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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Virology. 2015 Mar 14;481:124–135. doi: 10.1016/j.virol.2015.02.044

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

Reed F Johnson a,✉, Dima A Hammoud b, Matthew G Lackemeyer c, Srikanth Yellayi c, Jeffrey Solomon b, Jordan K Bohannon c, Krisztina B Janosko c, Catherine Jett c, Kurt Cooper c, Joseph E Blaney d, Peter B Jahrling a,c
PMCID: PMC4535421  NIHMSID: NIHMS673084  PMID: 25776759

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: Cowpox, Orthopoxvirus, Animal model, Computed tomography, CT, Pathogenesis, Aerosol inoculation

Introduction

Due to the eradication of Variola virus (VARV), the causative agent of smallpox, and the subsequent worldwide decrease in the number of vaccinated individuals, VARV constitutes a grave concern for public health if re-introduced either by accident or purposeful release. In addition, cowpox virus (CPXV) and monkeypox virus (MPXV) can cause lethal infections with case-fatality rates from MPXV infections reaching as high as 10% while case-fatalities from CPXV are rare (Jezek et al., 1988, 1983; Likos et al., 2005; Parker et al., 2007; Vorou et al., 2008). Rimoin et al. (2010) identified increased incidence of MPXV induced disease in the Democratic Republic of Congo, which further supports an increase in the orthopoxvirus naive human population. CPXV can be lethal to severely immunosuppressed individuals (Eis-Hubinger et al., 1990), but generally results in a localized infection. Based on the above considerations, investigations into orthopoxvirus pathogenesis, implementing animal models that reflect human disease, are needed to continue the development of countermeasures that has accelerated over the past decade (Chapman et al., 2010; Huggins et al., 2009; Jahrling et al., 2004; Jahrling, 2005; Sbrana et al., 2007).

The evaluation of medical countermeasures to VARV is dependent upon animal models that accurately reflect human disease. Animal models are also critical to our understanding of the pathogenesis of VARV, MPXV and CPXV. The current nonhuman primate (NHP) model of smallpox, intravenous MPXV inoculation of macaque species, does not faithfully reflect the route of human transmission, results in accelerated disease course, and skips the early events of infection that may play a role in disease staging and outcome (Earl et al., 2007, 2004, 2008; Johnson et al., 2011a; Jordan et al., 2009; Stittelaar et al., 2001). Due to successful eradication of smallpox and the rare and geographically remote nature of MPXV outbreaks, the best option for licensing new drugs and vaccines for orthopoxvirus induced diseases is extrapolation of data derived from well characterized animal models (Field et al., 2000; Geisbert and Jahrling, 2004; Kramski et al., 2010). The FDA “Animal Rule” was developed to establish guidelines for evaluation of medical countermeasures for biological threat agents in which studying in humans would be inhumane (Anon., 2009). The FDA animal rule requires that a countermeasure be evaluated in an animal model(s) in which the route of administration, quantification of exposure, incubation period, and disease progression mimics the human disease (Aebersold, 2012).

Unfortunately, existing orthopoxvirus models fail to completely mimic human disease, although potential VARV and MPXV counter-measures have been identified (Earl et al., 2004, 2008; Edghill-Smith et al., 2005a, 2005b; Hooper et al., 2004; Jahrling et al., 2004; Jordan et al., 2009; Sbrana et al., 2007; Stittelaar et al., 2001, 2005, 2006). Further complicating efforts to develop medical countermeasures to VARV and MPXV are the safety and security restrictions on the use of both viruses. MPXV is considered a Select Agent by the Centers for Disease Control and Prevention (CDC) and the CDC mandates use of biosafety laboratory-3 (BSL-3) containment. VARV research is restricted to BSL-4 containment at the CDC in the United States or the State Research Center of Virology and Biotechnology (Vector) in Russia. Furthermore direct experimentation with VARV requires World Health Organization approval. In addition, the future of VARV research is uncertain due to increasing international political interest in eradicating known VARV stocks (Damon et al., 2014; Tucker, 2011). Therefore, a more accessible NHP model that mimics all manifestations of human smallpox would serve as a distinct advantage to accelerate research and identify medical countermeasures. CPXV infection of NHPs may provide such a model.

CPXV is considered a BSL-2 pathogen and does not require Select Agent registration. CPXV is highly virulent in mice by intraperitoneal, intranasal, and aerosol routes of administration and has been used to study the efficacy of antivirals (Bray and Buller, 2004; Bray et al., 2000; Smee et al., 2008). CPXV also causes disease in NHPs. A CPXV outbreak in a European NHP holding facility was lethal to common marmosets (Martina et al., 2006; Matz-Rensing et al., 2006). Further evaluation of the CPXV isolate that induced lethal disease in that outbreak suggested that intranasal inoculation of common marmosets (Callithrix jacchus) provided some advantages over IV inoculation of MPXV in macaque species including incubation period, lower dose (lethal dose50 was < 103 PFU), and clinical presentation (Kramski et al., 2010). While establishing the common marmoset infection model was noteworthy, the size, availability, and lack of species-specific reagents hamper the further development of common marmosets as a NHP model. Therefore, evaluation of CPXV induced disease of monkeys or other NHP species may provide a suitable NHP model that more faithfully meets the Animal Rule requirements.

The route of transmission is believed to be critical to viral pathogenesis and disease presentation. Dixon hypothesized that the respiratory tract was initially infected by VARV with macrophages of the upper respiratory tract as the primary target (Dixon, 1962). The macrophages would then disseminate and seed peripheral lymphoid organs thus establishing disease (Fenner, 1977). Our initial CPXV NHP studies indicated that route of inoculation influences disease progression. We demonstrated that intravenous (IV) inoculation of CPXV Brighton Red (CPXV-BR) strain in cynomolgus monkeys (Macaca fasicularis) resulted in severe hemorrhagic disease that resembled human hemorrhagic smallpox, a rare, but nearly uniformly lethal presentation of disease (Dixon, 1962; Downie et al., 1969; Fenner, 1988; Johnson et al., 2011b). We also demonstrated that intrabronchial (IB) inoculation of CPXV-BR resulted in a systemic disease that shared many characteristics of smallpox (Smith et al., 2011). To further advance CPXV as a smallpox NHP model, we performed a small particle aerosol inoculation study. Several other groups have demonstrated that small particle aerosol (0.5 to 3 µm diameter) administration of MPXV results in disease that varied from group to group (Barnewall et al., 2012; Nalca et al., 2010; Zaucha et al., 2001). Our goal was to determine if small particle aerosol inoculation of CPXV more closely resembled human variola infection. Our data suggests that the rhesus monkeys develop a severe, lethal bronchopneumonia that could be quantified by computed tomography with little virus dissemination to other organ systems.

Results

Experimental design

Two groups of 4 rhesus monkeys were inoculated with 5 × 105 PFU or 5 × 104 PFU of CPXV-BR, by small particle aerosol inoculation. Briefly, subjects were anesthetized IM with ketamine, and placed on IV ketamine for a steady state anesthesia effect through the duration of the inoculation. Pre-inoculation aerosol characterization runs were performed on the CPXV-BR stock to determine estimated loss of titer to predict titer loss and aid determination of the duration of the exposure for individual subjects. The target doses selected for inoculation were 5 × 104 PFU and 5 × 105 PFU. The 5 × 104 PFU group received a presented dose of 4.32 × 104 PFU and the high dose 5 × 105 PFU group received a presented dose of 5.99 × 105 PFU. Subjects were given periodic physical exams as described in “Materials and methods”. Pre-inoculation physical exams and CTs were performed on days – 10, 3, 6, 9, 12, 15, 18, and 21. Physical exam with no CT was performed on day 0. On days when subjects met endpoint criteria, CT's, physical exams and blood withdrawals were performed prior to euthanasia and necropsy.

