key: cord-0857815-6ctcqqp5
authors: Cross, Robert W.; Prasad, Abhishek N.; Borisevich, Viktoriya; Geisbert, Joan B.; Agans, Krystle N.; Deer, Daniel J.; Fenton, Karla A.; Geisbert, Thomas W.
title: Crimean-Congo hemorrhagic fever virus strains Hoti and Afghanistan cause viremia and mild clinical disease in cynomolgus monkeys
date: 2020-08-13
journal: PLoS Negl Trop Dis
DOI: 10.1371/journal.pntd.0008637
sha: 704144bd337c537845dd31b87b346f3a5392204d
doc_id: 857815
cord_uid: 6ctcqqp5

BACKGROUND: Development of vaccines and therapies against Crimean-Congo hemorrhagic fever virus (CCHFV) have been hindered by the lack of immunocompetent animal models. Recently, a lethal nonhuman primate model based on the CCHFV Hoti strain was reported. CCHFV Hoti caused severe disease in cynomolgus monkeys with 75% lethality when given by the intravenous (i.v.) route. METHODOLOGY/PRINCIPAL FINDINGS: In a series of experiments, eleven cynomologus monkeys were exposed i.v. to CCHFV Hoti and four macaques were exposed i.v. to CCHFV Afghanistan. Despite transient viremia and changes in clinical pathology such as leukopenia and thrombocytopenia developing in all 15 animals, all macaques survived to the study endpoint without developing severe disease. CONCLUSIONS/SIGNIFICANCE: We were unable to attribute differences in the results of our study versus the previous report to differences in the CCHFV Hoti stock, challenge dose, origin, or age of the macaques. The observed differences are most likely the result of the outbred nature of macaques and low animal numbers often used by necessity and for ethical considerations in BSL-4 studies. Nonetheless, while we were unable to achieve severe disease or lethality, the CCHFV Hoti and Afghanistan macaque models are useful for screening medical countermeasures using biomarkers including viremia and clinical pathology to assess efficacy.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the genus Orthonairovirus (family Nairoviridae) and causes significant disease, CCHF, in humans [1, 2] . CCHFV has a broad geographic range and is endemic in more than 30 countries in Eurasia, Africa, and the Middle East. As a result of high case fatality rates, the ability to spread easily by human-tohuman contact, and the potential for aerosol release, CCHFV, like most hemorrhagic fever (HF) viruses, is classified by several US Government agencies as a Category A priority pathogen. The World Health Organization (WHO) has also recently placed CCHFV on their list of top 10 priorities for emerging virus research [3] . There are currently no FDA-approved CCHF vaccines or antiviral drugs for human use; ribavirin is indicated in some protocols, although controversy exists regarding its efficacy in treating CCHF [4] . CCHFV is usually transmitted to humans by bites from ixodid ticks of the genus Hyalomma, or through contact with blood or tissues of viremic livestock or patients [5] . Interestingly, while both wild and domestic animals develop viremia, humans and potentially nonhuman primates (NHPs) appear to be the only species where CCHFV infection causes overt disease. Human infection by CCHFV is characterized by headache, fever, myalgia, and petechial rash. In severe cases, circulatory shock and disseminated intravascular coagulation may occur with necrotic hepatitis. The global case fatality rate for CCHF has averaged around 30%; however, it varies geographically from a low of approximately 5% in Turkey [6] to a high of nearly 60% in the United Arab Emirates [7] .

Currently, there are no vaccines for CCHF available for human use. Development of vaccines has been hindered by the requirement to study CCHFV in high containment Biosafety Level (BSL)-4 facilities as well as the lack of available animal models. While the STAT-1 and IFNAR knockout mouse models are a significant advance in terms of animal models for CCHF [8] [9] [10] , there remains a clear need for immunocompetent animal models that faithfully reproduce human CCHF. Numerous attempts to develop such models have been made [11] . CCHFV-infection studies of mice, rats, guinea pigs, hamsters, rabbits, sheep, calves, and donkeys have demonstrated that these species exhibit low to undetectable viremia and clear the infection without overt signs of illness [12, 13] . Historically, a very small number of studies have assessed the pathogenic potential of CCHFV in NHPs, with none showing any promise regarding the development of a NHP model [12, 14, 15] . However, a lethal NHP model of CCHF using cynomolgus monkeys was recently reported [16] . In brief, this model utilizes the Hoti strain of CCHFV. Importantly, Haddock et al. reported that intravenous (i.v.) administration of 1x10 5 median tissue culture infectious dose (TCID 50 ) of CCHFV Hoti resulted in 75% lethality in macaques and that the disease observed faithfully reproduced human clinical manifestations. Here, we attempted to repeat these findings as well as to assess the pathogenic potential of a different strain of CCHF from Afghanistan in cynomolgus monkeys.

