Int. J. Exp. Path. (1995), 76, 227-236

Lethal experimental infections of rhesus monkeys byaerosolized Ebola virus

E. JOHNSON, N. JAAX, J. WHITE AND P. JAHRLINGUnited State Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA

Received for publication 13 January 1995Accepted for publication 24 May 1995

Summary. The potential of aerogenic infection by Ebola virus was estab-lished by using a head-only exposure aerosol system. Virus-containingdroplets of 0.8-1.2,um were generated and administered into the respira-

tory tract of rhesus monkeys via inhalation. Inhalation of viral doses as lowas 400 plaque-forming units of virus caused a rapidly fatal disease in 4-5days. The illness was clinically identical to that reported for parenteral virusinoculation, except for the occurrence of subcutaneous and venipuncturesite bleeding and serosanguineous nasal discharge. Immunocytochemistryrevealed cell-associated Ebola virus antigens present in airway epithelium,alveolar pneumocytes, and Inacrophages in the lung and pulmonary lymphnodes; extracellular antigen was present on mucosal surfaces of the nose,

oropharynx and airways. Aggregates of characteristic filamentous viruswere present within type I pneumocytes, macrophages, and air spaces ofthe lung by electron microscopy. Demonstration of fatal aerosol trans-mission of this virus in monkeys reinforces the importance of takingappropriate precautions to prevent its potential aerosol transmission tohumans.

Keywords: Ebola virus, aerogenic infection, rhesus monkey

The African filoviruses, Ebola virus and Marburg virus,are deadly pathogens in primates, yet their ecologyand epidemiology are largely unknown (Martini et a/.1968; Bremen et al. 1976). The environmental condi-tions that influence viral transmission from unknownreservoir species to humans are undefined. Theseviruses are thought to circulate undetected withinunknown reservoir species in sub-Saharan Africa,presumably as enzootic infections, until changingecological conditions lead to human infections. Inhumans, these pathogens cause sporadic, butdramatic, outbreaks of lethal haemorrhagic diseasewith case fatality rates ranging from 30 to 90% (Martini

Correspondence: N. Jaax, US Army Medical Research Instituteof Infectious Diseases, Frederick, Maryland 21702-5011, USA.

et al. 1968; Gear et a/. 1975; Francis et a/. 1978; Smith eta/. 1978; 1982).The route of infection for natural human exposures

can be inferred only from extremely limited epidemio-logical investigations conducted during the rare periodsof viral activity in human populations (Bremen et a/.1976). Frequently, human to human transmissionappears to be caused by direct contact with infectiousblood or body fluids. In humans, the highest virusconcentrations are found in blood; lower levels arepresent in throat washings and urine (WHO 1978a, b;Peters et a/. 1991). Presumably, the virus invades thebody through the conjunctiva, gastrointestinal tract, orbreaks in the skin. Experimental confirmation ofparenteral inoculation as a transmission mechanismhas been established by studies of non-human

© 1995 Blackwell Science Ltd 227

228 E. Johnson et a/.

primates (Baskerville et a/. 1978; Bowen et a/. 1978;Fisher-Hoch et a/. 1985). Limited experimental andepidemiological data suggest that aerosol transmissionof filoviruses from monkey to monkey or from monkey tohuman may also occur. In an outbreak of Ebola (Reston)within a US non-human primate quarantine facility, thevirus readily spread within animal rooms. While infec-tions in adjacent cages may have occurred by dropletcontact, infections in distant cages suggests aerosoltransmission, as evidence of direct physical contactwith an infected source could not be established(Dalgard et a/. 1992). The prominent pneumonic com-ponent of the disease in infected monkeys supports thisconclusion (Geisbert 1992), as does a serosurvey ofhuman contacts. Six animal care workers in closedaily contact with infected monkeys were seropositiveby indirect immunofluorescent antibody assay (IFA)(CDC 1990). Of the six, five had no known history ofparenteral or trauma-induced exposure to Ebola virus,which suggested the possibility of aerosol transmission.Numerous Marburg disease cases occurred in labora-tory workers who wore gloves while handling infectedmonkey tissues and/or were without visible woundswhen admitted to Marburg University Medical Clinic in1967 (Martini 1971). Experimental transmission ofMarburg virus between monkeys whose only contactwas by a nose-to-nose apparatus has also beenreported (Pokhodyaev et a/. 1991). Despite this circum-stantial evidence for aerosol spread of filoviruses, therehas been no previous experimental confirmation of theiraerosol transmission.