Small particle aerosol inoculation resulted in a severe respiratory disease

All 4 rhesus monkeys receiving 5 × 105 PFU met study endpoint criteria between days 7 and 9 post-inoculation (mean 8.25 days St. Dev. of 0.96 days). 50% of rhesus monkeys receiving 5 × 104 PFU met study endpoint criteria 9 days p.i., the remaining 50% survived to study end (28 days post-inoculation) (Fig. 1). Each subject that succumbed demonstrated severe respiratory distress and low peripheral oxygen saturation (Table 1). Respiratory rate peaks coincided with endpoint for 4/6 NHPs. Interestingly, the lower dose group demonstrated an increased average respiratory rate when compared to the higher dose but not for peripheral oxygen saturation (Table 1). The 5 × 105 PFU group demonstrated lower peripheral oxygen saturation than the 5 × 104 PFU group. The lowest observed peripheral oxygenation coincided with endpoint for 5/6 NHPs that met endpoint criteria. The increased respiratory rate and decreased peripheral oxygen saturation suggest that virus inoculation resulted in severe respiratory disease.

Fig. 1.

Fig. 1

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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.

Table 1.

Clinical parameters.

Dose
(PFU)		 Group
size		 Respiratory rate
 Peripheral O2 saturation
Mean peak (breaths
per minute) (range)		 Mean day of
peak (range)		 Proportion for which peak
coincided with endpoint		 Mean lowest
reading(%) (range)		 Mean day of lowest
reading (range)		 Proportion for which lowest
reading coincided with endpoint
5 × 104 4 112.5 (84–146) 9.75 (9–12) 2/2 82.25 (76–95) 9.75 (9–12) 2/2
5 × 105 4 81.25 (60–97) 8.25 (7–9) 4/4 73.5 (68–88) 8.25 (6–9) 3/4
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The hallmark skin lesion that was readily observed in human subjects as well as NHPs that were IV inoculated with MPXV was not a common observation in small particle aerosol CPXV-BR inoculated NHPs. Seven of eight study subjects did not develop skin lesions. One of eight developed less than 10 skin lesions on the face located around the eyes that were first observed from day 5 p.i. that continued to develop into typical pox-like lesions that were observable until study end (d28). Other clinical signs included: serous nasal discharge, tussis, inappetence, and dyspnea. Weights and temperatures did not significantly change throughout the experiment (data not shown).

CT indicates severe disease pathology

CT was employed for a real-time evaluation of disease progression and to determine if there was agreement to other clinical parameters. CT scans were performed pre-inoculation for baseline evaluation, then on day 3 after inoculation and every two to three days thereafter until study end. CT supported the clinical findings. Qualitatively, 5 × 105 PFU group subjects started showing CT abnormalities either at day 4 (n = 2) or day 6 (n = 2) p.i. (Fig. 2). Those abnormalities included bronchial wall thickening, peribronchovascular infiltrates, with overlapping foci of ground glass opacities, suggesting partial filling of the alveoli with fluid/exudate and/or partial collapse of the alveoli. Some of the infiltrates showed a denser consolidative pattern with three out of four subjects eventually showing frank consolidations with air bronchograms. Interestingly, the first animal to succumb to disease showed only minimal CT abnormalities at days 4 and 6 with no consolidations.

Fig. 2.

Fig. 2

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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).

In the 5 × 104 group subjects, CT abnormalities in two subjects started at day 6 while the other two showed abnormalities on day 9. The CT abnormalities were similar to the 5 × 105 group subjects including peribronchovascular infiltrates, bronchial wall thickening, and delayed consolidative patterns. The changes in general were less severe than the 5 × 105 group subjects, and lesser in the subjects that recovered (n = 2) compared to those that succumbed. The two subjects that survived to study end started showing improvement of CT abnormalities on day 15 p.i. There was further improvement over the following examinations up to day 21 p.i. when CT images appeared to approach near baseline appearance (data not shown). One of those two subjects eventually developed some peripheral cystic changes and infiltrates/fibrotic changes in the apices.

In both groups, the distribution of the infiltrates was relatively diffuse with no lobe predominance. Consolidations occurred mainly in the lower lung fields although some subjects showed apical consolidations. When we quantified the CT abnormalities, all high dose group subjects had slight increases in PCLH observed at day 3–6, followed by accelerated increased PCLH from day 6 to endpoint (Fig. 3A). The subjects in the 5 × 104 group on the other hand showed increased PCLH later in the course of disease, with the two subjects that succumbed showing higher PCLH with more accelerated increase (steeper slopes) than those that survived (Fig. 3B). In the two subjects that survived, there was a continuous increase in PCLH up to day 12 (figure). On day 15, PCLH values started to decline slowly in both surviving subjects, almost reaching baseline values on day 21.

Fig. 3.

Fig. 3

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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.

Histopathological and immunohistochemical findings of tissues

Microscopic analysis of hematoxylin and eosin stained slides revealed the following changes: three out of the four 5 × 105 dose group subjects showed necrotizing tracheitis with intraepithelial intracytoplasmic eosinophilic inclusions, while one out of the two 5 × 104 dose group subjects demonstrated the above lesions. 100% of subjects that succumbed, regardless of dose, had lung lesions characterized as bronchointerstitial necrotizing pneumonia with intraepithelial, intracytoplasmic inclusions and alveolar edema (Fig. 4A). Combined review of the CT data and the histopathology indicates that CT findings correspond to histopathology and support bronchointerstitial pneumonia (Fig. 4B).

Fig. 4.

Fig. 4

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(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.

Virology data suggests that CPXV-BR infection was limited to the respiratory tract

Quantitative PCR (qPCR) was used to determine genomic load in peripheral blood, nasal and oral swabs as described in “Materials and methods”. Unexpectedly, subjects in the 5 × 105 PFU group did not demonstrate detectable viral genome (viremia) in the blood until the subjects met endpoint criteria (Table 2). qPCR was also performed on oral and nasal swabs to establish if virus was being present at detectable quantities in secretions (Table 2). Virus genome was first detected in oral swabs by day 6 for the 5 × 105 PFU group and day 9 for the 5 × 104 PFU group were viral genome peaked at 7.17 log10 genome copies for the 5 × 105 PFU group and 5.5 log10 genome copies for the 5 × 104 PFU group. Peak viral genome load coincided with endpoint for 4/6 subjects that met endpoint criteria. Similar to the oral swabs, nasal swabs also demonstrated detectable viral genome. However, detection in the nasal swabs first occurred at days 3 and 6 post-inoculation for the high and low dose groups respectively. Peak viral genome in nasal swabs was 6.97 log10 genome copies for the 5 × 105 PFU group and 6.82 log10 genome copies for the 5 × 104 PFU group. Peak viral genome in nasals swabs coincided with endpoint for 6/6 subjects.

Table 2.

Viral load in blood and in oral and nasal swabs.