A CCHFV Afghanistan strain passage 4 (P4) isolate was obtained from the European Virus Archive (https://www.european-virus-archive.com). This virus was passed one time in SW-13 cells (ATCC CCL-105) to produce a P5 stock. Three different CCHFV Hoti stocks were obtained or prepared for this project. CCHFV Hoti (P7) was obtained from the European Virus Archive (https://www.european-virus-archive.com). In addition, the same CCHF Hoti P9 stock used in the previous study [16] that observed 75% lethality in macaques was kindly provided by Dr. Heinz Feldmann (NIAID/NIH, Rocky Mountain Laboratories, Hamilton, MT). This stock was passed one time in SW-13 cells (ATCC CCL-105) to produce a P10 stock.

Prior to challenge with CCHFV, all animals were anesthetized by intramuscular injection of ketamine. In the first study, two Chinese origin cynomolgus monkeys (one male and one female, 4-5 years of age, 3-4 kg) were exposed i.v. with a target dose of 2x10 5 pfu of cell culture passage 5 (P5) CCHFV Afghanistan while two Chinese origin cynomolgus monkeys (one male and one female,~4-5 years of age, 3-4 kg) were exposed i.v. with a target dose of 2x10 5 pfu of P10 CCHFV Hoti. In the second study, two Chinese origin cynomolgus monkeys (one male and one female,~4 years of age, 3.3-3.7 kg) and two Mauritius origin cynomolgus monkeys (both males,~4 years of age, 4.6-5.3 kg) were exposed i.v. with a target dose of 2x10 5 pfu of P10 CCHFV Hoti while one Chinese origin cynomolgus monkey (male,~4 years of age, 3.8 kg) and one Mauritius origin cynomolgus monkey (female,~4 years of age, 3.9 kg) were exposed i.v. with a target dose of 2x10 5 pfu of P9 CCHFV Hoti. In the third study, two Chinese origin cynomolgus monkeys (one male and one female, 3-8 years of age, 3-5.6 kg) were exposed i.v. with a target dose of 2x10 5 pfu of P7 CCHFV Hoti. In the fourth and final study, two Mauritius origin cynomolgus monkeys (both males, >18 years of age, 8.1-9.0 kg) were exposed i.v. with a target dose of 2x10 5 pfu of P5 CCHFV Afghanistan while one Mauritius origin cynomolgus monkey (male, >18 years of age, 9.6 kg) was exposed i.v. with a targer dose of 2x10 5 pfu of P10 CCHFV Hoti. At various times before and after virus challenge, animals from all four studies were monitored for clinical signs of illness including temperature, respiration quality, and clinical pathology.

The animal studies were performed at the Galveston National Laboratory, University of Texas Medical Branch at Galveston (UTMB) and were approved by the UTMB Institutional Animal Care and Use Committee. This facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and has an approved OLAW Assurance # D16-00202 (A3314-01). All steps were taken to ameliorate the welfare and to avoid the suffering of the animals in accordance with the ''Weatherall report for the use of nonhuman primates" recommendations. Animals were housed in adjoining individual primate cages allowing social interactions, under controlled conditions of humidity, temperature, and light (12-hour light/12-hour dark cycles). Food and water were available ad libitum. Animals were monitored (pre-and postinfection) and fed commercial monkey chow, treats and fruit twice daily by trained personnel. Environmental enrichment consisted of commercial toys. All procedures were conducted by trained personnel under the oversight of an attending veterinarian and all invasive clinical procedures were performed while animals were anesthetized with Ketamine or Telazol. All animals were monitored daily and scored for disease progression with an internal CCHF scoring protocol approved by the UTMB IACUC. The scoring changes measured from baseline included posture/activity level, attitude/behavior, food and water intake, weight, respiration and disease manifestations such as visible rash, hemorrhage, ecchymosis or flushed skin. A score of �9 indicated that an animal met the criteria for euthanasia. Animals were euthanized at the study endpoint using a sodium pentobarbitalbased commercial euthanasia solution.

