The members of the family Filoviridae
, first identified in 1967, are filamentous, enveloped, non-segmented, negative-sense RNA viruses with 19 kb genomes encoding seven structural gene products [1
]. These include marburgviruses and ebolavirus. The natural reservoirs of these negative-strand RNA viruses are thought to be various African fruit bats [2
]. Two marburgviruses, Marburg virus (MARV) and Ravn virus (RAVV) and four ebolaviruses, Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV) cause human disease [3
]. Filoviruses cause severe hemorrhagic fever in humans and non-human primates (NHP), causing epidemics at times involving hundreds of people. Filoviruses, which are classified as Category A Bioterrorism Agents by the Centers for Disease Control and Prevention (Atlanta, GA), have stability in aerosol form comparable to other lipid containing viruses such as influenza A virus, a low infectious dose (<10 pfu) by the aerosol route in NHPs, and case fatality rates as high as ~90% ([6
]). There are allegations that the former Soviet Union weaponized filoviruses [9
]. Filoviruses in aerosol form are therefore considered a possible serious threat to the health and safety of the public. A number of promising vaccines and post-exposure treatments have been developed but are not yet available [10
]. However, the efficacy of most of these vaccines and therapies has not been determined for an aerosol route of viral challenge (exceptions include [11
The mode of acquisition of viral infection in index cases is usually unknown. Secondary transmission of filovirus infection is typically thought to occur by direct contact with infected persons or infected blood or tissues. There is no strong evidence of secondary transmission by the aerosol route in African filovirus outbreaks. However, aerosol transmission is thought to be possible and may occur in conditions of lower temperature and humidity which may not have been factors in outbreaks in warmer climates [13
]. At the very least, the potential exists for aerosol transmission, given that virus is detected in bodily secretions, the pulmonary alveolar interstitial cells, and within lung spaces [14
Filovirus infection models have been developed in mice, hamsters, guinea pigs, pigs, and NHPs [15
]. Of these models however, only pigs and NHPs can be infected without virus adaptation. NHPs have demonstrated, sudden onset of fever, clinical signs involving multiple organ systems (gastrointestinal, respiratory, neurological, and vascular), liver dysfunction, coagulopathy resulting in hypovolemic shock, multi-organ system failure, and death. The typical route of inoculation for the animal models has been intramuscular (i.m.) or intraperitoneal (i.p.), despite a perceived threat by the aerosol route. Thus, well-characterized NHP models of aerosolized filovirus infections are needed for testing vaccines and therapeutics to satisfy the fulfillment of the Food and Drug Administration’s “Animal Rule” [18
Intentional direct aerosol exposure resulting in productive infection of NHPs with filoviruses has been demonstrated in a number of studies. These studies include aerosol infection of rhesus macaques (rhesus), African green monkeys (AGMs), and cynomolgus macaques (cynos) with MARV and EBOV; and vaccine studies testing vesicular stomatitis virus (VSV) or adenovirus-based vaccines against filovirus aerosol challenge of rhesus and cynos [8
]. In one study, rhesus infected by the aerosol route with EBOV had virus detected first in the lungs and bronchial washings (day 2), followed by the blood and liver (day 3) [20
]. Notably, infection of rhesus with EBOV by the aerosol route resulted in lesions consistent with those seen in i.p. infections. However, the animals that were infected with aerosolized virus also had lung lesions with a bronchocentric pattern and the heaviest antigen concentration in lung tissue adjacent to the bronchioles [13
The only published study utilizing aerosolized SUDV in NHPs demonstrated protection of cynos against aerosolized SUDV using a complex adenovirus based vector (CAdVax) vaccine system [11
]. An important finding of this study is that aerosolized SUDV is more likely than aerosolized EBOV to result in hemorrhagic pneumonia. Despite this, characterization of a NHP model of infection with aerosolized SUDV has not been reported. To this end, we exposed rhesus, cynos, and AGMs, to SUDV by the aerosol route, and monitored the clinical course of the disease. Three NHP species were chosen for this study for comparison, since each was previously used to characterize the disease course and vaccine and therapeutic efficacy tests by other routes of filovirus exposure. A primary goal of this study was to determine what differences, if any, exist in the clinical disease progression following airborne SUDV infection of these three NHP species. Clinical disease, temperature, heart rate, pressure, complete blood counts (CBC), blood chemistry, and viral load were monitored daily and the results of these observations are reported here. This report represents the first detailed characterization of the clinical disease course caused by aerosolized SUDV in rhesus, cynos, and AGMs.
