Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease

Abstract

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.

Keywords: Aerosol inoculation; Animal model; CT; Computed tomography; Cowpox; Orthopoxvirus; Pathogenesis.

Fig. 1

Kaplan–Meier analysis of the small particle aerosol inoculation of rhesus monkeys. All NHPs receiving 5 × 10⁵ PFU of CPXV-BR succumbed by day 9 post-inoculation. 50% of NHPs receiving 5 × 10⁴ PFU of CPXV-BR succumbed by day 9 post-inoculation.

Fig. 2

Representative computed tomography images of the progression of lung disease in a rhesus inoculated with 5 × 10⁵ PFU of CPXV-BR. Images were obtained at baseline, day 3, day 6, and day 9 post-inoculation. Over time, there is progressive development of peribronchovascular infiltrates and ground glass opacities (open black arrow), bronchial wall thickening (white arrowhead) and eventually (day 9) development of areas of consolidation p.i. (gray solid arrows).

Fig. 3

Analysis of CT images by measuring percent change in lung hyperdensity (PCLH) from baseline values. CT image analysis was performed as described in “Materials and methods”. Individual subjects are shown by group. (A) PCLH for 5 × 10⁵ PFU Group indicating that the PCLH increases as the part of virus induced disease. (B) PCLH for 5 × 10⁴ PFU Group indicating the rapid change in PCLH for the 2 subjects that succumbed and the delayed and less severe change for the surviving subjects.

Fig. 4

(A) Histopathology of CPXV-BR inoculated rhesus macaques. Representative manifestation of bronchointerstitial necrotizing pneumonia with alveolar edema, hemorrhage, fibrin, intraepithelial, intracytoplasmic viral inclusions, loss of bronchiolar epithelium, type II pneumocyte hyperplasia. (B) Radiopathologic correlates: CT changes and its pathological correlates seen during experimental aerosol CPXV infection. Inset (pathology) shows higher magnification of a bronchiole with peribronchiolar inflammation and edema. The CT inset shows bronchial wall thickening and surrounding infiltrates/edema.

Fig. 5

CPXV-BR demonstrated limited tissue distribution. At necropsy, tissue samples from select organs were homogenized and assayed for the presence of virus by plaque assay on VeroE6 cells. Mean virus titers are indicated for each tissue. Viral load in lungs are the mean titers of samples from the 6 lobes for each NHP. Liver titers are the mean of left, right, medial, and caudal liver lobes. Kidney and adrenal gland titers are the mean of both kidneys and adrenal glands. Bone marrow was collected from the femur for plaque assay. Bars represent standard deviation.

Fig. 6

The major PBMC populations increased in response to infection. Various PBMC cell populations were assayed periodically by TruCount analysis as described in “Materials and methods”.

Fig. 7

Correlation studies of the CT data. PCLH was compared to circulating monocyte number as well as O₂ saturation to determine if PCLH correlated to clinical and immunological data that increased or decreased as disease progressed. A. Correlation of O₂ Saturation and PCLH 5 × 10⁴ PFU Group. B. Correlation of O₂ Saturation and PCLH 5 × 10⁵ PFU Group. C. Correlation of monocyte number by TruCount and PCLH for 5 × 10⁴ PFU Group. D. Correlation of monocyte number by TruCount and PCLH for 5 × 10⁵ PFU Group. Pearson r test of correlation was performed using Graphpad Prism 6.0.