On procedure days, 100 μl of blood from K2-EDTA collection tubes was collected prior to centrifugation, and was added to 600 μl of AVL viral lysis buffer with 6 μL carrier RNA (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for at least 4 days for stabilization. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer and 1% Betamercaptoethanol (Qiagen) in a 2 mL cryovial using a tissue lyser (Qiagen) and 0.2mm ceramic beads. The tissues sampled included axillary and inguinal lymph nodes, liver, spleen, kidney, adrenal gland, lung, pancreas, urinary bladder, ovary or testis, and eye. All blood samples were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), and from tissues using the RNeasy minikit (Qiagen) according to the manufacturer's instructions supplied with each kit.

RNA was isolated from whole blood using the Qiamp viral RNA kit (Qiagen) using 100 μL of blood into 600 μL of viral lysis buffer AVL. For tissues, approximately 100 mg was stored in 1mL RNALater (Qiagen) for at least 4 days at 4˚C for stabilization. Tissues were removed from RNALater and homogenized in 600 μL RLT buffer and 1% β-mercaptoethanol (Qiagen) using a tissue lyser and 0.2mm ceramic beads. RNA was then isolated from tissue homogenates using the RNeasy minikit (Qiagen). Viral load was assessed using primers/probe targeting the nucleocapsid protein (N) on the S segment of CCHFV gene through real-time quantitative PCR (RT-qPCR), with the probes used being strain specific. The CCHFV-Afghanistan probe was 5'6-carboxyfluorescein (6FAM)-ATC TAC ATG CAC CCT GCT GTG C-3'-Black Hole Quencher 1 (3BHQ_1) (Integrated DNA Technologies, IDT) [17] and the CCHFV-Hoti probe was 5'(6FAM)-AGA AGG GGC TTG AGT GGT T-3'-Dabcyl Quencher 3 (Dab) (IDT) [18] CCHFV RNA was detected using the CFX96 detection system (BioRad Laboratories) in onestep probe RT-qPCR kits (Qiagen) with the following cycle conditions: 50˚C for 10 min, 95˚C for 10 s, and 45 cycles of 95˚C for 10 s and 54˚C for 30 s. Threshold cycle (CT) values representing CCHFV S genomes were analyzed with CFX Manager Software, and data are shown as genome equivalents (GEq). To create the GEq standard, RNA from strain specific CCHFV stocks was extracted. The number of CCHFV S genomes was calculated using Avogadro's number and the molecular weight of the CCHFV genome. Limit of detection was 1 × 10 3 GEq/ ml for CCHFV-Afghanistan and 1 × 10 4 GEq/ml for CCHFV-Hoti.

Virus titration was performed by plaque assay using SW-13 cells (ATCC CCL-105) from all plasma and tissue samples as previously described [19] . Briefly, increasing 10-fold dilutions of the samples were adsorbed to SW-13 cell monolayers in duplicate wells (200 μl) and overlaid with 1.25% Avicel RC-581 (FMC BioPolymer, Philadelphia, PA) in 1x Eagles minimum essentials medium (MEM) with 5% FBS and 1% P/S). After 3-4 days, the overlay was removed and the monolayer stained with a 1X crystal violet/10% buffered formalin solution. The limit of detection was 25 PFU/ml for plasma.

A plaque reduction neutralization test was employed to determine neutralizing antibody titers in serum. Sera was heat inactivated at 56˚C for 30 minutes and then serially diluted two-fold starting at a 1:10 dilution and each dilution were incubated with~100 PFU of the relevant CCHFV strain (Afghanistan or Hoti) for 1 h at 37˚C. The virus:serum mixtures were then added to individual wells of 6-well plates of confluent SW-13 cell (ATCC CCL-105) monolayers in duplicate. After a one-hour incubation, cells were overlaid with 0.8% agarose in 2x Modified Eagle Medium (EMEM, LifeTechnologies). Plates were stained with neutral red 2 days after infection, and plaques were counted 24 h after staining. Endpoint titers were determined by assessing the serum dilution at which there was a � 50% reduction in plaque counts compared to control wells.

Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed from blood collected in tubes containing EDTA using a laser based hematologic analyzer (Beckman Coulter). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), calcium, creatinine (CRE), C-reactive protein (CRP), gamma-glutamyltransferase (GGT), glucose, total protein, and uric acid by using a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis).

Circulating levels of 23 cytokines/chemokines in sera of CCHFV-infected macaques were measured using the Milliplex NHP cytokine magnetic bead panel plex (Millipore Sigma) following the manufacturer's instructions. Analytes measured included IL-1β, IL-1 receptor antagonist (IL1ra), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17, IL-18, IFN-γ, granulocyte colonystimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, tumor necrosis factor (TNF)-α, transforming growth factor-α (TGF-α), soluble CD40 ligand (sCD40L), and vascular endothelial growth factor (VEGF). Multianalyte profiling was performed using a Luminex-100 system; data were analyzed using Bio-Plex Manager software (Bio-Rad).