3. Experimental Section
Healthy adult African green monkeys (Chlorocebus aethiops), cynomolgus macaques (Macaca fascicularis), and rhesus macaques (Macaca mulatta) of both sexes were obtained from the NHP colony at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Baseline weights were between 3.5-5.2 kg for AGMs, 5.8–8.2 kg for cynos and 3.6–5 kg for rhesus. All NHPs were surgically implanted with subcutaneous PhysioTel D-70-PCT telemetry devices (Model # TL11M2-D70-PCT), Data Sciences International, St. Paul, MN) and allowed to recover for at least 45 days before being transferred to biosafety level 4 (BSL-4) containment facilities. Animals were acclimated to the BSL-4 containment facility for at least 1 week before virus exposure and baseline telemetry data were collected during this acclimation period.
3.2. Ethics and Animal Welfare
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The facility where this research was conducted (USAMRIID) is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (Frederick, MD). Research was conducted under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at USAMRIID. Animals were provided fresh fruits, vegetables, toys, and treats for environmental enrichment. Animals were anesthetized intramuscularly prior to any procedure and all efforts were made to minimize suffering. All animals were observed twice per day as described below in the “Post-exposure Monitoring” section of these methods. Early endpoint criteria, as also described in the “Post-exposure Monitoring” section below, were used to determine when animals should be humanely euthanized.
SUDV isolate Boniface was obtained from the USAMRIID repository. This stock was derived from a fatal human case from the 1976 outbreak in Sudan [24
]. After isolation from the human case, the virus was passaged once in guinea pigs and four times in Vero cells. The stock was tested and verified negative for endotoxin, mycoplasma, adventitious agents and contamination with other filoviruses. For aerosolization, virus was diluted to an appropriate concentration with Eagle’s Minimum Essential medium with non-essential amino acids (EMEM/NEAA) containing 10% fetal bovine serum (FBS) and 0.1% gentamicin.
3.4. Aerosol Exposure
The method used for aerosol exposures was previously described [25
]. In brief, the NHPs were anesthetized by i.m. injection with tiletamine/zolazepam (6 mg/kg) or ketamine-acepromazine (9 mg/kg of ketamine and 0.1 mg/kg of acepromazine). Whole body plethysomography was performed under the plane of anesthesia (Buxco Research Systems, Wilmington, NC) for calculation of the respiratory minute volume. Animals were then exposed to SUDV in a head-only aerosol chamber within a class III biological safety cabinet for a time-calculated aerosol exposure. Aerosols were generated by a three-jet Collison nebulizer (BGI, Inc, Waltham, MA) controlled by an automated exposure control system [26
]. The average mass median aerodynamic diameters (MMAD) of the generated aerosol particles was 1.4 µm with a geometric standard deviation (GSD) of 2.1, as measured by a Model 3321 Aerodynamic Particle Sizer (TSI, St. Paul, Minnesota). The temperature of the chamber during the spray was 22–28 °C. The steady-state humidity in the aerosol chamber ranged from 35–65%. The generated aerosol was sampled with an all-glass impinger (AGI) containing 10 mL of collection medium (EMEM/NEAA, 10% FBS, 0.1% gentamicin, and 0.001% antifoam A). Immediately after the exposure, AGI samples were analyzed by performing a plaque assay on Vero 76 cells. The inhaled dose was calculated for each NHP by the following formula: Dose Vm
x t x Ce
, where Vm
=respiratory minute volume, t=duration of the exposure and Ce
=aerosol concentration [25
]. The exposure time was calculated based on the minute volume of each NHP so that the same inhaled dose could be delivered to each animal with the dosage group. 1000 pfu is a standard filovirus challenge dose in the field and the original target doses for this study were 1000 and 100 pfu. However, in the first tier of this experiment the dose was 2-fold lower than the intended 1000 pfu target dose. To maintain consistency for the remaining 5 experimental tiersthe target doses were adjusted to 500 and 50 pfu.
3.5. Plaque Assay
SUDV samples were titrated in complete EMEM/NEAA supplemented with 10% FBS and 0.1% gentamicin. Dilutions were plated in 6-well dishes containing approximately 100% confluent Vero 76 cells. After a 1 h attachment incubation at 37°C, wells were overlaid with 1% agarose in Eagle’s Basal medium (EBME) (with 10% FBS and 0.1% gentamicin) and returned to the incubator for 7 days. On day 7, a 1% agarose secondary overlay containing 5% neutral red was added and after 2 more days at 37 °C, plaques were counted.
