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Emerging Infectious Diseases * Volume 2 * Number 4 * October-December 1996
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Dispatches

Experimental Inoculation of Plants and Animals with Ebola Virus

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    Thirty-three varieties of 24 species of plants and 19 species of
    vertebrates and invertebrates were experimentally inoculated with
    Ebola Zaire virus. Fruit and insectivorous bats supported
    replication and circulation of high titers of virus without
    necessarily becoming ill; deaths occurred only among bats that had
    not adapted to the diet fed in the laboratory.

The taxonomy of the Filoviridae is in a state of flux; the family includes
viruses currently designated Marburg, Ebola Zaire, Ebola Sudan, and Ebola
Ivory Coast, which are believed to be endemic to Africa, and Ebola Reston,
which putatively originates in the Philippines (1,2). The viruses are known
particularly for their propensity to cause fatal hemorrhagic disease of
humans with person-to-person spread, but their pathogenicity varies from
asymptomatic infection (Ebola Reston) to epidemics with death rates of 77%
to 88% (Ebola Zaire) (1,3). All of the viruses appear to be highly
pathogenic for nonhuman primates. Outbreaks of disease have occurred in
Europe and North America in monkeys imported from Africa and the
Philippines, sometimes with spread of infection to humans (1). Contact with
the tissues of dead nonhuman primates was a source of infection for humans
on at least two occasions in Africa (2,4). Nevertheless, the lethality of
the viruses for nonhuman primates suggests that, like humans, nonhuman
primates are incidental victims of infection and are not true reservoir
hosts (1). The source of filoviruses in nature remains unknown, but in some
instances, bats roosted in buildings or a cave visited or frequented by
people who subsequently were found to have primary cases of infection in
outbreaks of disease in Africa; one patient was bitten or stung by what is
presumed to have been an arthropod 7 days before coming down with Marburg
disease (1). Informal speculation has included the suggestion that
filoviruses may be plant viruses, perhaps even involving transmission by
arthropod vectors.

The search for the source of the viruses in nature has been hampered by the
erratic recognition of outbreaks of filovirus infection in widely separated
geographic locations at unpredictable intervals; therefore, comparatively
few field investigations have been reported (1). After the 1995 epidemic of
Ebola fever in Kikwit, Zaire, teams of scientists coordinated by the
Centers for Disease Control and Prevention, Atlanta, and the U.S. Army
Medical Research Institute of Infectious Diseases, Fort Detrick, collected
large numbers of vertebrate and arthropod specimens during June, July, and
August, 1995. Because evidence indicated that the outbreak had actually
started in January 1995, it was possible that the virus was no longer
circulating in its natural hosts in the vicinity of Kikwit by the time
ecologic studies were undertaken. To allow for the possibility that the
filoviruses manifest seasonal activity in their natural hosts, a team from
the National Institute for Virology (NIV) in South Africa visited Kikwit in
January 1996, to trap wild vertebrates and arthropods in the sites
investigated by the other teams in mid-1995. Testing of the field material
is a lengthy process, and no filoviruses have been detected so far. We
decided to narrow the search by performing pathogenicity studies with Ebola
virus in representatives of different classes and orders of living things,
including vertebrates, invertebrates, and even plants. The underlying
assumptions were that if a group of species is either refractory or
hypersusceptible to the virus, members of the taxon are unlikely reservoir
hosts of the virus, whereas members of taxa capable of circulating virus
for prolonged periods without becoming ill are suspected reservoirs.

The strain of Ebola Zaire virus used in the experiments, Zaire-95, had been
isolated from the blood of a patient in the 1995
                             [end of page 321]

epidemic in Kikwit and designated as the prototype strain of the outbreak.
Stocks were prepared from virus at pass level 4 in Vero V76 cell cultures
by freeze-thawing infected cultures, clarifying the supernatant culture
fluid at 3,000g, and storing it in small volumes at -70C. Virus titers
were determined by fluorescent focus assay in 8-chamber slide cultures as
described for rabies (5), except that Vero cells were used, and cultures
were stained with immune mouse ascitic fluid followed by
fluorescein-labeled anti-mouse immunoglobulin and read on day 3 to 5
postinoculation to detect infected foci. Inoculum volumes of 10l of
10-fold serial dilutions of stock virus or tissue suspensions were adsorbed
to cultures, and titers were expressed as fluorescent focus-forming units
(FFU) per ml.