Necropsy was performed on all subjects, and tissue samples of all major organs were collected for histopathologic and immunohistochemical (IHC) examination. Tissue sections were deparaffinized and rehydrated through xylene and graded ethanol. Slides went through heat antigen retrieval in a steamer at 95˚C for 20 mins in Sigma Citrate Buffer, pH6.0, 10× (Sigma Aldrich, St. Louis, MO). To block endogenous peroxidase activity, slides were treated with a 3% hydrogen peroxide and rinsed in distilled water. The tissue sections were processed for IHC using the Thermo Autostainer 360 (ThermoFisher, Kalamazoo, MI). Sequential 15 min incubations with avidin D and biotin solutions (Vector, Burlingame, CA) were performed to block endogenous biotin reactivity. Specific anti-CCHFV immunoreactivity was detected using a primary polyclonal rabbitα-CCHFV-NP antibody (IBT BioServices, Rockville, MD) at a 1:3200 dilution for 60 mins. A secondary biotinylated goat-α-rabbit-IgG (Vector Laboratories, Burlingame, CA) at 1:200 dilution for 30 mins followed by Vector Horseradish Peroxidase Streptavidin, R.T.U (Vector) for 30 mins. Slides were developed with Dako DAB chromagen (Dako, Carpenteria, CA) for 5 mins and counterstained with Harris hematoxylin for 30 seconds. Tissue sections from historical control STAT-1 knock out mice infected with lethal doses of either Hoti or Afghanistan CCHFV were used as positive controls. Tissue sections from uninfected macaques were used as negative controls.

We initially performed a small pilot study to confirm a previous report that CCHFV Hoti caused severe and usually lethal disease in cynomolgus monkeys when given by the i.v. route [16] , and to assess the pathogenic potential of CCHFV Afghanistan in comparison. We chose CCHFV Afghanistan because it was also isolated from a fatal human case, was a low cell culture passage isolate that had not been passaged in mice, and was from a different CCHFV clade than CCHFV Hoti [20] . In this initial study, two Chinese origin cynomolgus monkeys (subjects 1-H10-C1 and 1-H10-C2) were exposed to 2x10 5 pfu of P10 CCHFV Hoti by the i.v. route and two Chinese origin cynomolgus monkeys (subjects 1-A5-C1 and 1-A5-C2) were exposed to 2x10 5 pfu of P5 CCHFV Afghanistan by the i.v. route. All four macaques developed a transient viremia between days 2 and 5 after CCHFV challenge (Fig 1) . However, while some transient changes in clinical pathology, such as leukopenia and thrombocytopenia, were observed in all four animals (Tables 1 and 2) , none of the animals developed severe disease and all four animals survived to the study endpoint (day 28). Apart from subject 1-H10-C2, which exhibited minimal diffuse pallor of the liver, no significant findings were observed during necropsy of the animals in this study. Similarly, there was an absence of obvious histological lesions or CCHFV antigen labeling in all tissues analyzed, in contrast to tissues from murine historical controls which succumbed to disease from infection with CCHFV Afghanistan or CCHF Hoti (S1A-S1P and S1Q-S1T Fig). Neutralizing antibody titers against CCHFV were detected in all four macaques further confirming infection ( Table 3) .