This paper reports aerosol transmission of Ebolavirus to rhesus monkeys. The pathophysiologicalevents and histopathological lesions associated withEbola virus infections after aerosol exposures aredescribed.

Materials and methods

Biologic containment

Infectious material and animals were handled within themaximum containment (biological safety level 4) facili-ties at the United States Army Medical Research Insti-tute of Infectious Diseases, Frederick, MD. Laboratorypersonnel wore positive pressure protective suits (ILC,Dover, Frederica, Delaware) supplied with umbilical-fed, HEPA-filtered air.

Virus cultures and infectivity assays

Virus was propagated in Vero cells grown in Eagle's

minimum essential medium (EMEM) supplemented with10% heat inactivated fetal bovine serum, antibiotics(100U of penicillin and 100lxg of streptomycin sulphateper ml), and 2mM L-glutamine. Stocks of Ebola virus(Zaire type) were prepared as clarified, infected cell-culture fluids, passage level 4, and stored at -70°C.Viral titres were determined by plaque assay (Moe et a/.1981). The stocks routinely contained 6-7 log10 plaque-forming units (PFU) of infectious virus per ml.

Study design

Six rhesus monkeys (Macaca mulatta) were used toevaluate the aerosol infectivity of Ebola virus (Zaire).Rhesus monkeys were selected for aerosol exposure toEbola virus because this species was previously used todefine pathological changes following parenterallyinduced filovirus infections (Baskerville et a/. 1978;Bowen et a/. 1978; Fisher-Hoch et a/. 1985). All animalswere seronegative for filovirus-reactive antibody beforeEbola virus aerosol challenge.The monkeys were housed individually in stainless

steel cages. Monkeys were given water ad libitum andfed twice daily with Purina monkey chow supplementedwith fresh fruit. The animals were randomly assigned toone of three aerosol-exposure groups of two animalseach. Group one (monkeys 1465 and R280) was exposedto a low inhaled dose (2.6 log10 PFUs) of Ebola virus,group two (B64 and B65) was expQsed to a high dose(4.7 log10 PFUs) of Ebola virus, and group three (controlgroup, 121,N and 556A) was exposed to aerosolized,uninfected cell culture fluid.

All monkeys were checked daily for clinical signs ofillness. Monkeys were anaesthetized with intramuscularketamine HCI (5 mg/kg), and sterile femoral venousblood samples were collected on days 4, 7, and termin-ally for clinical chemistry and haematological evalua-tions. Values were compared with results obtained fromsamples drawn before exposure.

Aerosol exposure

Monkeys were anaesthetized with ketamine HCI (5 mg/kg) for the aerosol exposure phase of the experiment.While anaesthetized, each monkey was placed in dorsalrecumbency with its head extending through a rubberdam (designed to fit snugly around the neck withoutrestricting ventilation) into a 8000-cm3 exposure box.The monkey was placed in a gas tight, environmentcontrolled Hazelton chamber (240C and < 40% relativehumidity) that contained a modified Henderson appara-tus and a Collison nebulizer (Henderson 1952). The

© Blackwell Science Ltd, International Journal of Experimental Pathology, 76, 227-236

Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus 229

nebulizer, driven by compressed air at 20 PSI, gener-ated an aerosol flow rate of 16.5 I/min and disseminatedthe 25ml of test solution at 0.35ml/min. The medianmass diameter of the generated aerosol particlesranged from 0.8 to 1.2 im. The aerosol was mixedautomatically with a secondary 8.5 I/min air supply andcirculated through the exposure box. The aerosolchallenge dose was determined by a mid-exposuresample collected in an all-glass impinger calibrated tosample at a rate of 12.5 I/min. The aerosol specimen wasimpinged in supplemented EMEM containing an anti-foam emulsion. After a 10-minute exposure, the Hazel-ton chamber was flushed with clean air and the viruscontaminated contents exhausted through double HEPAfilters. The head and neck of the exposed animal weredecontaminated with 1.0% sodium hypochlorite and themonkey was placed in its cage. The viral titre per ml ofimpinger fluid was determined by plaque assay. Theexposure dose was calculated by multiplying the virusplaque-forming units (PFU) per litre of aerosol, theexposure period in minutes, and respiratory volume (I/min). Respiratory volume was determined on the basisof body weight and Guyton's formula (Guyton 1947).

