Page | 1 Temporal Characterization of Marburg Angola Infection Following Aerosol Challenge in ...

36 Pages·2015·1.53 MB·English

JVI Accepted Manuscript Posted Online 22 July 2015
J. Virol. doi:10.1128/JVI.01147-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Temporal Characterization of Marburg Angola Infection Following Aerosol Challenge in Rhesus
2 Macaques
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5 Kenny L. Lin1, Nancy A. Twenhafel1, John H. Connor2, Kathleen A. Cashman1, Joshua D.
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6 Shamblin1, Ginger C. Donnelly1, Heather L. Esham1, Carly B. Wlazlowski1, Joshua C. w
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7 Johnson1,3, Anna N. Honko1,3, Miriam A. Botto1 , Judy Yen2, Lisa E. Hensley1,3, and Arthur J. a
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8 Goff1# d

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10 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, tp
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11 USA1; Boston University School of Medicine and National Emerging Infectious Diseases jv
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12 Laboratory, Boston, Massachusetts, USA2; and Integrated Research Facility, National Institute of m
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13 Allergy and Infectious Diseases, Fort Detrick, Maryland, USA3 rg
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15 Running Head: Aerosol Marburg Angola Infection of Rhesus Macaques r
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16 2
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17 # Corresponding Author. Mailing address: United States Army Medical Research Institute of 0
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18 Infectious Diseases, 1425 Porter Street, Fort Detrick, MD, 21702. Phone: [phone]. E-mail: b
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19 [email protected] u
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20 t
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21 ABSTRACT
22 Marburg virus (MARV) infection is a lethal hemorrhagic fever for which no licensed vaccines or
23 therapeutics are available. Development of appropriate medical countermeasures requires a
24 thorough understanding of the interaction between the host and the pathogen and the resulting
25 disease course. In this study, fifteen rhesus macaques were sequentially sacrificed following
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26 aerosol exposure to MARV variant Angola, with longitudinal changes in physiology, w
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27 immunology, and histopathology used to assess disease progression. Immunohistochemical a
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28 evidence of infection and resulting histopathological changes were identified as early as day 3 d

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29 PE. The appearance of fever in infected animals coincided with the detection of serum viremia m

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30 and plasma viral genomes on day 4 postexposure (PE). High (>107 PFU/mL) viral loads were ttp
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31 detected in all major organs (lung, liver, spleen, kidney, brain, etc.) beginning day 6 PE. Clinical jv
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32 pathology findings included coagulopathy, leukocytosis, and profound liver destruction as m
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33 indicated by elevated liver transaminases, azotemia, and hypoalbuminemia. Altered cytokine g
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34 expression in response to infection included early increases in Th2 cytokines such as IL-10 and n

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35 IL-5 and late stage increases in Th1 cytokines such as IL-2, IL-15, and GM-CSF. This study r
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36 provides a longitudinal examination of clinical disease of aerosol MARV Angola infection in the 2
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37 rhesus macaque model. 0
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38 Importance. In this study we carefully analyzed the timeline of Marburg virus infection in b
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39 nonhuman primates in order to provide a well-characterized model of disease progression u
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40 following an aerosol exposure. t
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41 INTRODUCTION
42 Marburg virus (MARV) is a single-stranded negative sense RNA virus that belongs to the
43 family Filoviridae (1). The genus Marburgvirus is composed of a single species Marburg
44 marburgvirus, which includes the two subspecies MARV and Ravn virus. The first outbreak of
45 MARV occurred simultaneously in Germany and the former Yugoslavia in August 1967 when
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46 laboratory personnel were exposed to the virus through contact with infected tissues from w
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47 African green monkeys imported from Uganda (2, 3). Seven of the thirty-two confirmed human a
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48 cases (mostly primary exposures) succumbed to infection (4, 5). From 1975 – 1998, subsequent d

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49 MARV infections were limited to sporadic cases in select areas of Africa, until the occurrence of m

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50 two large outbreaks in the Democratic Republic of Congo from 1998 – 2000 and Angola from tp
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51 2004 – 2005 (6, 7). The case fatality rates for these outbreaks were 83% (128/154) and 90% jv
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52 (227/252) respectively and established MARV disease as an important public health threat (8, 9). m
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53 There are currently no licensed medical countermeasures to combat filovirus infections such g
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54 as MARV. Due to the high pathogenicity of MARV, it is classified as a Biological Select Agent n