Dose
(PFU)		 Group
size		 Viremiaa
 Oral swabb
 Nasal swabb
Mean peak
(log10 gene
copy/mL)
(range)		 Mean
day of
peak
(range)		 Proportion for
which peak
coincided with
endpoint		 Mean peak (log10
genome copies
copy/mL) (range)		 Mean
day of
peak
(range)		 Proportion for
which peak
coincided with
endpoint		 Mean peak
(log10 genome
copies/mL)
(range)		 Mean
day of
peak
(range)		 Proportion for
which peak
coincided with
endpoint
5 × 105 4 6.05 (5.2–6.43) 8 (7–9) 3/3 7.17 (4.5–7.76) 7.5 (6–9) 3/4 6.97 (5.4–7.4) 6.67 (3–9) 1/3
5 × 104 4 N/A N/A N/A 5.5 (4.90–5.78) 10 (9–12) 2/2 6.82 (6.04–6.95) 13 (9–21) 1/2
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a

Sample was not collected at necropsy for one NHP from the group that received 5 × 105 PFU of CPXV-BR.

b

Viral load was below the assay limit of detection for one NHP from the group that received 5 × 104 PFU of CPXV-BR.

Plaque assay to evaluate viral titer in tissues was performed on right adrenal gland, right axillary lymph node, bone marrow, heart, inguinal lymph node, kidney, liver, lung, and spleen as described in “Materials and methods”. Not surprisingly, the lungs demonstrated the highest titer of infectious virus 5.6 log10 PFU/g for the 5 × 105 PFU group and 4.4 log10 PFU/g for the 5 × 104 PFU group (Fig. 5). The plaque assay data indicates that virus infection was localized to the lungs until day of endpoint for the 5 × 104 PFU group as no infectious virus could be detected in the other tissues assayed. qPCR was not performed on the tissues because the viral load in blood would add to total genome detection with the assay and a simple subtraction of tissue viral load and blood viral load would be inaccurate due to differences in tissue blood perfusion. The 5 × 105 PFU group however, did demonstrate low levels of infectious virus in tissues outside of the lung. The heart, kidney, liver, and spleen demonstrated 2 log10 PFU/g to 3.5 log10 PFU/g of infectious virus. Comparison of the lung tissue titer data to mean peak viral genome load in the whole blood indicates that the infectious titers observed in the heart, kidney, liver and spleen are likely due to the appearance of infectious virus in the circulating blood at endpoint rather than monocyte/macrophage driven dissemination of virus to peripheral tissues.

Fig. 5.

Fig. 5

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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.

PBMC analysis indicates expansion of adaptive immune cells and innate immune cells

At physical exam and when subjects met endpoint criteria, concentrations of circulating CD14+ (monocytes), CD16+ (NK cells), CD4+ (helper T-cell), CD8+ (killer T-cell), and CD20+ (immature B-cell) cell populations were analyzed by TruCount methodology as described in “Materials and methods”. Analysis of the PBMC profiles indicated that all cell populations studied underwent expansion beginning day 6 p.i. for the 5 × 105 PFU group. Interestingly, a brief decline from pre-inoculation was observed in the CD8+ and CD4+ T-cells and B-cells prior to a rebound that occurred beginning day 6 p.i. for the 5 × 105 PFU group. The circulating CD14+ monocyte population underwent an average 7.8 fold (St. Dev 3.1) expansion from day zero to endpoint for the group. NK cell and CD4+ cells underwent a similar expansion, but only for one individual (Fig. 6).

Fig. 6.

Fig. 6

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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”.

The 5 × 104 PFU group demonstrated different PBMC population dynamics that parsed with outcome. One, subject which succumbed, demonstrated a mild decrease in CD14+ cells (0.77 fold increase) and the other a 4.6 fold increase in CD14+ cells in circulation. Neither subject demonstrated more than a 2 fold change for NK, CD4+, CD8+ T-cells, and B-cells. The two subjects which survived to study end demonstrated increases in CD20+ cells above pre-inoculation concentrations day 18 post-inoculation that was maintained to study end. CD4+, CD8 + , and NK cells peaked on day 18 post-inoculation (Fig. 6)

Cytokine/chemokine response to aerosol CPXV-BR infection

In keeping with our previous experiments, the cytokines/chemokines monocyte chemoattractant protein-1 (MCP-1 or CCL2), interleukin-6 (IL-6), IL-8, interferon-γ (IFN-γ), soluble CD40 ligand (sCD40L), granulocyte colony stimulating factor (G-CSF), IL-12, and vascular endothelial growth factor (VEGF) were evaluated for changes associated with disease progression and are summarized in Table 3. IL-6 demonstrated the greatest change of all of the cytokines analyzed with a 52.6 fold change that occurred on day 9 for all subjects from the 5 × 104 PFU group. IL-6 demonstrated an average 18.6 fold change for 5 × 105 PFU group which occurred on day 7.25 as an average across the group. IFN-γ concentration changes were 14.7 and 19 fold for the 5 × 105 PFU and 5 × 104 PFU groups respectively. MCP-1 concentration changed 5.2 fold for the 5 × 105 PFU group and did not change for the 5 × 104 PFU group. sCD40L steadily decreased for both groups throughout the study. The remainder of the cytokines did not demonstrate greater than 2 fold changes.

Table 3.

Plasma cytokine and chemokine dynamics.

Cytokine/chemokinea Dose Mean day of cytokine
peak (range)		 Mean peak concentration
(pg/mL) (range)		 Mean fold change
from day zero		 No. NHPs with peak concentration
coinciding with endpoint criteria
SCD40L 5 × 105 −5 (−14–8) 557.1 (23.8–1573.0) −0.71 1/4
5 × 104 −0.25 (−16–12) 554.1 (247.4–996.1) −3.3 0/2
IFNγ 5 × 105 6.5 (6–8) 66 (19.9–100.1) 14.7 2/4
5 × 104 7.5 (6–9) 119.3 (61.1–140.3) 19.7 2/2
IL-6 5 × 105 7.25 (6–9) 59.3 (23.2–93.1) 18.6 2/4
5 × 104 9 (no variation) 57.3 (17.1–107.3) 52.6 2/2
IL-8 5 × 105 1 (−14–9) 211.1 (43.5–371.7) 2.2 1/4
5 × 104 3.5 (−16–12) 107.5 (74.2–154.3) 1.43 1/2
IL-12 5 × 105 2.25 (0–3) 112.1 (62.4–225.1) 1.2 0/4
5 × 104 2.75 (−16 to −15) 53.0 (24.9–96.2) 1.0 0/2
G-CSF 5 × 105 5.3 (3–9) 198.0 (62.63–519.62) 2.1 1/4
5 × 104 7.5 (0–15) 91.6 (40.88–126.24) 0.98 0/2
MCP-1 5 × 105 5.75 (0–9) 598.6 (246.7–1263) 5.2 2/4
5 × 104 9 (no variation) 335.7 (229.1–388.7) 1.0 2/2
VEGF 5 × 105 3.8 (3–6) 467.4 (94.04–1126) 1.3 0/4
5 × 104 −3.5 (−16–3) 201.7 (103.5–291.51) 1.6 0/2
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a

Sample was not collected at necropsy for one NHP from the group that received 5 × 105 PFU of CPXV-BR