One-month-old potted seedlings of 33 varieties of 24 species of weeds and
crop plants used in plant virology (Table 1), plus colonized leafhoppers,
were obtained from Dr. G. Pietersen of the Plant Protection Research
Institute (PPRI) at Rietondale, Pretoria, South Africa. The plants were
selected because collectively they could provide culture substrates for a
broad spectrum of the known viruses of economically important plants. They
were kept under suitable lighting for 10 hours each day and watered as
necessary to sustain growth in the laboratory. Pigeons were obtained from
the South African Institute for Medical Research in Johannesburg; 1- to
3-month-old hatchling snakes from the Transvaal Snake Park, Midrand;
cockroaches from a colony at the Bureau of Standards in Pretoria; and
Mastomys natalensis and NIH mice from colonies at NIV. All other animals
used in the studies (Table 2) were collected in the Kruger National Park,
South Africa, with the permission of the National Parks Board as part of a
long-standing research project on hemorrhagic fever viruses. The animals
were translocated from the park under permit from the Department of
Veterinary Services, and the experiments were conducted in a biosafety
level 4 containment laboratory at NIV with clearance from the Department of
Agriculture, Conservation and Environment of Gauteng Province, and the
Animal Ethics Committee of NIV. All animals were fed a diet similar to
their natural diets and were provided with fresh drinking water daily.

         Table 1. Plants experimentally inoculated with Ebola virus
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         Scientific name                     Common name
         ----------------------------------------------------------
         Arachis hypogaea                    Groundnut
         Beta vulgaris                       Beetroot
         Chenopodium amaranticolor           Goosefoot weed
         Chenopodium quinoa                  Goosefoot weed
         Cucumis sativus                     Cucumber
         Cucurbita pepo                      Pumpkin
         Glycine max(a))                     Soybean

         Gomphrena globosa                   Weed
         Gossypium hirsutum                  Cotton
         Lupinus albus                       Lupin
         Lycopersicon esculentum             Tomato
         Macroptilium atropureum             Siratro bean
         Nicotiana benthamiana               Wild tobacco
         Nicotiana clevelandii               Wild tobacco
         Nicotiana glutinosa                 Wild tobacco
         Nicotiana langsdorfi                Wild tobacco
         Nicotiana rustica                   Wild tobacco
         Nicotiana tabacum                   Tobacco
         Phaseolus vulgaris(a)               French bean

         Pisum sativum                       Green pea
         Triticum aestivum                   Wheat
         Vicia faba                          Broadbean
         Vigna unguiculata(b)                Cowpea

         Zea mays                            Maize
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         (a)Five varieties inoculated
         (b)Two varieties inoculated

Vertebrates were inoculated subcutaneously with 0.1 ml of stock virus
diluted 1:10 in cell culture medium, and back titration of the inoculum
indicated that each animal received a dose of 40,000 or 10 to the 4.6th power FFU virus.
Invertebrates were inoculated with undiluted stock virus and received
approximately 1.0l containing 4,000 or 10 to the 3.6th power FFU of virus,
except for leafhoppers, which received about 0.3l inoculum. The arthropods
were inoculated intrathoracically (6), except for ants and millipedes, 
which were inoculated into the hemocoel through the membranous integument
between tergites. To simulate mechanical transmission, undiluted stock
virus mixed with Carborundum powder was rubbed gently with cotton buds 
onto two leaves on each of the plant varieties on experiment; to 
simulate vector-borne transmission, a second plant of each type was
 inoculated with 1.0l virus suspension into the phloem of the stem, 
using the same apparatus as for arthropods. A third plant
                             [end of page 322]

