Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures

Abstract

In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

Keywords: BALB/c; antiviral; ectromelia; medical countermeasure; smallpox; surrogate model; vaccine.

Figure 1

Lethal dose (LD) range survival data for determination of LD₅₀ and LD₉₀. 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.

Figure 2

Kaplan–Meier Plot for ECTV-Mos and sham animals. Two groups were assessed for data (n = 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.

Figure 3

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.

Figure 4

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.

Figure 5

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 × 10³ to 3.39 × 10⁴ 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.

Figure 6

Animal qPCR titers in tissue. (A) 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) 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.

Figure 6

Animal qPCR titers in tissue. (A) 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) 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.

Figure 7

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 10³ (4.5 days PI) to 10⁶ PFU/mL (7.5 days PI).

Figure 8

Viral load in tissues as measured by the plaque assay. (A) the first positive viral titer displayed in the spleen was observed at 3.5 days PI. Titers ranged from 10⁴ to 10⁹ PFU/g up to 7.5 days PI; (B) viral titers in the liver were first observed at 3.5 days PI. Titers ranged from 10⁴ to 10⁹ 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.

Figure 9

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.