3.6. Post-Exposure Monitoring
NHPs were examined and evaluated by study personnel for clinical signs of disease at least two times per day after exposure. Animals were observed for responsiveness and appearance and, for the morning observations, anesthetized with tiletamine/zolazepam (3 mg/kg; i.m) for the remainder of the assessment. The following assessments were made and scores recorded: telemetry device temperature (0 = no change from baseline; 1=↑≥1.5 °C; 2=≥↑2 °C; 20=↓4 °C), responsiveness/appearance (0 = normal; 2 = depression, mild unresponsiveness, becomes active when approached; 5 = head down, tucked abdomen, hunched, flushed face, grimace; 10= moderate unresponsiveness, weakness, persistent prostration but rises when approached; 20=severe unresponsiveness & prostrate), petechial rash (0 = none; 1 = barely visible; 2 = moderate; 3 = widespread), weight loss (0 = no change from baseline; 2 = ↓10%; 3 = ↓15%; 20 = ↓20%), bleeding (0 = none; 2 = blood withdraw site; 3 = rectal), urine (0 = normal; 3 = none), gastrointestinal signs (0 = normal; 1 = little stool; 2 = diarrhea or no stool; 3 = vomiting), appetite (0 = eating; 1 = no food 1 day; 2 = no food ≥ 2 days), breathing (0 = normal; 2 = tachypnea/dyspnea; 3 = labored; 20 = agonal), and dehydration (0 = not present; 2 = moderate; 3 = severe). Animals that met the clinical criteria (as defined in the approved research protocol) were humanely euthanized. The early endpoint criterion for humane euthanasia was a cumulative total score of ≥15. A necropsy was performed on each animal and tissue samples were collected for histological and virological analyses, the results of which will be a topic of a future publication.
3.7. Telemetry Data Collection and Analysis
PhysioTel D-70-PCT (TL11M2-D70-PCT) radiotelemetry devices (Data Sciences International (DSI), St. Paul, MN, USA) collected temperature, arterial pressure, lead II electrocardiogram (ECG), heart rate and activity readings for a 4 minute period every hour beginning at least 3 days prior to challenge and continuing up to 14 days post-exposure. Data was recorded by the DataQuest A.R.T 4.1 system (DSI). Pre-exposure temperature data was used to develop a baseline period to fit an autoregressive integrated moving average (ARIMA) model. Forecasted values for the post-exposure period were based on the baseline extrapolated forward in time using SAS ETS (vers. 9.2). Residual changes were determined by subtracting the predicted value from the actual value recorded for each time point. For temperature, residual changes greater than 1.5 degrees were used to compute fever duration (number of hours of significant temperature elevation), fever hours (sum of the significant temperature elevations), and average fever elevation (fever hours divided by fever duration in hours). The diurnal changes in heart rate were assayed by calculating the standard deviation of the heart rate (SD-HR) for each 24 h.
3.8. Clinical Laboratory Analysis
While the NHPs were anesthetized, blood samples were collected from a femoral vein of each animal. Approximately 1.5 mL, less than 1% of the total blood volume, was collected each time in accordance with USAMRIID animal welfare policies. On the day of aerosol challenge (day 0), the samples were collected before exposure to virus; these samples served as a baseline. Each surviving animal had samples collected daily, days 1-10 post-exposure. One rhesus that survived the viral challenge in the low viral dose group also had samples collected on days 12 and 14. Complete blood counts (CBCs) and blood chemistries were analyzed with Beckman Coulter ACT-Diff hematology (Brea, California) and Abaxis Piccolo chemistry (Union City, California) analyzers. Analysis of serum chemistry included the following concentration measurements: glucose (GLU), blood urea nitrogen (BUN), creatinine, uric acid (UA), total calcium (CA), albumin (ALB), total protein (TP), alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), gamma-glutamyltransferase (GGT), and amylase (AMY).
3.9. Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)
RNA was isolated from blood samples using the Ambion MagMAX Viral RNA Isolation Kit following the protocol that is included with the kit (Austin, Texas). SUDV specific primers (F1051 and R1130) (Invitrogen) and p1079S-MGb probe (MGB), as published in Trombley et al.