of each type served as control. Plants were observed daily and those that
wilted or developed apparent lesions were harvested; some material was
fixed in 2.5% glutaraldehyde in buffer for examination by electron
microscopy, and the rest was stored at -70C for virus assay. The process
of embedding, sectioning, and examining the plant tissues by electron
microscopy was performed by H.J. van Tonder of PPRI. Animals were
sacrificed and assayed for virus content either in pools or individually,
before inoculation and at intervals postinoculation (Table 2). Serum and
pooled visceral organs were tested separately, and individual organ samples
were preserved at -70C and in Formalin fixative for more detailed study
later. Urine and feces samples were tested on some occasions. Materials for
virus assay were prepared as 10% suspensions in culture medium, and in
parallel with attempts to titrate infectivity, 0.1 ml volumes were injected
into Vero cell monolayers in 25 cm-square flasks, which were subcultured
thrice at weekly intervals before specimens were recorded as negative.

                      Table 2. Results of experimental infection of various animals with Ebola virus
NOTE: Wide table, print with condensed font or landscape mode.
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                                                                   Day postinfection
Species                              Pool
     Common name                     size  1    2   3   4   5   6   7   8   9   10  11  12  13  14  21  28
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Columba livia             
     Domestic pigeon                 1     0/1* 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/2 0/2
Hyperolius viridiflavus   
     Painted reed frog               2     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/2
Bufo regularis            
     Common toad                     1                  0/1     0/1                     0/1         0/1
Chiromantis xerampelina   
     Grey tree frog                  1                      0/1     0/1                         0/1 0/1
Hemidactylus mabouia      
     Tropical house gecko            1     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1     0/1
Lamprophis fuliginosus    
     Brown house snake               1     0/1  0/1 1/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 0/1 0/1 0/1 0/2 0/2
Geochelone pardalis       
     Leopard tortoise                1                          0/1     0/1     0/1                     0/1
Kinixys belliana          
     Hinged-back tortoise            1                          0/2     0/2     0/2                     0/2
Tadarida condylura          
     Angola free-tailed bat          1                  1/2 1/2         2/2 1/1         1/1 
Tadarida pumila           
     Little free-tailed bat          1     1/1  1/1 1/1     0/1 1/2 0/2 0/1     1/1 1/1 1/1 0/1 1/1 0/4 0/5
Epomophorus wahlbergi     
     Wahlbergs epauletted fruit bat 1          0/1     1/1     1/1 1/1 1/1     1/1 1/1 1/1         1/1 0/1
Mastomys natalensis ss    
     Multimammate mouse              1     0/3  0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
Mus musculus              
     NIH mouse                       1          1/1 0/1 0/1         1/1 0/1 1/1 0/1 0/1         0/1 0/2
Periplaneta americana     
     American cockroach              2     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 
Austria agallia           
     Leafhopper                      50                                                                 0/1
Messor barbarus capensis   
     Myrmicine ant                   5              0/1     0/1     0/1  
Stegodyphus dumicola      
     Social spider                   2     0/1  1/1 0/1 0/1 0/1 0/1 0/1 0/1                     1/1
Alloporus sp.             
     Millipede                       1     0/1  0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 
Achatina sp.              
     African landsnail               1              0/1     0/1                                         0/1
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*Virus isolations/pools tested

Thirteen plants either wilted or developed lesions on the leaves ascribed
to mechanical injury during the inoculation process, but no infectivity
could be recovered from the tissues, and no evidence of virus infection was
observed by electron microscopy.
                             [end of page 323]

All animal experiments (Table 2) were performed in parallel on a single
occasion except for a second experiment with insectivorous bats (Tadarida
spp.). Insectivorous bats had difficulty in adapting to a laboratory diet,
and consequently 10 of 18 died in the first experiment. Although virus was
recovered from the blood and organs of some, no histopathologic lesions
were observed, and no evidence of widespread infection was detected by
immunohistochemical technique (7). All of the bats that died had not been
eating well, and hence more insectivorous bats (T. pumila) were collected
from the same colony as before and adapted to a diet of mealworms over a
period of 3 weeks before inoculation. The bats in the second experiment
received the same dose of virus as the previous group, and no deaths
occurred before the 12 animals in the study, including nine that were kept
21 to 28 days, were sacrificed (Table 2). None of the other vertebrates
died, although some of the ants, cockroaches, and spiders died, possibly
from desiccation as a result of injury during inoculation.

