A characterization of aerosolized Sudan virus infection in African green monkeys, cynomolgus macaques, and rhesus macaques

Abstract

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and "Cuevavirus". Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4-5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.

Figure 1

Survival, clinical disease scores, and rash in NHPs exposed to aerosolized SUDV. A) Percent survival of NHP exposed via two different doses of aerosolized SUDV. B) Average clinical disease scores and C) average rash score over time after exposure of three different species of NHP to aerosolized SUDV (Figures B and C share a legend). There were six animals per group. Error bars represent the standard error of the mean. The dashed line on figure B represents the cutoff score, above which, animals were humanely euthanized.

Figure 2

Telemetry: Temperature, Heart Rate, Arterial Pressure. A) Temperature, B) heart rate and C) pressure were measured by internally implanted telemetry devices once every hour throughout the study in NHPs exposed to two different doses of aerosolized SUDV. There were six animals per group. The values shown represent the average for each group. The straight dashed lines indicate the baseline maximum and minimum average diurnal values before exposure. D) The standard deviation of the heart rate (SD-HR) was calculated, expressed as beats per minute (bpm). For all species and doses examined, the SD-HR decreased significantly post-exposure, suggesting a loss of the diurnal rhythm (p <0.01). Telemetry was not calculated for the rhesus 50 pfu group due to equipment failure.

Figure 3

Average white blood cell count (WBC), percentage of lymphocytes and platelet number. Graphs are shown for the group averages of A) WBC, B) lymphocytes, C) platelets in three NHP species after exposure to two different doses of aerosolized SUDV. Average measurements on day 0 were taken before exposure serve as a baseline. There were six animals per group. Error bars represent the standard error of the mean. The horizontal dashed line indicates the average baseline (day 0) for the 12 animals in both dosage groups. WBC increased significantly above baseline for cynos and rhesus (p<0.05). All groups had significant decrease in lymphocytes late in infection (p<0.01) except the rhesus 500 pfu group (p >0.05). Platelets decreases were significant for cyno, AGM 500 pfu and rhesus 50 pfu groups (p <0.01).

Figure 4

Blood urea nitrogen (BUN), creatinine, total calcium, albumin, and total protein averages. Graphs are shown for the group averages of A) BUN, creatinine, and total calcium, and B) albumin and total protein in three NHP species after exposure to two different doses of aerosolized SUDV. Average measurements on day 0 were taken before exposure serve as a baseline. There were six animals per group. Error bars represent the standard error of the mean. The horizontal dashed line indicates the average baseline (day 0) for the 12 animals in both dosage groups. BUN increases were significant for African green monkeys (AGM), rhesus 50 pfu, and cyno 50 pfu groups (p <0.05). Creatine increases were significant for AGM groups and rhesus 50 pfu groups (p <0.001). Calcium decreases were significant for all groups (p <0.01) except the AGM 50 pfu group. Albumin decreases were significant for all groups (p <0.01). Decreases in protein were significant for all groups (p ≤0.01) except for AGM 500 pfu and cyno 50 pfu groups.

Figure 5

Average alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALKP), gamma-glutamyltransferase (GGT), and amylase concentrations in NHPs exposed to aerosolized SUDV. Graphs are shown for the group averages of A) ALT, B) AST, C) ALKP, D) GGT, and E) amylase in three NHP species after exposure to two different doses of aerosolized SUDV. Average measurements on day 0 were taken before exposure serve as a baseline. There were six animals per group. Error bars represent the standard error of the mean. The horizontal dashed line indicates the average baseline (day 0) for the 12 animals in both dosage groups. AGM groups had significant changes in all parameters (p ≤0.01) except the AGM 50 pfu group for amylase. Cyno groups had significant increases primarily in ALKP (p ≤0.01) and the 50 pfu group for amylase (p <0.01). Rhesus groups had significant changes in AST and amylase (p ≤0.01) but not for other parameters.

Figure 6

Average SUDV viral load in blood samples drawn daily from NHPs exposed to aerosolized SUDV. SUDV genomes were quantified by real-time RT-PCR and a standard curve was used to report the results in terms of pfu/mL. Graphs are shown for group averages of SUDV pfu/mL of blood drawn each day on the day of (day 0) and following exposure to aerosolized SUDV. There were six animals per group. Error bars represent the standard error of the mean. Rhesus peak viral load was significantly less than AGMs (p <0.01) but not cynos.