, were used to amplify the target (nucleoprotein gene) [23
]. The reactions were assembled using the Invitrogen SuperScript One-Step RT-PCR Kit plus bovine serum albumin (Ambion) and run on a Roche Light Cycler (Roche Applied Science, Indianapolis, ID) as described previously [23
]. A standard curve was made to correlate the RT-PCR results to standards with known plaque-forming unit (pfu)/mL and the results are reported as pfu/mL.
3.10. Statistical Tests
For comparison of mean time-to-moribund condition and temperature variables between groups, t-tests with stepdown bootstrap adjustment were used. Kaplan Meier survival analysis and log-rank tests with step-down Sidak adjustment were used to compare survival curves among groups. Comparison of survival rates among groups was performed with Fisher’s exact tests with step-down bootstrap adjustment. T-tests were performed to see if significant increases or decreases had occurred throughout the disease course. These were 2-tailed paired t-tests. All tests were done for each group, comparing the last day at which all animals in a group were still alive with the baseline taken on day 0 before exposure. In several cases, this value was not significant because animals within the same group progressed at different times, with some animals from a group reaching the criteria for euthanasia days before others. Therefore, t-tests were also performed between the baseline data and the days at which each animal from a group was euthanized. If either test showed significance, this value was reported. Baseline and minimum values for SD-HR were compared for each species using paired t-tests. The days to the minimum SD-HR and the days to moribund condition were also compared for each species using paired t-tests. An alpha value of 0.05 was used as the criterion for statistical significance.
Very limited data exists on SUDV infection of NHPs, with several publications describing the infection of one to three cynos as controls for vaccine studies, utilizing i.m. or aerosol routes. Cynos succumbed on days 6-8 by i.m. inoculation and days 7-9 by aerosol inoculation [11
]. A single rhesus control inoculated with SUDV by the i.m. route succumbed on day 17 post-exposure [29
]. The macaques in these studies had the presence of a rash, lymphopenia, thrombocytopenia, and increases in ALT, AST and BUN but few other clinical findings were described. Thus, the study reported here, utilizing the aerosol exposure route, represents the first detailed characterization of the disease caused by SUDV in AGMs, cynos and rhesus.
To summarize, exposure to aerosolized SUDV results in a disease course with numerous similarities among AGMs, cynos, and rhesus: with decreased platelets, kidney and liver injury, likely leading to reduced blood volume, followed by altered blood pressure and heart rate in the final stage. Dyspnea and fever develop in all three species. In addition, all three species have increases in WBC and BUN and decreases in lymphocytes, albumin and calcium throughout the disease course. Some important differences include more prominent increases in ALT, AST and ALKP in AGMs. AGMs also had the earliest fever onset, fewest occurrences of petechia and less alteration in heart rate and blood pressure. Cynos, which are typically used in vaccine studies, had shorter survival time on average, the highest frequency of petechia and the greatest alteration of heart rate. In contrast, rhesus, a species often used to test filovirus therapeutics, had the longest average survival and less severe clinical disease. Accordingly, the smallest increases in ALT, AST and ALKP and lowest viral load in the blood occurred in rhesus. The reason for the difference in peak viral load could be worthy of exploration, particularly whether it is related to the longer survival time or the recovery of one rhesus.
Disturbances in the heart rate and blood pressure coincided with the onset of fever and these changes were more pronounced in rhesus and cynos than AGMs. Increased heart rate and decreased blood pressure could be caused by a variety of factors; in addition to being a generalized response to infection and fever, any vascular leakage or coagulopathy could also be contributory. Perturbations in heart rate coinciding with fever in cynos infected with aerosolized Marburg virus,
has also been reported [22
]. Additionally, changes in blood pressure and increased pulse and respiratory rate are more pronounced in EBOV infected rhesus succumbing to infection than survivors [30
In all three species of NHP studied, a loss of the diurnal fluctuation in heart rate and reduced heart rate variability, an indicator of poor prognosis in cardiovascular diseases, was observed after viral challenge with SUDV [31
]. To the authors’ knowledge, this is the first report of such a change in in ebolavirus infection. Cardiac abnormalities, such as diffuse myocarditis and tachycardia, have been observed in humans infected with MARV and EBOV [10
]. However, similar losses of diurnal heart rate fluctuation have been reported previously in both cynos and AGMs after infection with pneumonic plague, suggesting that such a change in heart rate as observed in the present study is not unique to ebolavirus infections [32
It is possible that the WBC counts initially increased as a normal host response to infection and that the subsequent decrease was caused by destruction of the WBCs due to direct viral infection of the cells and/or by “bystander apoptosis” of lymphocytes [34
]. In another study, rhesus infected with EBOV by the aerosol route developed leukocytosis on day 4 post-exposure with peak counts between ~10,000 and 15,000 cells/µl followed by leucopenia later in infection [13
]. Likewise, consistent with the results of this study, lymphocytopenia is typically observed later in the filoviral disease course, including a study of rhesus infected with SUDV by the i.m. route [29
]. Another hallmark of filovirus infections is marked coagulopathy [16
]. Because of limitations of the blood volume collected, coagulation was not directly measured in this study. However, the significant decreases in platelet numbers that were observed in all three NHP species are consistent with what typically occurs in disseminated intravascular coagulation (DIC). The presence of a petechial rash in many of the animals and hemorrhaging from body orifices and/or venipunture sites in some monkeys were also suggestive of coagulopathy.