The virus replicated in bats: titers of 10 to the 4.6th power - 10 to
the 7.0th power FFU/ml were recorded in sera and titers of 10 to the
2.0th power - 10 to the 6.5th power FFU/ml in pooled viscera of fruit bats.
In addition, virus was recovered from the feces of a fruit bat on day 21
postinoculation. Virus was also recovered on a few occasions from snakes,
and NIH mice and spiders (Table 2), but this was at a minimal titer of
10 to the 1.0st power FFU/ml and could represent residual infectivity 
from inoculation. Histopathologic and immunohistochemical investigations
have thus far been limited to some sets of the bat organs, and the only
virus antigen detected was present in the endothelial cells of lung tissue
of a bat sacrificed on day 8 postinoculation (Figure). Four insectivorous
bat sera collected on day 28 postinoculation and four samples from 
noninfected bats were tested for Ebola virus antigen or antibody by 
enzyme-linked immunoassay (8) using conjugated chicken anti-rodent 
immunoglobulin; antibody was found in only one of the infected bats.

             [Image] [Figures not available in ASCII version]

             Figure 1. Ebola virus antigen-positive cells
             (red) in lung of an insectivorous bat as
             determined by immunohistochemistry. Note
             prominent endothelial immunostaining. (Rabbit
             anti-Ebola virus serum, napthol/fast red with
             hematoxylin counterstain, original magnification
             x 250).

Although they do not provide conclusive evidence that bats are potential
reservoir hosts of filoviruses or that the other animals are not, the
findings demonstrate the validity of the experimental approach to the
search for the source of the viruses in nature. If it can be shown, for
instance, that a further two to three species of birds of widely divergent
orders or families are refractory to the virus, birds can be accorded low
priority in field studies, and efforts can be concentrated on animals
capable of circulating virus. Even if evidence is obtained that certain
animals become infected in the field, it would remain desirable to study
the nature of the infection they undergo in the laboratory to determine
whether they can harbor virus for prolonged periods and transmit it to
other animals. Additionally, materials derived from the experiments can be
used to develop, test, and perfect methods for detecting infectious virus,
viral antigen, nucleic acid, or antibodies in different species, and to
establish whether or not demonstrable immune response develops in
ostensibly refractory animals. There are no accepted methods for
demonstrating antibody in many wild vertebrates; anti-bat immunoglobulin is
being produced as part of the present project.

The two tadarids studied here, and many other bats, have a distribution
that overlaps the sites of known filovirus outbreaks in Africa, and the
migratory habits of some species would facilitate dissemination of
                             [end of page 324]

virus (9). The presence of virus in lung tissue implies that respiratory or
oral spread of infection could occur in the confined spaces where bats
roost, and isolation of virus from feces suggests the existence of
mechanisms for transmission of infection to other animals. However, much
remains to be learned about the nature of the infection in bats, including
the sites of virus replication, persistence, and the behavior of the virus
in further genera and species.

Robert Swanepoel,* Patricia A. Leman,* Felicity J. Burt,* Nicholas A.
Zachariades,* Lawrence E.O. Braack, Thomas G. Ksiazek, Pierre E. Rollin,
Sherif R. Zaki, and Clarence J. Peters
*National Institute for Virology, Sandringham, South Africa; National
Parks Board, Skukuza, South Africa; Centers for Disease Control and
Prevention, Atlanta, Georgia, USA

References

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  8. Ksiazek TG, Rollin PE, Jahrling PB, Johnson E, Dalgard DW, Peters CJ.
     Enzyme immunosorbent assay for Ebola virus antigens in tissues of
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