CY results correlate with other study parameters

Correlation tests were performed to determine if clinical or immunological factors correlated with CT findings and thus establish biomarkers for disease progression. Pearson r tests were run to correlate peripheral oxygenation and increase in monocyte number with changes in PCLH (Fig. 7). Oxygen saturation inversely correlated with PCLH with a p-value of 0.0088 and R2 = 0.9259 for the 5 × 104 PFU group and p = 0.0008 and R2 0.9109 ((Fig. 7A) for the 5 × 105 PFU group (Fig. 7B). These data suggest that as lung abnormalities increased the oxygenation of peripheral tissue decreased which is reflected in the observed outcomes. The PCLH and circulating monocyte number suggest that as circulating monocyte number increased, PCLH also increased. Interestingly, peripheral circulating monocyte number (CD14+ by our TruCount assay) positively correlated with PCLH. Pearson r test was also used to determine correlation between circulating monocyte number and PCLH. Monocyte counts positively correlates with PCLH with a p-value of p=0.0086 and R2 = 0.7102 for the 5 × 104 PFU group and p = 0.0083 and R2 of 0.9283 (Fig. 7C) for the 5 × 105 PFU group (Fig. 7D). The strength of correlation was weaker for the lower dose group, likely due to not as rapid of a change in monocyte number and a peak followed by a decrease in the two subjects that survived.

Fig. 7.

Fig. 7

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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.

Discussion

The goal of this experiment was to determine if small particle aerosol inoculation of CPXV-BR into rhesus monkeys resulted in disease that more accurately recapitulates human smallpox than other orthopox macaque models to develop a more accessible animal model for VARV infection. An NHP model utilizing CPXV provides several advantages over MPXV, CPXV is a BSL-2 pathogen, not a select agent, and is better suited for follow up studies in mice, in which both BALB/C and C57BL/6 background knockout mice can be used for pathogenesis studies. Two groups of 4 rhesus were inoculated with 5 × 105 and 5 × 104 PFU of CPXV-BR. All 4 subjects receiving 5 × 105 PFU succumbed by day 9 p.i. and 2/4 subjects receiving 5 × 104 PFU succumbed by day 9 p.i. Disease progression was monitored by sampling during periodic physical exam and CT. The data suggest that CT can be used to monitor disease progression, and that CPXV-BR results in a severe bronchointerstitial necrotizing pneumonia, limited typical pox-like lesional disease, and limited viral dissemination. When compared to our previous CPXV-BR studies, small particle aerosol inoculation results in lethal disease, although presentation is remarkably different. Our CPXV-BR IB study demonstrated that 5 × 106 PFU was uniformly lethal with dissemination of virus to other tissues and our CPXV-BR IV inoculation resulted in a severe hemorrhagic smallpox like uniformly lethal disease with 5 × 105 PFU of CPXV-BR (Johnson et al., 2011b; Smith et al., 2011).

Similar to previous experiments with MPXV, CPXV-BR aerosol inoculated subjects did not develop severe skin lesions (Barnewall et al., 2012; Zaucha et al., 2001) when compared to minimum uniformly lethal dose intravenous inoculation of MPXV (Johnson et al., 2011a). However, Nalca et al. (2010) observed severe lesional disease in two of their MPXV aerosol inoculated subjects. In contrast to IB CPXV and MPXV inoculation of cynomolgus monkeys, small particle aerosol inoculated rhesus developed few if any lesions (Johnson et al., 2011a; Smith et al., 2011). When small particle inoculation of CPXV-BR is compared to IB MPXV at the highest dose evaluated (5 × 106 PFU 66% lethal) we observed a comparable increased respiratory rate but a severe decrease in peripheral oxygenation, indicating that the widespread lung involvement observed in small particle aerosol CPXV-BR inoculated NHPs was more detrimental and the increased respiratory rate compensated for the lung damage observed in MPXV IB inoculated subjects. Respiratory rate and O2 saturation were not measured for CPXV-BR IB inoculated subjects, so no comparisons can be made.

Although method of quantification varies between laboratories, several inferences can be made about viral load and distribution in tissues. CPXV-BR small particle aerosol resulted in limited viral dissemination. Viral load in the blood (viremia) by qPCR could not be detected until day of necropsy in small particle CPXV-BR 5 × 105 PFU inoculated subjects. Peak viremia was on average 6.05 log10 genome copy/ml, lower than previously observed by intravenous inoculation (Johnson et al., 2011a). In IB inoculated NHPs viremia could be detected between days 2 and 6 across the 4 doses tested (Smith et al., 2011). Barnewall et al. (2012) and Nalca et al. (2010) could detect viremia beginning day 4 p.i., similar to our MPXV IB study (Johnson et al., 2011a). CPXV genome could be detected in oral and nasal swabs with peaks of 7.2 and 6.9 log10 genome copies/ml of swab respectively with peaks occurring between days 3 and 21 similar to throat swab data from aerosol inoculated MPXV NHPs described by Nalca et al. (2010).

Although skin lesions are commonly considered an indication of dissemination of poxviruses due to secondary viremia, in this experiment, subjects did not have detectable viremia until necropsy by qPCR that is sensitive to 25 genome copies (Sofi Ibrahim et al., 2003). However subjects developed rhinorrhea that was detectable as early as day 3 post-inoculation. Due to irritation of the area it is more likely that the subject that developed skin lesions did so through self-inoculation after scratching its nose and rubbing or scratching its face rather than through a secondary viremia.

When compared to the MPXV aerosol, IB and CPXV-BR IB models, CPXV-BR small particle does not readily disseminate to other tissues. Viral load in tissues suggest that there was limited dissemination of virus from the lungs in small particle inoculated subjects. Infectious virus was less than 4 log10 per gram of tissue for spleen, kidney, liver and heart. In our CPXV-BR IB model we observed between 5 and 8 log10 PFU per gram of comparable tissue (Smith et al., 2011). Also, the qPCR data suggests that the viral load in the tissue was most likely due to the blood component of the tissue as our subjects were not perfused prior to tissue collection. It is possible that the difference observed between these inoculation routes is due to mechanical insult and localized deposition of concentrated virus during IB inoculation that compromises lung architecture that may facilitate entry of the virus into the bloodstream, whereas during aerosol inoculation, the virus is widely dispersed following the airflow of the lung and should not result in damage to the respiratory epithelium.

Characterization of the immune response to small particle inoculation demonstrated similarities and differences when compared to IV and IB inoculation. Subjects receiving 5 × 105 and 5 × 104 PFU of CPXV-BR small particle aerosol did not demonstrate the severe changes in PBMC and cytokine dynamics as was observed in IV inoculated subjects. When compared to equivalent doses by IB CPXV-BR inoculation, MCP-1 and IL-6 changes were similar on a mean peak fold change basis, indicating that changes in these cytokines may play a role in disease outcome. Unfortunately comparisons between PBMC dynamics cannot be made between IB and small particle aerosol inoculated subjects. When comparing the CPXV-BR IV to CPXV-BR small particle aerosol, the rapid increase in PBMCs that occurred in the IV inoculated subjects was also observed. In particular monocytes demonstrated a rapid change that correlated with the increase in PCLH. Characterizing the nature of the PBMC response is beyond the scope of the current work and investigation of T-cell, NK cell, and monocytes provides grounds for future hypothesis driven research that may provide insight into CPXV-BR pathogenesis and maturation of an effective immune response.