Haematology, serum enzyme and coagulation assays

Total and differential white blood cells (WBC) werecounted by using a laser-based haematological analy-ser. Leucocyte differentials were confirmed manually ona Wright stained blood smear. Platelets were countedwith a Coulter Model ZBI particle counter. Lactic aciddehydrogenase (LDH), serum alanine aminotransferase(ALT), blood urea nitrogen (BUN), creatinine (CRE),alkaline phosphatase (ALP), and creatinine phospho-kinase (CK) levels were measured with a Multi Stat IlIlPlus chemical analyser.

Histopathology and immunocytochemistry examination

Complete necropsies were performed on all virusexposed animals. One monkey (1465) died on day 8post-exposure and was necropsied immediately. Theother three monkeys (B54, B65, and R280) werehumanely killed when they became moribund on days7, 8 and 9 post exposure, respectively. They wereexsanguinated while deeply anaesthetized with intra-venous sodium pentabarbitol, and perfused with auniversal fixative (neutral phosphate-buffered 4.0%paraformaldehyde containing 1.0% glutaraldehyde) forlight and ultrastructural microscopy. Representativetissue specimens from the non-perfused animal thatdied naturally were selected and fixed by immersion in

10% neutral buffered formalin. The tissues were pro-cessed and embedded in paraffin according to estab-lished procedures (Prophet et a/. 1992). Histologysections were cut at 5-6,um on a rotary microtome,mounted on glass slides, and stained with Harris'shaematoxylin and eosin in a Stainomatic specimenstainer. Phosphotungstic acid haematoxylin (PTAH)stain was used to detect fibrin deposits (Prophet et a/.1992). For immunohistochemical staining, the paraffinembedded tissues were sectioned at 5bm and mountedon silane coated glass slides. After deparaffinizationand hydration, the sections were digested with proteaseand treated with Ebola virus reactive monoclonal anti-bodies (AEll, 8A11 and Bli) prepared from hybridomacells provided under contract by the Centers for DiseaseControl, Atlanta, Georgia. Biotinylated horse anti-mouseimmunoglobulin was reacted with the bound mono-clonal antibody and the reactive product visualized byan alkaline phosphatase-labelled streptavidin method(Jahrling et a/. 1990). Archived necropsy material pre-viously collected from fatal monkey infections inducedby low doses (1-10PFU) of parenterally inoculatedEbola virus (Zaire strain) and Marburg virus (Musokistrain) were processed in parallel to serve as positiveand negative controls, respectively, for Ebola virusantigen.

Electron microscopy

Tissues were fixed in universal fixative and sliced into1-mm cubes, washed three times in phosphate buffer,and post-fixed in phosphate buffered 1.0% osmiumtetroxide. The fixed tissues were washed in distilledwater, stained en bloc in 1.0% uranyl acetate, dehy-drated, and embedded in Epon 812. Ultrathin sections,cut on an LKB Ultratome, were stained with uranylacetate and lead citrate and examined in a Jeol 100CX electron microscope at 80 kV.

Results

Clinical disease

Clinical disease occurred in all four Ebola virus exposedmonkeys, but not in either of the control monkeys. Theclinical disease in the aerosol challenged monkeys wassimilar to the syndrome previously described in parent-erally inoculated rhesus monkeys (Baskerville et ai.1978; Bowen et a/. 1978; Fischer-Hoch et a/. 1985).However, we observed serosanguineous nasal dis-charge, subcutaneous haemorrhage, and prolongedbleeding after venipuncture in these aerosol infected

© Blackwell Science Ltd, International Journal of Experimental Pathology, 76, 227-236

230 E. Johnson et a/.

monkeys, which was not reported in previous studies(Baskerville et al. 1978; Bowen et al. 1978; Fisher-Hochet a/. 1985). The monkeys remained active and clinicallynormal until the onset of fever (39.9-40.2oC) between 4and 6 days post exposure. The fever was accompaniedby anorexia, lethargy and development of a cutaneousrash involving the face, trunk and proximal limbs. Onehigh-dose animal (1465) died within 3 days of the onsetof clinical signs (8 days post exposure). The other threewere humanely killed when they became moribund on

days 7, 8 and 9 post-exposure.Haemotological parameters were altered in three of

four Ebola virus exposed rhesus monkeys (Figure 1).