Given our unexpected result that CCHFV Hoti did not cause severe or lethal infection in either of the two Chinese origin cynomolgus macaques in the initial study, we performed a second study to further explore this model. The goal of this second study was to assess the effect of different experimental conditions from the previously published experiment [16] , including the origin of the macaques employed as well as the passage history of the CCHFV Hoti challenge material. Our first study employed Chinese origin cynomolgus monkeys which are the most frequently used type of cynomolgus monkey used for infectious disease research [21] , and are commonly used as uniformly lethal models of other important HF virus infections such as those caused by Ebola, Marburg, and Lassa viruses [22] [23] [24] [25] [26] . We hypothesized that a different origin of macaque, such as Mauritius origin cynomolgus monkeys, may be differentially susceptible to CCHFV given the more homologous genetic background of Mauritius-derived animals [27] , which is thought to contribute to narrowed variability in development of immunity [28, 29] . Furthermore, marked variation in time to death to lethal infection has been observed in Ebola virus-infected Mauritius macaques compared with those of Chinese origin [30] . In addition, the CCHFV Hoti challenge material in our initial study was passaged one additional time than the virus stock previously reported to cause severe and lethal disease in macaques [16] . Therefore, we also utilized the identical stock of CCHFV Hoti virus from Haddock et al. [16] in this study. In brief, our second study employed six cynomolgus macaques: three of Chinese origin (subjects 2-H10-C1, 2-H10-C2, and 2-H9-C1) and three of Mauritius origin (subjects 2-H10-M1, 2-H10-M2, and 2-H9-M1). As we were limited in having only a very small volume of the lower passage (P9) CCHFV Hoti stock used in the previous reported work [16] , we challenged one Chinese origin animal (subject 2-H9-C1) and one Mauritius origin animal (subject 2-H9-M1) with 2x10 5 pfu of this material by the i.v. route. We then challenged two Chinese origin macaques (subjects 2-H10-C1 and 2-H10-C2) and two Mauritius origin macaques (subjects 2-H10-M1 and 2-H10-M2) with 2x10 5 pfu by the i.v. route with the same P10 CCHFV Hoti stock used in our first study. Consistent with the results of our initial study, all six macaques developed a transient viremia between days 2 and 5 after CCHFV challenge which was accompanied by similar changes in clinical pathology, yet none of the animals developed severe disease ( Table 2) . As before, no significant lesions were observed upon postmortem examination or histopathological/immunohistochemical analysis of tissues from any of the animals, and all six macaques in this study developed neutralizing antibodies. ( Table 3) .

As our first two studies produced consistent findings and as it appeared that discrepancies between our findings and those of the previous report [16] were not likely due to the origin of the macaques, we further explored the potential influence of the passage history of the CCHFV Hoti stock in being responsible for the lesser severity of disease that we observed. To do so, we obtained the lowest passage of CCHFV Hoti stock that we could procure (P7) and used this stock to challenge two Chinese origin cynomolgus macaques (subjects 3-H7-C1 and 3-H7-C2) to 2x10 5 pfu by the i.v. route. Consistent with the results of our first two studies, both animals developed a transient viremia; however, the duration of viremia was shortened to between days 2 and 3 after CCHFV challenge (Fig 1) . Again, neither of the animals exhibited any gross pathological or histological lesions, and CCHFV antigen was absent in all tissues analyzed. As with the previous two studies, changes in hematology or serum biochemistry did not coincide with the development of severe disease ( Table 2) . Both animals survived to the study endpoint (day 28) and developed neutralizing antibodies to the challenge virus ( Table 3) .

In a final attempt to model lethal disease in humans and conceivably explain the discrepancy in our results versus those in Haddock et al. [16] , we considered the age of the macaques used in these studies. In both the previous report and in our three studies, all of the animals ranged in age from 3 to 8 years. We procured three aged (> 18 years) Mauritius origin cynomolgus macaques. Two animals (subjects 4-A5-M1 and 4-A5-M2) were exposed to 2x10 5 pfu of P5 None. Subject survived to study endpoint (d28).

Thrombocytopenia (d2-7); monocytopenia (d2, 28); granulopenia (d14); leukocytosis (d10); lymphocytosis (d10, 14); thrombocytosis (d14); granulocytosis (d3, 28); > 3-fold " in ALT (d3-7); > 3-fold " in AST (d3-7); > 3-fold " in CRP (d28)

1-H10-C2 F Chinese~5 10 d0, 2, 3, 5, 7, 10, 14, 21, 28

Fever (d2, 3); anorexia (d1, 3); petechial rash (d3, 4, 7, 8); periorbital edema (d7-9); dark ocular discharge (d7). Subject survived to study endpoint (d28).

Lymphopenia (d2, 5); thrombocytopenia (d5, 7); monocytopenia (d5); granulopenia (d5-14); lymphocytosis (d10, 14); hypoalbuminemia (d5-14); hypoproteinemia (d7); > 2-fold " in AST (d3-10); > 2-fold " in ALP (d3-7)

2-H10-C1 F Chinese~4 10 d0, 2, 3, 5, 7, 10, 14, 21, 28

Anorexia (d1-7); petechial rash (d2-4). Subject survived to study endpoint (d28).