Because bronchopneumonia was reported as the most common complication of human smallpox (Chapman et al., 2010) CT was used during the course of this experiment to evaluate disease progression. Changes in Hounsfield units were used to quantify changes associated with lung pathology from baseline values (Solomon et al., 2014). The findings from this simple, yet effective, quantitative analysis correlated with the severity of disease qualitatively. CT findings of diffuse lung pathology were seen including bronchial wall thickening, peribronchovascular infiltrates, and patchy ground glass opacities. There were also areas of consolidation with air bronchograms. Segmental collapse occurred less commonly, more so in subjects receiving 5 × 105 PFU CPXV-BR. In this study, the CT findings retrospectively seemed to provide some prognostic information. For example, delayed presentation of lung infiltrates in both groups was associated with a milder lung disease and better prognosis, especially in the lower dose group. These data suggest that CT can be used as non-invasive biomarker of infectious disease progression. In the latter, both subjects that survived started developing abnormalities that were observed on day 9, while the non-survivors showed infiltrates earlier on day 6. Subjects with consolidations generally fared worse than those without. The general pathology however appeared phenotypically similar in both groups and in both survivors and non-survivors, with the only difference being the magnitude of lung involvement.

Correlation of lung PCLH and monocyte increases suggest that monocytes may play a role in disease progression, however histopathology did not indicate a qualitative increase in the number of alveolar macrophages in the specimens examined. Nor were the functional capabilities of monocytes or tissue macrophages evaluated in this study. Correlation between PCLH and peripheral tissue oxygenation indicates that the increase in lung pathology, due to inflammation or direct cytolysis, likely lead to poor blood oxygenation by decreasing the available surface area for oxygen exchange.

Overall, we have further demonstrated that route of inoculation of CPXV-BR alters disease presentation but not necessarily outcome. Small particle aerosol inoculation results in inoculation deep into the lungs, likely at the alveolar level. Dixon suggested that upper airway inoculation would result in infection of macrophages which would then disseminate the virus to regional lymph nodes resulting in spread of disease (Dixon, 1962). The lack of dissemination of virus into the periphery suggests that the depth of inoculum penetration stages disease that is localized. It is possible that changing the inoculation deposition depth may alter disease presentation. Aerosol inoculation would provide an improvement to existing models as it more accurately reflects human transmission of VARV. Similarities to naturally occurring MPXV and VARV induced disease include: inappetence, respiratory tract involvement, and instigation of a pro-inflammatory response that may further disease severity. In contrast, an ideal model would involve development of skin lesions and the involvement of multiple organ systems, and the observed lung disease is likely more severe than what was commonly observed in smallpox cases. As such, this model could be used to evaluate countermeasures for models mimicking a purposeful aerosol release of VARV or MPXV.

Materials and methods

Virus and cells

CPXV-BR was propagated in B-SC-1 cells at a multiplicity of infection (MOI) of 0.1 for 2 days. B-SC-1 cells were maintained in Modified Eagle's medium (MEM) (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Sigma St. Louis MO) and 1% penicillin/streptomycin at 37 °C with 5% CO2. BSC-1 cells were maintained in minimum essential medium (MEM) supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C with 5% CO2. Virus was recovered by scraping infected cells followed by a low speed centrifugation, 500 g for 10 min (min) at 4 °C then two rounds of freeze-thaw at −80°C, sonication (Misonix ultrasonic processor) for 120 s (s) at 40% power on ice followed by another round of low speed centrifugation (500 g for 10 min (min) at 4 °C ). Virus containing supernatant was cushioned over 36% sucrose, resuspended in PBS, titered and stored at − 80 °C. Inocula were prepared by rapid thaw of the virus stock sonication (Misonix ultrasonic processor, Misonix NY, USA) for 120 s (s) at 40% power on ice and dilution in PBS to the desired concentration.

Challenge and monitoring of NHPs

Eight rhesus monkeys (Macaca mulatto), ranging in weight from 4.7 to 6.6 kg were screened prior to enrollment for simian retrovirus (SRV), simian T-lymphotrophic virus (STLV), MPXV, VACV, and CPXV by quantitative polymerase chain reaction (qPCR). NHPs were also screened for detectable neutralizing antibody activity against VACV. The eight subjects were divided into two groups of 2 males and 2 females each. Groups received a target dose of 5 × 105 PFU (high dose) and 5 × 104 PFU (low dose) by small particle aerosol inhalation described below. The average weight at study start for the high dose was 5.65 kg with a standard deviation (St. Dev.) of 0.82 kg, and the low dose was 5.74 kg with a St. Dev. of 0.23 kg. Average age for the high dose group was 4.5 yr with a St. Dev. of 0.57 yr and for the low dose group was 6.5 yr with a St. Dev. of 3.3 yr. All animal procedures were approved by the National Institute of Allergy and Infectious Diseases (NIAID) Division of Clinical Research Animal Care and Use Committee, and adhered to National Institutes of Health (NIH) policies. The experiments were carried out at the NIAID Integrated Research Facility, an AAALAC and AALAS accredited facility. Prior to and post aerosol inoculation, CT, physical exams, including temperature (data not shown), weight (data not shown), and lesion counts, were performed, and blood draws and swabs from oral and nasal cavities were taken on days-18 to −14, 0, 3, 6, 9, 12, 15, 18, 21 and 28 days post-inoculation (p.i.). Subjects were maintained on isoflurane during CT procedures. NHPs were monitored at least twice daily and euthanized when they met established endpoint criteria. A pre-established scale was used to evaluate subject health and disease progression, these criteria included: (1) overall clinical appearance, (2) labored breathing, (3) activity and behavior, (4), responsiveness, and (5) core body temperatures. Moribund clinical endpoint criteria were met and NHPs humanely euthanized when clinical signs included: severe respiratory distress, severe recumbency, non-responsiveness, and hypothermia were observed. At the time of necropsy, additional blood and oral and nasal swabs were collected. In addition, select tissues were excised for virological and histopathological analysis as described below.

Small particle aerosol inoculation

Rhesus monkeys (n = 8) were exposed to CPXV-BR using a 16 liter, head only aerosol exposure chamber and an aerosol management platform (AeroMP, Biaera Technologies, USA) within a Class III biosafety cabinet (BSC) (Germfree, FL, USA). Several characterization studies ensuring viral stability and viability were conducted prior to the aerosol challenges. Virus dilutions were prepared in Eagle's Minimum Essential Medium (Lonza, MD, USA). The subjects were anesthetized with ketamine and received up to a 30 min single aerosol challenge. Aerosol particles were generated by a 3-jet Collison nebulizer (BGI Inc., MA, USA) operating at 7.5 LPM (25–30 PSIG), which produced small particles ranging from 0.5 to 3 µm in size, thus allowing for dissemination deep within the alveolar region of the respiratory tract. Aerodynamic particle size was measured by sampling approximately halfway through each NHP exposure using an Aerodynamic Particle Sizer (APS, model 3321, TSI Inc.). An all glass impinger (AGI) (Ace Glass Inc., NJ, USA) operating at a continuous flow rate of 6 LPM was used to determine the aerosol concentration within the chamber. An air wash period of 5 min between exposures allowed the particles to decay. The exposure chamber (− 0.1 in. WC) and Class III BSC (−1.0 in. WC) both maintained negative airflow throughout the challenge. Prior to an exposure, minute volume values were obtained from the NHPs using plethysmography acquisition equipment (Buxco-DSI, MN, USA). An averaged respiratory minute volume (mL/min) for a 3 min collection period was obtained and used to calculate the presented dose. A presented dose was calculated using the simplified formula D = R × Ca × T, where D is the presented or estimated inhaled dose (PFU), R is the respiratory minute volume (L/min), Ca is the aerosol concentration (PFU/L), and T is the duration of the exposure (min). These formulas have been outlined previously (Hartings and Roy, 2004). Aerosol inoculation is summarized in Table 4. Four NHPs received a presented dose of 4.32E+04 PFU (low dose referred to as 5 × 104 PFU) and four NHPs received a presented dose of 5.99E+05 PFU (high dose referred to as 5 × 105 PFU).