These three animals developed a leucocytosis by day 4with white blood cell counts ranging from 8700 to 15200celIS/MM3 . The leucocytosis was due to an absoluteneutrophilia that ranged from 9400 to 12400 granulo-cytes per mm3 on day 7 (excluding monkey B65), whichis consistent with other reports (Fisher-Hoch et a/. 1983;Fisher-Hoch 1985). There was a concoimitant absolutelymphopenia. The mean platelet count fell from a pre-exposure level of 288 x i03 to 124 x 103 permm3 day 7post exposure.

Clinically significant elevated clinical chemistryvalues were observed in all four monkeys after Ebolavirus aerosol exposure. All biochemistry parameters

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232 E. Johnson et al.

measured were normal on day 4 post exposure. On day7 post exposure, 11-16-fold increases in LDH levels,accompanied by a fivefold increase in ALT, were pre-sent in all animals (Figure 2). Terminal values of ALPand CK were markedly elevated. CRE and BUN levelswere only mildly elevated in the infected animals on day7 post exposure. Near death, however, these levelswere markedly elevated.

Histopathology

Lungs and pulmonary lymph nodes. All Ebola virusaerosol exposed monkeys had a mild to moderate,patchy, interstitial pneumonia, clearly oriented aroundterminal bronchioles (bronchocentric pattern). Addi-tional pulmonary lesions included diffuse leucocytosis,vasculitis, multifocal septal necrosis, thrombosis, hae-morrhage, oedema, and increased numbers of alveolarmacrophages. Large, amorphous, pleomorphic, acido-philic intracytoplasmic inclusions of 5-20-im were pres-ent in circulating and alveolar macrophages of thelungs. With the exception of the bronchocentric patternevident in this study, all lesions were consistent withthose described in previously published reports(Baskerville et a/. 1978; Bowen 1978); the broncho-centric pattern was consistent with an inhalation modeof infection. When examined by immunocytochemistry,the strongest viral antigen reactivity in the lungs ofaerosol exposed monkeys also occurred in a broncho-centric pattern, with the heaviest antigen concentrationoccurring adjacent to terminal bronchioles. Bronchialand bronchiolar epithelium, alveolar pneumocytes, andalveolar macrophages showed positive Ebola virusantigen staining (Figure 3). Copious extracellularEbola virus antigen was also present in secretions onthe mucosal surfaces of the nose, oropharynx andairways. Severe lymphoid depletion, necrosis, vascu-litis, thrombosis and variable degrees of haemorrhage,from mild to severe, were present in the tracheo-bronchial (TBL) lymph nodes of aerosol exposedmonkeys; intracytoplasmic inclusions were present incirculating and fixed macrophages. The lesions in theTBL nodes were consistent with mesenteric lymph nodelesions reported in other studies (Baskerville et at. 1978;Bowen et al. 1978). Immunocytochemical staining of TBLlymph nodes of aerosol infected monkeys revealedcopious extracellular and intracellular (within macro-phages) Ebola virus antigen accumulation.

Extrapulmonary lesions in these aerosol exposedmonkeys did not differ significantly from those pre-viously reported (Baskerville et at. 1978; Bowen et at.1978). The principal extrapulmonary histological lesions

were mild to moderate randomly patterned necrosis inthe liver, pancreas, intestines, adrenal gland and bonemarrow; moderate to severe necrosis of both the redand white pulp of the spleen, mesenteric, peripherallymph nodes and gut-associated lymphoid tissue(GALT); and fibrin deposition and thrombi throughoutthe vascular system in all organs. Large, amorphous,pleomorphic, acidophilic intracytoplasmic inclusions -of5-20pLm were present in circulating macrophages ofvarious organs, hepatocytes and adrenocortical cells,as well as the previously described locations.A detailed immunohistochemical examination of

