A</b>) the first positive viral titer displayed in the spleen was observed at 3.5 days PI. Titers ranged from 10<sup>4</sup> to 10<sup>9</sup> PFU/g up to 7.5 days PI; (<b>B</b>) viral titers in the liver were first observed at 3.5 days PI. Titers ranged from 10<sup>4</sup> to 10<sup>9</sup> PFU/g up to 7.5 days PI. Approximately 76% of liver samples collected at 3.5 days PI or later were positive for virus via the plaque assay.</p> "> Figure 9
<p>Kaplan–Meier curve showing abnormal clinical chemistry parameters for ECTV-infected animals. Blood/urea/nitrogen (BUN) and BUN/creatinine ratio levels yielded abnormal results in the majority of evaluated animals (50% or greater), where levels were abnormally high (normal BUN levels, 17.0–60.0 mg/dL). A proportion of the animals demonstrated abnormal AST (15%) and ALT (3%) levels, while 43.5% of animals demonstrated abnormal creatinine levels.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Viral Material
2.3. Animals
2.4. Lethal Dose
2.5. Natural History of ECTV Infection Following IN Challenge of BALB/c Mice
2.5.1. Study Design
2.5.2. Body Weight and Temperature Collection
2.5.3. Clinical Observations
2.5.4. Viremia and Viral Titers
2.5.5. Hematology and Clinical Chemistry
2.5.6. Pathology
2.5.7. Statistical Analysis
3. Results
3.1. Determination of ECTV-Mos Lethal Dose by Intranasal Challenge
3.2. Natural History of Infection
3.2.1. Mortality Assessment
3.2.2. Body Weight
3.2.3. Body Temperature
3.2.4. Clinical Observations
3.3. ECTV Disease Progression
3.3.1. Viral Titers via qPCR
3.3.2. Viral Titers via Plaque Assay
3.3.3. Hematology and Clinical Chemistry
3.3.4. Pathology
4. Discussion
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Group | Inoculation | No. of Animals (M/F) | Oropharyngeal Secretions Collection Times - Swabs (Hours PI) | qPCR/Plaque Assay Collection Times (Hours) | Hematology/Clinical Chemistry Collection Times (Hours PI) |
---|---|---|---|---|---|
1 | ECTV | 4/4 | 12, 24 | 12, 24 | NA |
2 | SHAM | 2/2 | |||
3 | ECTV | 4/4 | 36, 48 | 36, 48 | |
4 | SHAM | 2/2 | |||
5 | ECTV | 4/4 | 60, 72 | 60, 72 | |
6 | SHAM | 2/2 | |||
7 | ECTV | 4/4 | 84, 96 | 84, 96 | |
8 | SHAM | 2/2 | |||
9 | ECTV | 4/4 | 108, 120 | 108, 120 | |
10 | SHAM | 2/2 | |||
11 | ECTV | 4/4 | 132, 144 | 132, 144 | |
12 | SHAM | 2/2 | |||
13 | ECTV | 4/4 | 156, 168 | 156, 168 | |
14 | SHAM | 2/2 | |||
15 | ECTV | 4/4 | 180, 192 | 180, 192 | |
16 | SHAM | 2/2 | |||
17 | ECTV | 2/2 | NA | NA | 0 |
18 | ECTV | 2/2 | 12 | ||
19 | ECTV | 2/2 | 24 | ||
20 | ECTV | 2/2 | 36 | ||
21 | ECTV | 2/2 | 48 | ||
22 | ECTV | 2/2 | 60 | ||
23 | ECTV | 2/2 | 72 | ||
24 | ECTV | 2/2 | 84 | ||
25 | ECTV | 2/2 | 96 | ||
26 | ECTV | 2/2 | 108 | ||
27 | ECTV | 2/2 | 120 | ||
28 | ECTV | 2/2 | 132 | ||
29 | ECTV | 2/2 | 144 | ||
30 | ECTV | 2/2 | 156 | ||
31 | ECTV | 2/2 | 168 | ||
32 | ECTV | 2/2 | 180 | ||
33 | ECTV | 2/2 | 192 | ||
34 | ECTV | 4/4 | NA | ||
35 | SHAM | 4/4 |
Severity of Condition | Clinical Condition/Definition |
---|---|
General Appearance | |
Mild (1) | 1 of 3 of the following signs: Lethargy, Hunched Posture, Ruffled Fur |
Moderate (2) | 2 of 3 of the following signs: Lethargy, Hunched Posture, Ruffled Fur |
Severe (3) | 3 of 3 of the following signs: Lethargy, Hunched Posture, Ruffled Fur or Lesions present |
Dehydration | |
Mild (1) | Slight decrease in skin turgor, skin will not tent |
Moderate (2) | Moderate decrease in skin turgor; skin will tent and slowly (several seconds) return to normal |
Severe (3) | Skin visibly wrinkled, dry; greatly decreased skin turgor, skin will tent |
Dyspnea | |
Mild (1) | Undefined |
Moderate (2) | Demonstrates respiratory abnormalities |
Severe (3) | Demonstrates labored breathing |
Ocular Abnormalities | |
Mild (1) | Clear discharge from one or both eyes |
Moderate (2) | Clear discharge from one or both eye; partial closure of one eye |
Severe (3) | Clear discharge from one or both eyes; and/or partial closure of both eyes |
Group | Target Dose (PFU) | Back Titer (PFU) | Number of Animals Succumbed/Total Number of Animals | Mortality Proportion (Binomial Exact 95% Confidence Interval) |
---|---|---|---|---|
1 | 5 | 2.44 | 10/16 | 0.63 (0.35, 0.85) |
2 | 50 | 27.50 | 14/16 | 0.88 (0.62, 0.98) |
3 | 200 | 119.50 | 15/16 | 0.94 (0.70, 1.00) |
4 | 500 | 292.50 | 16/16 | 1.00 (0.79, 1.00) |
5 | 1000 | 700 | 16/16 | 1.00 (0.79, 1.00) |
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Garver, J.; Weber, L.; Vela, E.M.; Anderson, M.; Warren, R.; Merchlinsky, M.; Houchens, C.; Rogers, J.V. Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures. Viruses 2016, 8, 203. https://doi.org/10.3390/v8070203
Garver J, Weber L, Vela EM, Anderson M, Warren R, Merchlinsky M, Houchens C, Rogers JV. Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures. Viruses. 2016; 8(7):203. https://doi.org/10.3390/v8070203
Chicago/Turabian StyleGarver, Jennifer, Lauren Weber, Eric M. Vela, Mike Anderson, Richard Warren, Michael Merchlinsky, Christopher Houchens, and James V. Rogers. 2016. "Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures" Viruses 8, no. 7: 203. https://doi.org/10.3390/v8070203