Table 4.

Aerosol exposure summary.

Group NHP I.D./sex Minute ventilation
(L/min.)a 		Aerosol concentration
(PFU Log10/L)		 Exposure
time (min)		 Inhaled dose
(PFU Log10)b 		AVG STDEV %CV MMAD Mass
GSD		 Endpoint
(study day)
5 × 104 PFU A6E005c-F 1.03 3.18 30 4.67 4.64 4.03 24.53 1.44 2.03 28
Z9E010-M 0.81 3.23 30 4.62 1.46 2.03   9
A9E029cd-M 1.12 3.21 30 4.74 1.41 2.06 28
A2E030-F 0.87 3.05 30 4.47 1.5 2.17   9
5 × 105 PFU A8E024-M 1.00 4.27 30 5.71 5.67 5.08 20.21 1.57 2.07   7
Z9E009-F 0.79 4.29 30 5.66 1.59 2.06   9
Z8E005-F 0.93 4.36 30 5.80 1.62 2.01   8
A9E028-M 1.00 4.27 30 5.75 1.61 2.09   9
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a

Minute ventilation from plethysmography prior to aerosol challenge.

b

Inhaled dose=Minute ventilation × aerosol concentration × exposure time.

c

Subjects that survived.

d

Subject that developed skin lesions on the face and specifically around the eyes.

Computed tomography (CT)

We acquired high resolution breath-hold chest CT data with a hybrid Philips Precedence 16P single-photon emission computed tomography (SPECT/CT) scanner (Philips USA) specifically designed to function in a BSL-4 environment. The experiments were carried out in a BSL-4 facility due to location of the equipment rather than due to biosafety concerns. CT parameters were as follows: 140 kVp; 300 mAs; 160 mm axial field of view; 0.8 mm slice thickness and 0.4 mm increment covering the whole lung. Reconstruction was performed on a 512 × 512 matrix using a lung filter.

CT image analysis

For each CT scan, the lungs were segmented using a region-growing approach (MIM Software, OH USA). A histogram-based analysis was performed within the segmented lung. Percent change in the volume of hyper-dense lung tissue (PCLH) was determined as described previously (Solomon et al., 2014). Briefly, for each animal, we first analyzed the image histogram of the baseline pre-inoculation scan. The 5% cutoff was then determined as the value below which 95% of voxels had the lowest Hounsfield unit (HU) values. As the disease progresses, a larger number of voxels will have higher HU values, due to consolidation/infiltrates that would replace the normal aerated lung tissues. These disease changes are reflected on the histogram by more voxels with intensities above the 5% cutoff value. We then determined the percentage change in hyperdense volume from baseline [(Vn – Vb)/Vb] × 100. Of the 8 subjects imaged, two subjects from the lower dose group survived and the 6 remaining succumbed after 4 imaging scans including baseline. We tracked the percentage change in hyper-dense volume for all subjects. The quantitative analysis was correlated with a blinded, qualitative evaluation of CT lung pathology over time, performed by a radiologist (DH).

Hematology and serology

Complete blood cell differential count (CBC/diff) was determined from blood samples collected in ethylenediaminetetraacetic acid (EDTA)-coated blood tubes and analyzed using a Sysmex XT2000V™ (Sysmex America, IL USA).

To determine absolute numbers of peripheral blood mononuclear cells (PBMCs), EDTA whole blood samples were collected from NHPs and analyzed using TruCount™ tubes (BD Biosciences, San Jose, CA). A commercial antibody cocktail containing markers for CD45, CD3, CD4, CD8, CD14, CD16, CD20 (BD Biosciences, CA, USA), and CD159(NKG2a) (Beckman Coulter, CA, USA) was brought up to a volume of 50 µL in phosphate buffered saline (PBS) plus 2% fetal bovine serum (FBS) and 50 µL of cocktail was transferred into a TruCount™ tube. 50 µL of EDTA-anticoagulated whole blood was then added to the tube, gently mixed by vortex and incubated for 20 min at room temperature. Samples were lysed and fixed using BD FACS Lysis Buffer (BD Biosciences CA, USA) and then analyzed on the BD Fortessa Flow Cytometer. Data were analyzed using BD FACSDiva software v6.1.3 (BD Biosciences, CA, USA). Cells were first gated on CD45, then CD3 + and CD3 − populations were selected against CD45. CD3 + populations were then gated against CD4 and CD8 subpopulations. CD3- populations were gated against CD14, NKG2a and CD20 subpopulations.

Quantification of viremia by quantitative PCR

Viral load in whole blood was determined by quantitative PCR as described previously (Johnson et al., 2011a). The concentration CPXV-BR in oral and nasal swabs and tissue samples was determined by qPCR Swabs were placed in 1 mL of sterile single strength (1 × ) PBS, soaked for 30 min-2 h, and stored at −80 °C. Whole blood and nasal and oral swabs were thawed, extracted with Trizol and screened for the presence of CPXV-BR using primers specific for the HA gene (Sofi Ibrahim et al., 2003) using an ABI 7900HT. The limit of detection was 10 gene copies/mL.

Plaque assay

Briefly, tissue samples were excised at necropsy, flash frozen, and stored at − 80 °C. A weight/volume homogenate between 10% and 30% was generated and serial 10 fold dilutions were made and incubated on confluent B-SC-1 cells overlayed with 1.6% tragacanth. Following incubation, Tragacanth overlays were removed, the monolayers were stained with crystal violet (0.1% crystal violet, 20% ethanol 10% formalin v/v), and plaques were enumerated. Viral load in lungs were the average titers of samples from the 6 lobes for each NHP. Liver titers were the average of left, right, medial, and caudal liver lobe titers. Kidney and adrenal gland titers were the average of kidneys, and adrenal gland titers.

Cytokine and chemokine analysis

The concentrations of 8 cytokines and chemokines in serum samples were analyzed using the Millipore Non-Human Primate Cytokine Panel (Millipore MA, USA). Plasma samples were transferred to a 96-well plate and incubated with antibody-coated beads directed against different cytokines or chemokines. Following incubation, the beads were washed, incubated with anti-cytokine and chemokine antibodies, and incubated with Streptavidin-R-phycoerythrin (SAV/RPE). Beads were assayed on the FlexMap 3D System (Luminex, Austin, TX).

Histopathology and immunohistochemistry

All subjects were necropsied on the day they met moribund endpoint criteria and gross lesions were recorded. Forty one tissues from all major organ systems were collected and fixed in 10% neutral buffered formalin. Samples were embedded in paraffin, sectioned at 5–6 µm, and stained with hematoxylin and eosin (H&E) according to established protocols. Immunohistochemistry (IHC) was performed as previously described (Johnson et al., 2011b). H&E and IHC sections were examined by light microscopy by the veterinary pathologists (SY).