extrapulmonary necropsy tissues was performed andwill be addressed in a separate publication. Briefly,copious extracellular and intracellular Ebola virus anti-gen was present in the liver, adrenal gland, bonemarrow, spleen, testes and kidney. Large antigendeposits were also present in phagocytic cells (suchas Kupffer cells and splenic macrophages), cellularthrombi and inflammatory foci.The presence of virions in the tissues of aerosol

infected monkeys was confirmed by electron micro-scopy. Extracellular filamentous virions were abund-ant, and were present in lung alveoli (Figure 4), theinterstitium of parenchymal organs, the space of Disseand bile canaliculi of the liver, and were associated withfibrin thrombi in numerous organs. Type 1 alveolarpneumocytes contained nucleocapsid inclusions withcharacteristic viral particles budding through plasmamembranes into the air space (Figure 5). Nucleocapsidinclusions and budding viral particles were also seen inmononuclear cells within the interstitial component ofthe adrenal glands, spleen and lymph nodes; alveolarsepta and alveolar macrophages of the lungs; and inhepatocytes.

Discussion

This study demonstrates aerosol transmission of Ebolavirus to non-human primates. Inhalation doses as low as400 PFU of virus caused a fatal illness clinically similarto that previously reported for monkeys infected byparenteral inoculation (Baskerville et a/. 1978; Bowenet at. 1978; Fisher-Hoch et a/. 1985). The illness wascharacterized by fever, anorexia and a petechial rash.Fibrin deposition and fibrin thrombi throughout thevascular system in all monkeys suggested thatdisseminated intravascular coagulation (DIC) mayhave also played a role in the clinical manifestationsof Ebola virus infection.Our aerosol infectivity findings for Ebola virus support

Dalgard's and Pokhodyaev's observations that suggested

© Blackwell Science Ltd, International Journal of Experimental Pathology, 76, 227-236

Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus 233

Figure 3. Lung from an Ebola virus aerosol exposed rhesusmonkey stained with a mixture of Ebola virus reactivemonoclonal antibodies. Antigen reactivity occurred in abronchocentric pattern oriented around terminal bronchioles.Pulmonary macrophages within alveoli were also positivelystained. xlQO.

a role for aerosol transmission of filoviruses inmonkeys. Epidemiology studies of human disease out-breaks in sub-Saharan Africa did not suggest thataerosol transmission of filoviruses was likely in thatsetting. Virus did not spread easily from person toperson during the Ebola virus epidemics in Africa, andattack rates were highest in individuals who were indirect physical contact with a primary case (Bres 1978).The rates were 3.5 times higher in people who providednursing care than in those who were in casual contactwith a primary case; no cases occurred in childrenwhose only known exposure to the virus was sleepingin the huts occupied by their fatally ill parents. Althoughcoughing was common among the human Ebola hae-morrhagic fever cases in Africa, there was no directevidence for aerogenic spread of Ebola virus in humanpopulations. Several potential explanations might

account for this situation. It is possible that the quantityand di"stribution of virus within most patients' respiratorytracts may have been below the level needed to estab-lish effective aerosol transmission. This possibility issupported by histologic examination of archived tissuesfrom one human reference case of Marburg on file inthis institute; examination by immunocytochemistry andelectron microscopy revealed no positive Marburg viralantigen staining or any ultrastructural evidence of virusin the lung tissue of that case (personal communication,N. Jaax). We also demonstrated aerosol transmission ofEbola virus at lower temperature and humidity than thatnormally present in sub-Saharan Africa. Ebola virussensitivity to the high temperatures and humidity inthe thatched, mud, and wattel huts shared by infectedfamily members in southern Sudan and northern Zairemay have been a factor limiting aerosol transmission ofEbola virus in the African epidemics. Both elevatedtemperature and relative humidity (RH) have beenshown to reduce the aerosol stability of viruses(Songer 1967). Our experiments were conducted at240C and < 40% RH, conditions which are known tofavour the aerosol stability of at least two other Africanhaemorrhagic fever viruses, Rift Valley fever and Lassa(Stephenson et a/. 1984; Anderson et a/. 1991). If thesame holds true for filoviruses, aerosol transmission isa greater threat in modern hospital or laboratorysettings than it is in the natural climatic ranges ofviruses. The route of infection or the degree of pul-monary involvement of the primary cases may also bean important factor to consider when evaluating thenatural aerosol transmissibility of the filoviruses.While both parenteral and aerosol exposure to Ebolavirus cause a systemic disease involving all organs,monkeys exposed to viral aerosols during our studydeveloped strong immunoreactivity for Ebola virus anti-gen in airway epithelium, in oral and nasal secretions,and in bronchial and tracheobronchial lymphoid tissue.By electron microscopy, viral replication after aerosolexposure occurred in the lungs and tracheobronchiallymph nodes, and extracellular virus accumulated inalveoli of the lung. Copious extracellular Ebola virusantigen was present in secretions on the mucosalsurfaces of the nose, oral cavity and pulmonary airwaysof aerosol exposed monkeys, strong evidence to supportthe potential for secondary spread of Ebola virus byaerosol. As previously stated, aerosol spread wasimplicated in the spread of disease among the monkeysat Reston (Dalgard et a/. 1992; Geisbert 1992), and mayhave occurred between monkeys and animal handlerswho were in close contact with infected monkeys (CDC1990). Finally, host sensitivity to filovirus infections may