Lymphopenia (d2-5); thrombocytopenia (d3-7); monocytopenia (d2-7); granulopenia (d3-10); leukocytosis (d14); granulocytosis (d2-5, 14-28); > 2-fold " in CRE (d5); hypoalbuminemia (d5-10); hyperamylasemia (d2); hypoamylasemia (d3-7, 21,28); > 3-fold " in ALT (d2-14); > 3-fold " in AST (d2-10); > 3-fold " in CRP (d3) parentheses. Lymphopenia, granulopenia, monocytopenia, and thrombocytopenia are defined by a �35% drop in numbers of lymphocytes, granulocytes, monocytes, and platelets, respectively. Leukocytosis, lymphocytosis, monocytosis, and granulocytosis are defined by a two-fold or greater increase in numbers of white blood cells over baseline. Anorexia is defined as �25% decrease in baseline food intake. Fever is defined as a temperature more than 2.5˚F over baseline, or at least 1.5˚F over baseline and � 103.5˚F. Hypothermia is defined as a temperature �3.5˚F below baseline. Hyperglycemia is defined as a two-fold or greater increase in levels of glucose. Hypoglycemia is defined by a�25% decrease in levels of glucose. Hypoalbuminemia is defined by a �25% decrease in levels of albumin. Hypoproteinemia is defined by CCHFV Afghanistan by the i.v. route and one animal (subject 4-H10-M1) was exposed to 2x10 5 pfu of P10 CCHFV Hoti by the i.v route. Two animals (subjects 4-H10-M1and 4-A5-M1) developed viremia between days 2 and 7, with levels of infectious virus fluctuating between detectable and undetectable between days 3 and 7 in subject 41-873 (Fig 1) . Subject 4-A5-M2 exhibited low level viremia on day 3 only. Likewise, despite some perturbations in hematology and serum biochemistry profiles, none of the animals developed severe disease ( Table 2 ). All three animals had detectable neutralizing antibody titers by day 21 post-infection ( Table 3) .

For all studies, a panel of serum cytokines/chemokines was assayed from blood collections on procedure days. Subjects were grouped by challenge virus strain for comparison. Similar to what was observed in the work by Haddock et al. [16] , most analytes remained relatively static from baseline (day 0) through the study endpoint (day 21 or day 28), or showed marked variation between individual animals. However, several circulating analytes including IFN-gamma, IL-10, IL-6, IL-15, sCD40L, IL-1RA, MCP-1, and IL-18 were modestly changed over the course of infection but then returned to baseline shortly after the acute phase of disease (approximately day 5-7), indicative of a controlled response to infection (Figs 2 and 3) . Early in infection, MCP-1 was markedly increased in parallel with induction of IL-18, a recognized inducer of myloid cell activation with a predilection for NK cells [31, 32] . In line with potential myloid activation (including natural killer and dendritic cells), early increases in IL-15 was followed by controlled induction and cessation of the potent antiviral IFN-γ, followed by reduction in viral genomes detected in blood [33, 34] (Figs 1 and 2) . Early production of proinflammatory cytokine IL-6 occurred with a paralleled production of the anti-inflammatory IL-1RA from days 2-7, followed by increased levels of circulating IL-10, another potent anti-inflammatory cytokine. Interestingly, there was a marked depression in circulating sCD40L, a marker of platelet and T-cell activation that appeared to trend with the transient thrombocytopenia and lymphocytopenia observed early in infection (Fig 3, Tables 1 and 2) . Vascular leakage is a hallmark of many viral HFs including CCHFV [35] [36] [37] . Modest increases in VEGF and IL-17, which are associated with vascular function, coincided with decreases in circulating albumin and total protein in all animals to varying degrees of severity, with all of these markers returning to baseline by study endpoint (Fig 4, S2 Fig). Early studies on VEGF have determined it to be an incredibly potent agonist of vascular leakage, up to 50,000 times as potent as histamine [38] . IL-17, while not known to directly cause vascular leakage, is associated with enhanced nitric oxide production, a recognized potent vasomodulator [39, 40] .

CCHF is the most widespread tick-borne viral disease of humans, causing high rates of morbidity and mortality. Prevention and containment of CCHF outbreaks presents logistical challenges for several reasons. Tick vectors are prevalent and widespread, and CCHFV infection of domestic animals goes largely unnoticed. The problem is further amplified by the lack of an Note that due to differences in the sampling schedule between studies, data is not available for Afghanistan animals at the day 2 timepoint.

https://doi.org/10.1371/journal.pntd.0008637.g003 PLOS NEGLECTED TROPICAL DISEASES effective and available vaccine. In order to advance the development of candidate vaccines and treatments for emerging viral pathogens like CCHFV, animal models that mimic human disease are needed. Until recently, there were no animal models for CCHF. Over the last decade several immunocompromised mouse models of CCHF have been developed [8, 9] . While these models may have some utility for triaging antivirals and have been used to assess CCHFV vaccines [11, 19, [41] [42] [43] [44] [45] [46] [47] , they are not ideal, as these mice do not have intact immune systems and thus may not satisfy regulatory requirements for vaccine licensure under the US FDA Animal Rule [48] .