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55 by the U.S. Department of Health and Human Services (HHS) and a Category A Bioterrorism r
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56 Agent by the Centers for Disease Control and Prevention (CDC) (1). As such, any MARV 2
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57 research must be conducted in highly regulated Biosafety Level 4 (BSL-4) containment 0
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58 laboratories. b
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59 Data from the ongoing Ebola virus outbreak in Western Africa has suggested that filoviruses u
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60 have the potential to be transmitted by airborne droplets, highlighting the need to study the t
61 disease course following aerosol exposure. It has also been demonstrated that Ebola can be
62 transmitted from pigs to non-human primates when they are co-housed (10). While the
63 respiratory route of infection may not be the primary means of filovirus transmission, the virus
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64 can be spread by contact with large droplets containing virus (i.e. a sneeze). Moreover there are
65 continual concerns over a potential bioterrorist attack. It was previously thought that filoviruses
66 would not remain viable for long in an uncontrolled environment if there was ever a purposeful
67 release. However, both EBOV and MARV can be detected for up to ninety minutes in a dynamic
68 aerosol spray (11).
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69 Well-characterized animal models of infection are critical to allowing the development of w
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70 vaccines and antiviral compounds that protect against MARV infection (12). A number of a
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71 MARV studies have been conducted using various exposure routes, doses, virus strains (Ci67, d

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72 Angola, Musoke), and animal models (mice, guinea pigs, Syrian golden hamsters, and nonhuman m

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73 primates) (3, 13-19). From these tests, nonhuman primate (NHP) models in cynomolgus or tp
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74 rhesus macaques most accurately recapitulate the clinical aspects of Marburg virus disease jv
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75 (MVD) observed in human infections (14). m
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76 The aim of this work was to provide a well-defined animal model of aerosolized MARV g
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77 Angola infection for the use in future vaccine and therapeutic assessment studies. We report the n

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78 findings of a sequential sacrifice study of fifteen rhesus macaques exposed to aerosolized MARV r
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79 Angola variant Ang1379c. Disease progression was evaluated based on clinical parameters, 2
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80 virology, serology, inflammatory responses, gross necropsy, histopathology and 0
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81 immunohistochemistry. Assessment of these parameters at 2 day intervals provided not only b
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82 valuable insight into the timeline of viral dissemination, but also immunological changes in u
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83 response to infection (Connor et al., companion manuscript). Our results demonstrate that the t
84 disease caused by Marburg virus delivered by the aerosol route is similar to disease resulting
85 from intramuscular exposure.
86
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87 MATERIALS AND METHODS
88 Animals. Fifteen healthy, adult rhesus macaques (Macaca macaca) weighing between 5 - 9 kg
89 were obtained from World Wide Primates (Miami, FL). Prior to the start of study, the animals
90 were acclimated to a BSL-4 laboratory. The animals were found to be negative for standard viral
91 agents (Herpes B, STLV-1, SIV, SRV 1, 2, and 3) and were negative for filovirus antibodies.
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92 NHPs were randomized to five groups of 3 animals each using Excel (Microsoft, Redmond, w
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93 WA), with one group of animals to be sacrificed on each of the following days: 1, 3, 5, 7, and 9 a
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94 post-exposure (PE). On days -8 and -7 prior to infection, animals received a physical d

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95 examination, including body weight, rectal temperature, and clinical observations. Baseline m

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96 parameters for hematology, serum chemistry, coagulation, and cytokine expression were also tp
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97 obtained on these days. The data shown in the graphed figures represents the average of all of the jv
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98 NHPs that samples were obtained from at that time point. Standard deviation of those data points m
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99 is also shown. g
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100 n

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101 Virus. The MARV Angola isolate used for this study was from a patient specimen collected in r
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102 2005 [Marburg virus H.sapiens-tc/ANG/2005/Angola-1379c (order Mononegavirales, family 2
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103 Filoviridae, species Marburg marburgvirus)] (7). The reference sequence, Ang1379c, was 0
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104 determined from RNA isolated directly from clinical material and also from the virus isolate b
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105 (Ang1379v) after one passage on Vero E6 cells. A sample was obtained from the Centers for u
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106 Disease Control and Prevention, Atlanta, Georgia, USA following two passages in VeroE6 cells. t
107 A virus seed stock (passage 3) was previously propagated in Vero E6 cells, analyzed for sterility,
108 purity, and morphology using electron microscopy, real-time PCR, and tests for endotoxin and
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109 mycoplasma. Nucleotide sequencing was conducted on the seed stock, and plaque assay was
110 used to determine the final virus concentration.
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112 Aerosol exposure. Animals were exposed to a small-particle aerosol target dose of 1000 plaque
113 forming units (pfu) of MARV Angola on day 0 using the United States Army Medical Research
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114 Institute of Infectious Diseases (USAMRIID) head-only Automated Bioaerosol Exposure System w
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115 (ABES-II). Prior to exposure, each animal was anesthetized by intramuscular injection of telazol a
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116 (tiletamine hydrochloride/zolazepam hydrochloride 3mg/kg, Fort Dodge Laboratories, Fort d