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234 E. Johnson et a/.

Figure 4. Extracellular mature virions(V) in the alveolar sac (AS) and in aninterstitial space(IS) between epithelialcells Type I or 11 (Ep or 11) and thebasal lamina. Note the viralnucleocapsids in the cytoplasm ofepithelial cells (single arrow) and aportion of a septal cell (double arrow).An erythrocyte (E) is also apparent inthis field. En, Endothelial cell. x15000.

also be a significant factor influencing the degree ofaerosol transmissibility. Asian (Macaca fascicularis)and African (Cercopithecus aethiops) monkeys areknown to be disseminating hosts for filoviruses (Gearet al. 1975; CDC 1990). Virus replicates extensively inEbola virus infected non-human primates, reaching highconcentrations in tissues (consistently greater than6 log10/g of tissue or serum for Reston virus; P. Jahrlingunpublished). Quantitative data for viraemia levels inhumans are lacking, as are estimates of minimal infec-tious aerosol doses. Certainly, filoviruses have infectedhumans with no known history of parenteral or ocularinoculation of the virus (Martini 1971; CDC 1990), and arespiratory route of infection cannot be ruled out(Simpson 1977). Those case may have resulted fromexposures to unusual concentrations of aerosolizedvirus not often encountered naturally. In that regard,retrospective examination of any available archivedhuman tissues by immunocytochemistry could prove tobe helpful.

Regardless, we have shown that Ebola virus (Zairestrain) can be transmitted by aerosol in an experimentalprimate model. In light of the pathogenicity of humanfilovirus infections, health care personnel at risk ofexposure should use precautions to minimize the riskof aerosol exposure while managing acutely ill haemo-rrhagic fever cases of unknown or filoviral aetiology.The risk can be markedly reduced by adhering to soundand practical infectious disease management proce-dures. This requires curtailing aerosol generating pro-cedures; using protective clothing, including gloves andmasks; adequately decontaminating potentially infec-tious material; and conducting virus diagnostic proce-dures in regional containment facilities capable of safelyhandling highly hazardous infectious agents (Compertset a/. 1978; CDC 1988).

In conducting this research, the investigators adheredto the Guide for the Care and Use of Laboratory Animals,prepared by the Committee on the Care and Use ofLaboratory Animals of the Institute of Laboratory Animal

© Blackwell Science Ltd, International Journal of Experimental Pathology, 76, 227-236

Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus 235

A

Figure 5. Note extracellular maturevirions (V) in alveolar sac (AS) and a

large cytoplasmic inclusion (doublearrow) containing viral nucleocapsids ina type epithelial cell (Ep).Nucleocapsid filaments (single arrow)are also seen in the cytoplasm of the

pithelial cell that extends over a type 11epneumocyte. En, Endothelial cell.x30000.

Resources, National Research Council (NIH PublicationNo. 86-23, revised 1985).The views, opinions, and/or findings contained in this

report are those of the authors and should not beconstrued as an official Department of the Army posi-tion, policy, or decision unless so designated by otherdocumentation.

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