The recent report of an immunocompetent NHP model of CCHF was a major advance in being able to assess the efficacy of candidate countermeasures [16] . In order to confirm this CCHF model and further develop additional CCHF NHP models we exposed eleven cynomolgus macaques to CCHFV Hoti and four cynomolgus macaques to CCHFV Afghanistan. Surprisingly, under near identical test conditions we were unable to reproduce the severe disease and lethality seen in the previous study. Despite developing transient viremia and seroconversion, indications that infection with CCHFV did occur, all animals in our study only developed mild clinical disease when challenged with either the Hoti or Afghanistan strains of CCHFV. However, it should be noted that in the Haddock et al. study [16] , the authors did observe asymptomatic infection in some animals challenged with CCHFV Hoti by a different route (sub-cutaneous). In addition, a recent report by Smith et al. also assessed the pathogenic potential of the Hoti and Afghanistan strains in cynomolgus macaques, and similarly observed a lack of severe or lethal disease [49] . However, the interpretation of their findings regarding the host response to infection is complicated by the fact that a substantial portion of the animals in their study were found to be latently infected with Mycobacterium tuberculosis. While the results of this previous study may have utility in assessing co-infection, a direct comparison to the study performed by Haddock et al. [16] is challenging given this confounding factor. In an attempt to understand the discrepancy between our study and Haddock et al. [16] , we employed a number of different experimental conditions including using CCHFV Hoti stocks with different passage histories and employing cynomologus macaques of different origin as well as using aged (>18 years) animals. None of these altered test conditions affected the clinical outcome observed in the initial experiment. While we cannot exclude other unknown variables for the different results between our studies and those in Haddock et al. [16] , the most likely explanation is the outbred nature of cynomologus macaques, where small genetic differences from animal to animal may impact the outcome, as has been postulated for variable challenge outcomes with other viruses, such as SARS coronavirus, where a similar inconsistency in disease modeling between studies in macaques is apparent [50] . CCHFV infection of humans results in an average case fatality rate of 30% with fatality rates as low as 5% for outbreaks in some regions. Therefore, while it would be advantageous for screening of vaccines and therapies to have a uniformly lethal NHP model, it may not be easily achievable. There are other examples of viral infection of NHPs that produce a broad spectrum of disease from mild illness to severe disease and fatal infection such as with Lassa virus infection of rhesus and cynomolgus macaques [51] . The use of small numbers of animals in BSL-4 studies common to most BSL-4 facilities for logistical, financial, and ethical considerations may also contribute to disparate results among studies. This may be particularly more noticeable in animal models where disease manifestations vary and there is not uniform severe illness or lethality.

There is also great utility in models of varied lethality in that it affords the opportunity to study disease processes associated with early signals of disease as well as markers of survival and or persistence. For example, cytokines-chemokines and comparisons with viral clearance and comparison with human disease. Indeed, aberrations in levels of MCP-1, IL-1RA, IL-6, IFN-γ, IL-10, VEGF-A, and sCD-40L have all been detected in humans [52] [53] [54] . While only a small number of clinical studies have been performed, taking information from animal models has power in identifying new biomarkers which may play important roles in human disease severity and recovery from infection. For instance, elevated levels of circulating nitric oxide intermediates have been detected in human CCHF disease [55] which raises potential connection with elevated IL-17 levels in this study observed in this study.

In summary, the CCHFV Hoti and CCHFV Afghanistan macaque models should prove valuable for assessing promising interventions. While we did not observe severe or lethal CCHF disease, other NHP models, such as influenza [56] , SARS [50] , and Zika [57] also use biomarkers of infection, such as viremia and changes in clinical pathology parameters, to assess efficacy of medical countermeasures. 