Increases in BUN can be caused by increased production of urea due to protein catabolism associated with fever, necrosis, and/or hemorrhage into the intestinal tract. Both BUN and creatinine are removed from blood by the kidneys and excreted in the urine; this is dependent on adequate blood flow to the kidneys and proper renal function. Although the fevers present in the monkeys in this study may have accounted for some of the increases seen in the BUN, another explanation for the increased concentrations of BUN and creatinine observed in these animals is decreased renal excretion, caused by reduced blood volume due to dehydration and/or virus-induced vascular leakage. Decreased renal excretion is supported by cage-side observations of decreased urine output in this study.
All of the NHPs in this study had significant hypoalbuminemia that was possibly caused by a combination of decreased hepatic production of albumin and albumin loss through hemorrhage and/or vascular leakage. For both groups of rhesus, the decreases seen in total protein correlated with the decreases in albumin and calcium. However the total protein concentrations in the cynos and AGMs were not as clearly linked to the degree of hypoalbuminemia and it is possible that increased production of acute phase reactant proteins and immunoglobulins by these animals partially offset the decreased albumin and calcium concentrations.
Aerosolized SUDV, reported here, and previously published characterization of aerosolized EBOV and MARV in NHPs, are summarized and compared in Table 3
]. Interestingly, cynos succumb earliest to either aerosolized SUDV or EBOV and have the greatest decreases in lymphocytes. Increases in WBCs and decreases in platelets occur in aerosolized SUDV, EBOV and MARV infection of each NHP species tested. Liver and kidney damage occur in both SUDV and MARV aerosol infected NHPs, as do blood pressure and heart rate perturbations, although these parameters were not measured in the EBOV study. Overall, the disease course among the three NHP species infected with aerosolized SUDV was similar, albeit with some nuanced differences that were previously highlighted. This, the first characterization of the clinical disease course of aerosolized SUDV in NHPs, can serve as a foundation for testing vaccines and therapeutics.
Comparison of aerosol exposure of NHPs to filoviruses.
Comparison of aerosol exposure of NHPs to filoviruses.
|Virus||SUDV||EBOV ||MARV |
|Fever Onset (d.p.e.)||3.7||4.5||3.9||5.2||3.3||5.8||5.0|
|Rash||50% (barely visible)||100% (widespread)||50% (moderate)||None||100% (prominent)||100% (prominent)||100%(Mild to moderate)|
|Viremia (peak)||3.5 x 107||1 x 108||5.5 x 105||~8 x 107||~7 x 106||~7 x 107||6 x 107|
|Renal ||BUN ↑85%||BUN ↑133%||BUN ↑49%||N.D.||N.D.||N.D.||Fibrin thrombi and acute degeneration|
|Liver (↓protein, ↓albumin)||Yes||Yes||Yes||N.D.||N.D.||N.D.||N.D.|
|Liver ||1.3:1 (AST:ALT)||3.1:1 (AST:ALT)||3.8:1 (AST:ALT)||N.D.||N.D.||N.D.||Mild lesions|
|Lymphocytes||↓62%||↓73%||↓39%||↓46%||↓76%||↓39%||Reported # not %|
|Heart Rate (Tachycardia)||Mild||Severe||Moderate||N.D.||N.D.||N.D.||Present|
|Blood Pressure (Perturbation)||Yes||Yes||Yes||N.D.||N.D.||N.D.||N.D.|