Acknowledgments

This work was supported by the NIAID Division of Intramural Research. We are grateful to Marisa St. Claire, Russell Byrum, Dan Ragland, and the entire EVPS and IRF team for their contributions to these studies. We thank Jiro Wada for his contribution to the preparation of this manuscript. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract no. HHSN272200700016I. J.K.B. and K.B.J. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: C.J., an employee of Tunnell Government Services, Inc.; M.G.L., an employee of Lovelace Respiratory Research Institute; and H.H. and I.I., both employees of MRI Global.

References

  1. Aebersold P. FDA experience with medical countermeasures under the animal rule. Adv. Prev. Med. 2012;2012:507571. doi: 10.1155/2012/507571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anon Annual Report to Congress. Department of Defense, Chemical and Biological Defense Program. 2009 [Google Scholar]
  3. Barnewall RE, Fisher DA, Robertson AB, Vales PA, Knostman KA, Bigger JE. Inhalational monkeypox virus infection in cynomolgus macaques. Front. Cell. Infect. Microbiol. 2012;2:117. doi: 10.3389/fcimb.2012.00117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bray M, Buller M. Looking back at smallpox. Clin. Infect. Dis. 2004;38:882–889. doi: 10.1086/381976. [DOI] [PubMed] [Google Scholar]
  5. Bray M, Martinez M, Smee DF, Kefauver D, Thompson E, Huggins JW. Cidofovir protects mice against lethal aerosol or intranasal cowpox virus challenge. J. Infect. Dis. 2000;181:10–19. doi: 10.1086/315190. [DOI] [PubMed] [Google Scholar]
  6. Chapman JL, Nichols DK, Martinez MJ, Raymond JW. Animal models of orthopoxvirus infection. Vet. Pathol. 2010;47:852–870. doi: 10.1177/0300985810378649. [DOI] [PubMed] [Google Scholar]
  7. Damon IK, Damaso CR, McFadden G. Are we there yet? The smallpox research agenda using variola virus. PLoS Pathog. 2014;10:e1004108. doi: 10.1371/journal.ppat.1004108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dixon CW. Smallpox. London: Churchill; 1962. [Google Scholar]
  9. Downie AW, Fedson DS, Saint Vincent L, Rao AR, Kempe CH. Haemorrhagic smallpox. J. Hyg. 1969;67:619–629. doi: 10.1017/s0022172400042078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Earl PL, Americo JL, Wyatt LS, Anne Eller L, Montefiori DC, Byrum R, Piatak M, Lifson JD, Rao Amara R, Robinson HL, Huggins JW, Moss B. Recombinant modified vaccinia virus Ankara provides durable protection against disease caused by an immunodeficiency virus as well as long-term immunity to an orthopoxvirus in a non-human primate. Virology. 2007 doi: 10.1016/j.virol.2007.02.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Earl PL, Americo JL, Wyatt LS, Eller LA, Whitbeck JC, Cohen GH, Eisenberg RJ, Hartmann CJ, Jackson DL, Kulesh DA, Martinez MJ, Miller DM, Mucker EM, Shamblin JD, Zwiers SH, Huggins JW, Jahrling PB, Moss B. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature. 2004;428:182–185. doi: 10.1038/nature02331. [DOI] [PubMed] [Google Scholar]
  12. Earl PL, Americo JL, Wyatt LS, Espenshade O, Bassler J, Gong K, Lin S, Peters E, Rhodes L, Jr., Spano YE, Silvera PM, Moss B. Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus. Proc. Natl. Acad. Sci. USA. 2008;105:10889–10894. doi: 10.1073/pnas.0804985105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edghill-Smith Y, Bray M, Whitehouse CA, Miller D, Mucker E, Manischewitz J, King LR, Robert-Guroff M, Hryniewicz A, Venzon D, Meseda C, Weir J, Nalca A, Livingston V, Wells J, Lewis MG, Huggins J, Zwiers SH, Golding H, Franchini G. Smallpox vaccine does not protect macaques with AIDS from a lethal monkeypox virus challenge. J. Infect. Dis. 2005a;191:372–381. doi: 10.1086/427265. [DOI] [PubMed] [Google Scholar]
  14. Edghill-Smith Y, Golding H, Manischewitz J, King LR, Scott D, Bray M, Nalca A, Hooper JW, Whitehouse CA, Schmitz JE, Reimann KA, Franchini G. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat. Med. 2005b;11:740–747. doi: 10.1038/nm1261. [DOI] [PubMed] [Google Scholar]
  15. Eis-Hubinger AM, Gerritzen A, Schneweis KE, Pfeiff B, Pullmann H, Mayr A, Czerny CP. Fatal cowpox-like virus infection transmitted by cat. Lancet. 1990;336:880. doi: 10.1016/0140-6736(90)92387-w. [DOI] [PubMed] [Google Scholar]
  16. Fenner F. The eradication of smallpox. Prog. Med. Virol. 1977;23:1–21. [PubMed] [Google Scholar]
  17. Fenner F. The clinical features of smallpox. In: Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID, editors. Smallpox and its Eradication. Geneva: World Health Organization; 1988. [Google Scholar]
  18. Field HE, Barratt PC, Hughes RJ, Shield J, Sullivan ND. A fatal case of Hendra virus infection in a horse in north Queensland: clinical and epidemiological features. Aust. Vet. J. 2000;78:279–280. doi: 10.1111/j.1751-0813.2000.tb11758.x. [DOI] [PubMed] [Google Scholar]
  19. Geisbert TW, Jahrling PB. Exotic emerging viral diseases: progress and challenges. Nat. Med. 2004;10:S110–S121. doi: 10.1038/nm1142. [DOI] [PubMed] [Google Scholar]
  20. Hartings JM, Roy CJ. The automated bioaerosol exposure system: preclinical platform development and a respiratory dosimetry application with nonhuman primates. J. Pharmacol. Toxicol. Methods. 2004;49:39–55. doi: 10.1016/j.vascn.2003.07.001. [DOI] [PubMed] [Google Scholar]
  21. Hooper JW, Thompson E, Wilhelmsen C, Zimmerman M, Ichou MA, Steffen SE, Schmaljohn CS, Schmaljohn AL, Jahrling PB. Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J. Virol. 2004;78:4433–4443. doi: 10.1128/JVI.78.9.4433-4443.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huggins J, Goff A, Hensley L, Mucker E, Shamblin J, Wlazlowski C, Johnson W, Chapman J, Larsen T, Twenhafel N, Karem K, Damon IK, Byrd CM, Bolken TC, Jordan R, Hruby D. Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246. Antimicrob. Agents Chemother. 2009;53:2620–2625. doi: 10.1128/AAC.00021-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jahrling PB, Hensley LE, Martinez MJ, Leduc JW, Rubins KH, Relman DA, Huggins JW. Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox. Proc. Natl. Acad. Sci. USA. 2004;101:15196–15200. doi: 10.1073/pnas.0405954101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jahrling PB, Huggins JW. In: Orthopoxviruses in Biodefense: Research Methodology and Animal models. Swearengen JR, editor. Boca Raton, FL: CRC Press; 2005. [Google Scholar]
  25. Jezek Z, Grab B, Szczeniowski MV, Paluku KM, Mutombo M. Human monkeypox: secondary attack rates. Bull. World Health Organ. 1988;66:465–470. [PMC free article] [PubMed] [Google Scholar]