Crimean-Congo haemorrhagic fever

Crimean-Congo hemorrhagic fever virus: new outbreaks, new discoveries

Annual review of diseases prioritized under the Research and Development Blueprint

Ribavirin for treating Crimean Congo haemorrhagic fever

Crimean-Congo Hemorrhagic Fever: Tick-Host-Virus Interactions

Crimean-Congo hemorrhagic fever in Turkey: Current status and future challenges

An outbreak of Crimean-Congo hemorrhagic fever in the United Arab Emirates, 1994-1995. The American journal of tropical medicine and hygiene

Pathogenesis and immune response of Crimean-Congo hemorrhagic fever virus in a STAT-1 knockout mouse model

Crimean-Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice

Lethal Crimean-Congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy. The Journal of infectious diseases

Hemorrhagic fever of bunyavirus etiology: disease models and progress towards new therapies

A comparative study of the Crimean hemorrhagic fever-Congo group of viruses

Viremia and antibody response of small African and laboratory animals to Crimean-Congo hemorrhagic fever virus infection. The American journal of tropical medicine and hygiene

Experimantal Congo virus (Ib -AN 7620) infection in primates

Diversity, replication, pathogenicity and cell biology of Crimean Congo hemorrhagic fever virus

A cynomolgus macaque model for Crimean-Congo haemorrhagic fever

A one-step multiplex real-time RT-PCR for the universal detection of all currently known CCHFV genotypes

Novel one-step real-time RT-PCR assay for rapid and specific diagnosis of Crimean-Congo hemorrhagic fever encountered in the Balkans

Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever

Phylogeography of Crimean Congo Hemorrhagic Fever Virus

The genetic composition of populations of cynomolgus macaques (Macaca fascicularis) used in biomedical research

Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection

Mechanisms underlying coagulation abnormalities in ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/ macrophages is a key event

Evaluation in nonhuman primates of vaccines against Ebola virus

Pathogenesis of Marburg hemorrhagic fever in cynomolgus macaques

Pathogenesis of Lassa fever in cynomolgus macaques

Mitochondrial DNA of the Mauritian macaques (Macaca fascicularis): An example of the founder effect

Mauritian Cynomolgus Macaques Share Two Exceptionally Common Major Histocompatibility Complex Class I Alleles That Restrict Simian Immunodeficiency Virus-Specific CD8+ T Cells

Unusually High Frequency MHC Class I Alleles in Mauritian Origin Cynomolgus Macaques

Implementation of a non-human primate model of Ebola disease: Infection of Mauritian cynomolgus macaques and analysis of virus populations

Inflammatory monocytes require type I interferon receptor signaling to activate NK cells via IL-18 during a mucosal viral infection

Cytokine-Mediated Activation of NK Cells during Viral Infection

IL-15 complexes induce NK-and T-cell responses independent of type I IFN signaling during rhinovirus infection

Interleukin-15 and the regulation of lymphoid homeostasis

Vascular events in viral hemorrhagic fevers: a comparative study of dengue and hantaviruses

Viral hemorrhagic fever-a vascular disease?

Evidence of vascular endothelial damage in Crimean-Congo hemorrhagic fever

Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis

Nitric oxide, S-nitrosation, and endothelial permeability

Role of IL-17 in LPS-induced acute lung injury: an in vivo study

Immunogenicity of combination DNA vaccines for Rift Valley fever virus, tick-borne encephalitis virus, Hantaan virus, and Crimean Congo hemorrhagic fever virus

Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

Immunization of Knock-Out α/ β Interferon Receptor Mice against High Lethal Dose of Crimean-Congo Hemorrhagic Fever Virus with a Cell Culture Based Vaccine

A novel vaccine against Crimean-Congo Haemorrhagic Fever protects 100% of animals against lethal challenge in a mouse model

A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease

Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice

Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice. Vector Borne Zoonotic Dis

Product Development Under FDA's Animal Rule: Understanding FDA's Expectations and Potential Implications for Traditional Development Programs

Persistent Crimean-Congo hemorrhagic fever virus infection in the testes and within granulomas of non-human primates with latent tuberculosis

Animal models for SARS and MERS coronaviruses

Experimental studies of arenaviral hemorrhagic fevers

Cytokines as biomarkers of Crimean-Congo hemorrhagic fever

Increased Levels of VEGF-A and HIF-1α in Turkish Children with Crimean-Congo Hemorrhagic Fever

Characterization of Biomarker Levels in Crimean-Congo Hemorrhagic Fever and Hantavirus Fever with Renal Syndrome

Serum nitric oxide levels in patients with Crimean-Congo haemorrhagic fever. Scandinavian journal of infectious diseases

A single dose of an ISCOM influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect Cynomolgus macaques against distant drift variants of influenza A (H3N2) viruses

A Recombinant Subunit Based Zika Virus Vaccine Is Efficacious in Non-human Primates

The authors would like to thank the UTMB Animal Resource Center for husbandry support of laboratory animals, Drs. Kevin Melody and Chad Mire for assistance with the animal studies, Kira Zapalac for assistance with RNA extractions, and Natalie Dobias for expert histology and immunohistochemistry support.