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117 Dodge, IA). A full body plethysmograph (Buxco Research Systems, Wilmington, NC) was used m

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118 to ascertain each animal’s respiratory rate and capacity. The animal was then placed in the tp
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119 ABES-II system for the exposure time calculated based on that individual animal’s jv
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120 plethysmograph data. The aerosol dose was calculated by multiplying the volume of inhaled m
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121 exposure material (length of exposure X respiratory minute volume) by concentration of virus in g
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122 the aerosol. Using a Collison nebulizer (BGI Inc., Waltham, MA), a target aerosol of 1 – 3 µm in n

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123 diameter was generated. Hank’s buffered saline with 1% fetal calf serum and 0.001% antifoam A r
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124 was used in an all-glass impinger (AGI, Ace Glass, Vineland, NJ) to collect a sample of 2
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125 aerosolized virus during exposure. Starting virus concentrations and exposure dose (AGI 0
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126 material, Table 1) were confirmed through plaque assay. b
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127 u
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128 Post exposure monitoring. A group of animals (n=3) was euthanized on each of the following t
129 days: 1, 3, 5, 7 PE. One animal succumbed on day 8 PE and the remaining two were sacrificed
130 on day 9 PE. To ensure that hematological, serological, and physiological data was available for
131 each day of the study (days when euthanasia was not scheduled), physical examinations and
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132 blood collection were also performed for select surviving animals on days 2, 4, 6, and 8 PE. At
133 the time of euthanasia, body weight, rectal temperature, and blood collection were performed on
134 each animal.
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136 Necropsy. Following euthanasia, complete necropsies were conducted on each animal in an
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137 ABSL-4 laboratory. The following tissues were collected from each animal for viral genome, w
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138 viral titer analysis, and histopathology: axillary lymph node (LN), inguinal LN, mandibular LN, a
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139 mesenteric LN, tracheobronchial LN, lung, liver, spleen, brain, kidney, bone marrow, heart, d

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140 adrenal gland, pancreas, and ovary. Tissues were fixed by immersion in containers of 10% m

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141 neutral buffered formalin for a minimum of 21 days for histopathologic examination. tp
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142 jv
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143 Histology and molecular pathology. Following fixation, tissue samples were embedded in m
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144 paraffin sections and further analyzed for immunohistochemistry (IHC), histology, and TUNEL g
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145 staining. TUNEL staining was assessed using ApopTag In Situ apoptosis detection kit (Millipore n

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146 Corporation, Billerica, MA) as per manufacturer’s protocol as previously reported (13). IHC was r
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147 performed using an immunoperoxidase kit EnVision System (Dako Inc, Carpinteria, CA) 2
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148 according to manufacturer’s protocol. Anti-MARV GP Angola antibody (USAMRIID) or 0
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149 Caspase 3 antibody (Cell Signaling Technology, Beverly, MA) labeling were performed as b
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150 previously described (13). Histology sections were stained with hematoxylin and eosin. u
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151 t
152 Serum chemistry, hematology, and coagulation tests. Whole blood was collected in Serum
153 Clot Activator Vacuette tubes (Greiner Bio-One, Monroe, NC) for serum separation. Serum
154 tubes were allowed to clot for a minimum of 30 minutes and centrifuged at 1800 ×(cid:1859) for 10
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155 minutes at ambient temperature. Serum was separated within 1 hour of collection and analyzed
156 on a Piccolo Point-of-Care Blood Analyzer (Abaxis, Union City, CA) using a Piccolo Chem13
157 Panel disc. The parameters examined included creatinine (CRE), blood urea nitrogen (BUN),
158 albumin, total protein, aspartate aminotransferase (AST), and alanine aminotransferase (ALT).
159 Whole blood was also collected in tripotassium ethylenediaminetetraacetic acid-coated
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160 vacuette tubes (Greiner Bio-One, Monroe, NC) for hematological analysis. Complete blood w
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161 count analysis was performed on whole blood within 4 hours of blood collection using a a
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162 HemaVet 950FS Hematology Analyzer (Drew Scientific, Oxford, CT). The parameters analyzed d

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163 included white blood cell counts, neutrophil counts, lymphocyte counts, red blood cell counts, m

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164 percent hematocrit, and platelet counts. tp
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165 Coagulation abnormalities and presence of D-dimers were assessed on plasma separated jv
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166 from whole blood. The coagulation parameters aPTT and PT were determined using a m
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167 ThromboScreen (Fischer Diagnostics, Middletown, VA) per manufacturer’s protocol. D-dimers g
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168 were analyzed using an Asserachrom D-Dimer Enzyme Immunoassay kit (Diagnostica Stago n