  26. Jezek Z, Gromyko AI, Szczeniowski MV. Human monkeypox. J. Hyg. Epidemiol. Microbiol. Immunol. 1983;27:13–28. [PubMed] [Google Scholar]
  27. Johnson RF, Dyall J, Ragland DR, Huzella L, Byrum R, Jett St C, Claire M, Smith AL, Paragas J, Blaney JE, Jahrling PB. Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route. J. Virol. 2011a;85:2112–2125. doi: 10.1128/JVI.01931-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnson RF, Yellayi S, Cann JA, Johnson A, Smith AL, Paragas J, Jahrling PB, Blaney JE. Cowpox virus infection of cynomolgus macaques as a model of hemorrhagic smallpox. Virology. 2011b;418:102–112. doi: 10.1016/j.virol.2011.07.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jordan R, Goff A, Frimm A, Corrado ML, Hensley LE, Byrd CM, Mucker E, Shamblin J, Bolken TC, Wlazlowski C, Johnson W, Chapman J, Twenhafel N, Tyavanagimatt S, Amantana A, Chinsangaram J, Hruby DE, Huggins J. ST-246 antiviral efficacy in a nonhuman primate monkeypox model: determination of the minimal effective dose and human dose justification. Antimicrob. Agents Chemother. 2009;53:1817–1822. doi: 10.1128/AAC.01596-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kramski M, Matz-Rensing K, Stahl-Hennig C, Kaup FJ, Nitsche A, Pauli G, Ellerbrok H. A novel highly reproducible and lethal nonhuman primate model for orthopox virus infection. PLoS One. 2010;5:el0412. doi: 10.1371/journal.pone.0010412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Likos AM, Sammons SA, Olson VA, Frace AM, Li Y, Olsen-Rasmussen M, Davidson W, Galloway R, Khristova ML, Reynolds MG, Zhao H, Carroll DS, Curns A, Formenty P, Esposito JJ, Regnery RL, Damon IK. A tale of two clades: monkeypox viruses. J. Gen. Virol. 2005;86:2661–2672. doi: 10.1099/vir.0.81215-0. [DOI] [PubMed] [Google Scholar]
  32. Martina BE, van Doornum G, Dorrestein GM, Niesters HG, Stittelaar KJ, Wolters MA, van Bolhuis HG, Osterhaus AD. Cowpox virus transmission from rats to monkeys, the Netherlands. Emerg. Infect. Dis. 2006;12:1005–1007. doi: 10.3201/eid1206.051513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matz-Rensing K, Ellerbrok H, Ehlers B, Pauli G, Floto A, Alex M, Czerny CP, Kaup FJ. Fatal poxvirus outbreak in a colony of New World monkeys. Vet. Pathol. 2006;43:212–218. doi: 10.1354/vp.43-2-212. [DOI] [PubMed] [Google Scholar]
  34. Nalca A, Livingston VA, Garza NL, Zumbrun EE, Frick OM, Chapman JL, Hartings JM. Experimental infection of cynomolgus macaques (Macaca fascicularis) with aerosolized monkeypox virus. PLoS One. 2010;5:1–12. doi: 10.1371/journal.pone.0012880. http://dx.doi.org/10.1371/journal.pone.0012880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parker AK, Parker S, Yokoyama WM, Corbett JA, Buller RM. Induction of natural killer cell responses by ectromelia virus controls infection. J. Virol. 2007;81:4070–4079. doi: 10.1128/JVI.02061-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rimoin AW, Mulembakani PM, Johnston SC, Lloyd Smith JO, Kisalu NK, Kinkela TL, Blumberg S, Thomassen HA, Pike BL, Fair JN, Wolfe ND, Shongo RL, Graham BS, Formenty P, Okitolonda E, Hensley LE, Meyer H, Wright LL, Muyembe JJ. Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo. Proc. Natl. Acad. Sci. USA. 2010;107:16262–16267. doi: 10.1073/pnas.1005769107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sbrana E, Jordan R, Hruby DE, Mateo RI, Xiao SY, Siirin M, Newman PC, AP DAR, Tesh RB. Efficacy of the antipoxvirus compound ST-246 for treatment of severe orthopoxvirus infection. Am. J. Trop. Med. Hyg. 2007;76:768–773. [PubMed] [Google Scholar]
  38. Smee DF, Gowen BB, Wandersee MK, Wong MH, Skirpstunas RT, Baldwin TJ, Hoopes JD, Sidwell RW. Differential pathogenesis of cowpox virus intranasal infections in mice induced by low and high inoculum volumes and effects of cidofovir treatment. Int. J. Antimicrob. Agents. 2008;31:352–359. doi: 10.1016/j.ijantimicag.2007.11.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith AL, St Claire M, Yellayi S. Intrabronchial inoculation of cynomolgus macaques with cowpox virus. The Journal of General Virology. 2012;93(Pt 1):159–164. doi: 10.1099/vir.0.036905-0. http://dx.doi.org/10.1099/vir.0.036905-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sofi Ibrahim M, Kulesh DA, Saleh SS, Damon IK, Esposito JJ, Schmaljohn AL, Jahrling PB. Real-time PCR assay to detect smallpox virus. J. Clin. Microbiol. 2003;41:3835–3839. doi: 10.1128/JCM.41.8.3835-3839.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Solomon J, Johnson R, Douglas D, Hammoud D. New image analysis technique for quantitative longitudinal assessment of lung pathology on CT in infected Rhesus macaques . Proceedings of the IEEE Computer-Based Medical Systems. 2014 [Google Scholar]
  42. Stittelaar KJ, Kuiken T, de Swart RL, van Amerongen G, Vos HW, Niesters HG, van Schalkwijk P, van der Kwast T, Wyatt LS, Moss B, Osterhaus AD. Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine. 2001;19:3700–3709. doi: 10.1016/s0264-410x(01)00075-5. [DOI] [PubMed] [Google Scholar]
  43. Stittelaar KJ, Neyts J, Naesens L, van Amerongen G, van Lavieren RF, Holy A, De Clercq E, Niesters HG, Fries E, Maas C, Mulder PG, van der Zeijst BA, Osterhaus AD. Antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection. Nature. 2006;439:745–748. doi: 10.1038/nature04295. [DOI] [PubMed] [Google Scholar]
  44. Stittelaar KJ, van Amerongen G, Kondova I, Kuiken T, van Lavieren RF, Pistoor FH, Niesters HG, van Doornum G, van der Zeijst BA, Mateo L, Chaplin PJ, Osterhaus AD. Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus. J. Virol. 2005;79:7845–7851. doi: 10.1128/JVI.79.12.7845-7851.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tucker JB. Breaking the deadlock over destruction of the smallpox virus stocks. Biosecur. Bioterror.: Biodef. Strateg. Pract. Sci. 2011;9:55–67. doi: 10.1089/bsp.2010.0065. [DOI] [PubMed] [Google Scholar]
  46. Vorou RM, Papavassiliou VG, Pierroutsakos IN. Cowpox virus infection: an emerging health threat. Curr. Opin. Infect. Dis. 2008;21:153–156. doi: 10.1097/QCO.0b013e3282f44c74. [DOI] [PubMed] [Google Scholar]
  47. Zaucha GM, Jahrling PB, Geisbert TW, Swearengen JR, Hensley L. The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca fascicularis) Lab. investing. J. Tech. Methods Pathol. 2001;81:1581–1600. doi: 10.1038/labinvest.3780373. [DOI] [PMC free article] [PubMed] [Google Scholar]

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