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169 Inc, Troy Hills, NJ). Samples were read on a Spectramax M5 microplate reader (Molecular r
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170 Devices, Sunnyvale, CA) with Softmax Pro 4.7 software (Molecular Devices). 2
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171 Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using 0
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172 Histopaque-1077 prefilled Accuspin tubes (Sigma-Aldrich, St. Louis, MO). Total cell counts and b
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173 percent cell viability were determined using trypan blue staining solution and a Countess u
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174 automated cell counter (Life Technologies, Grand Island, NY). After cells were counted, 250 µL t
175 of viable cells were mixed with 750 µL of TRI Reagent LS (Sigma-Aldrich, St. Louis, MO) and
176 stored at -70 + 10℃.
177
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178 Plaque assay. Viral load was determined from plaque assays performed on the viral seed stock,
179 the virus challenge working stock, samples of aerosol exposure (via AGI), serum samples, and
180 tissues collected at necropsy. Tissues were homogenized in 500 µL of cell culture media with a
181 Tissuelyser II (QIAGEN, Valencia, CA) at 30 Hz for 1 minute. All experimental samples were
182 serially diluted and plated in duplicate onto confluent Vero E6 cells in 6-well plates. Following
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183 45 – 60 minutes of initial absorption (without cell culture media), 2 mL of 1X Avicel overlay w
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184 (2.5% preparation mixed 1 to 1 with 2X cell culture media) were added to each well and the a
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185 plates incubated at 37℃/5% CO . The plates were stained on day 7 with 1 mL of 0.4% crystal d
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186 violet at room temperature. Plaques were counted on day 8. m

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187 tp
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188 Viral genome quantification. Viral RNA was quantified from plasma and tissues treated with jv
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189 ethylenediaminetetraacetic acid (EDTA) collected at necropsy by qRT-PCR using an ABI 7500 m
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190 Fast Dx (Life Technologies, Grand Island, NY). Plasma and homogenized tissue samples were g
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191 inactivated with 1 part to 3 parts TRI Reagent LS (Sigma-Aldrich, St. Louis, MO) and stored at - n

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192 70 + 10℃. RNA was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA). r
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193 qRT-PCR was performed utilizing SuperScript II One-Step RT-PCR System (Life Technologies, 2
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194 Grand Island, NY) with primers and probes specific for the MARV matrix (VP40) gene. Forward 0
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195 primer: 5′ - CCA GTT CCA GCA ATT ACA ATA CAT ACA - 3′. Reverse primer: 5′ - GCA b
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196 CCG TGG TCA GCA TAA GGA - 3′. Probe: CAA TAC CTT AAC CCC C – MGBNFQ (minor u
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197 groove binder-tagged nonfluorescent quencher). Samples were analyzed in triplicate; a pfu t
198 equivalent (pfu/mL eq) was calculated by taking the average of the reported concentration values
199 and multiplying by the Trizol extraction, elution volumes, and the reaction volume. The
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200 calculations were standardized using an inactivated viral stock of known concentration extracted,
201 eluted and analyzed identically to the samples to generate a standard curve (20).
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203 Cytokine analysis by multiplex antibody bead assay. Previously frozen plasma samples were
204 thawed and assayed for protein cytokine and chemokine concentrations using a Millipore 23-plex
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205 NHP cytokine kit (EMD Millipore Corporation, Billerica, MA) with a Bio-plex analyzer (Bio- w
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206 Rad, Hercules, CA). Standard curves were optimized using Bio-plex Manager 5.0 (Bio-Rad), and a
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207 data were exported to Excel for analysis. d

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208 m

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209 RESULTS tp
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210 To examine the course of disease following exposure of macaques to Marburg Anglola jv
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211 virus through an aerosol rout, fifteen rhesus macaques were exposed to a target dose of 100 m
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212 pfu by the aerosol route. The actual dose delivered ranged from 3.09 x 103 to 6.25 x 103 plaque- rg
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213 forming units (pfu) (Table 1). Following exposure on day 0, one group of animals (n=3) was n

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214 sacrificed on days 1, 3, 5, and 7 post-exposure (PE). One animal succumbed on day 8 PE and r
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215 therefore only two were sacrificed on day 9 PE. The animals that were alive on days 2, 4, 6, and 2
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216 8 PE received physical examinations and phlebotomy. 0
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217 b
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218 Clinical Signs of Infection. The most common clinical findings following aerosol exposure to u
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219 MARV included fever, lymphadenopathy, and maculopapular rash. Elevated body temperatures t
220 were initially noted beginning on day 4 PE and reached a peak of 40℃ on day 5 PE (Figure 1A).
221 After the animals broke with fever general activity decreased as well as food consumption. As
222 the animals became moribund, a decline in body temperature to below baseline levels was
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