THE JOURNAL OF INFECTIOUS DISEASES. VOL. 147, NO.2. FEBRUARY 1983
Biologic Differences Between Strains of Ebola Virus from Zaire and Sudan
J. B. McCormick, S. P. Bauer, L. H. Elliott,
P. A. Webb, and K. M. Johnson
From the Special Pathogens Branch, Division of Viral
Diseases, Centers for Disease Control, Atlanta, Georgia; the
Vector-Borne Diseases Division, Centers for Disease Control,
Fort Collins, Colorado; and the U.S. Army Medical
Research Institute for Infectious Diseases, Fort Detrick,
Frederick, Maryland
iologic observations as well as data concerning
genetic, molecular, and immunologic properties
of virus strains, which we believe define the geographic origin of the original epidemics of EHF.
Ebola virus made a devastating entry into the
world in 1976 at two different geographic points:
Nzara and Maridi in Sudan and Yambuku in
Zaire, resulting in over 400 deaths due to clinical
hemorrhagic fever with an associated negative
economic impact on the two affected regions [1,
2] . The viruses isolated were morphologically
similar to Marburg virus but did not share anti:,.
gens with that virus [3]. The initial interpretation
of this extraordinary simultaneous occurrence of
highly lethal epidemics in two separate areas was
that there must have been spread of the causative
agent from one area to the other [4]. Extensive
searching, however, for possible links between
disease epidemics in Sudan and Zaire failed to
yield any direct evidence linking the two epidemics. Furthermore, the lack of established
communications and trade routes between the two
areas, which are 850 km apart, prompted the suggestion that the epidemics were unrelated except in
time.!
A single case of Ebola hemorrhagic fever (EHF)
in Zaire in 1977 and a second outbreak of EHF in
Nzara, Sudan, in 1979, lent further credence to the
existence of discrete endemic foci. In this article
and in three others [5-7], we present epidem-
Materials and Methods
Background. The Ebola virus strains were
first isolated in 1976 during two epidemics. Ebola
virus has since been characterized as a negativestrand RNA virus with an unusual length of
850-920 nm [8]. It has recently been suggested as a
member of a newly proposed taxon, Filoviridae
[9].
The Sudan epidemic began in June 1976 in the
southwestern part of the country. It was the result
of person-to-person transmission of disease in
Nzara followed by a massive outbreak of nosocomial origin after a death due to EHF in a large
regional hospital in Maridi. Over 200 cases occured (mortality, 50070) [1].
The epidemic of hemorrhagic disease in Zaire
began at the end of July 1976 [2]. The 'index case
was never identified, but it was clear that the
spread of disease resulted from nosocomial spread
of virus in the inpatient and outpatient services of
a local hospital, largely as a result of contaminated hypodermic needles [2] . Disease was
spread into many villages within a 50- to 75-km
radius of the hospital. Three hundred eighteen
cases were identified, and 280 deaths occurred
(mortality, 88070).
Thus, the mortality in Zaire was substantially
higher than that in Sudan, an observation further
substantiated in 1979 when a second outbreak in
Received for publication July 6, 1982, and in revised form
September 29, 1982.
Please address requests for reprints to Dr. J. B. McCormick,
Special Pathogens Branch, Division of Viral Diseases, Centers
for Disease Control, Atlanta, Georgia 30333.
1 J. B. McCormick, "Travel in Northern Zaire and Southern
Sudan in Search of Cases of African Hemorrhagic Fever,"
report to the International Commission of the World Health
Organization, November 1976.
264
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Studies of three outbreaks of Ebola hemorrhagic fever in Zaire and Sudan have shown
that human mortality resulting from Zaire strains of Ebola virus was 90% while that
resulting from Sudan strains was 55070-65070. Zaire strains were much easier to isolate in
cell culture than all of the Sudanese agents; also, fewer than 10 infectious particles of a
Zaire strain were lethal for suckling mice, whereas 10,000 infectious particles of a Sudan
strain failed to kill any of these animals. These biologic data indicate that these antigenically related viruses are different, a conclusion supported by genetic, biochemical,
and immunologic data.
Ebola Virus from Zaire and Sudan
2 R. B. Baron, J. B. McCormick, and O. EIZubeir, "Risk of
Acquiring Ebola Virus Infection During an Outbreak in Sudan
in 1979," manuscript submitted for publication.
ulated into Vero-76 cells. Virus presence was confirmed by IFA testing after 10 days of incubation
at 37 C.
The viruses used in these studies were isolated
from the serum or blood of acutely ill patients in
Zaire or Sudan. With the exception of the BON
strain, all were isolated in cell cultures. The MAY
and ECK strains were isolated in 1976 from patients in Zaire. The BND strain was isolated from
a patient in Tandala, Zaire, in 1977. The BON
strain was isolated in guinea pigs from the serum
of a patient in Sudan in 1976 (supplied by Dr. E.
Bowen, Porten Down, England) [12, 13]. Strains
MAL and KUM were isolated from the 1979
Sudan outbreak. 3 All of the viruses were used for
these studies after only one to four passages in E-6
Vero cells (a cloned line of Vero 76 that is maint'lined at the CDC). Virus materials used outside
the Maximum Containment Laboratory were
purified, inactivated, and tested for safety before
being brought out [14].
Suckling mouse studies. The third passage in
Vero cells of a strain from Zaire (MAY) (infectivity titer, 106 . 7 TCIDso/ml) and the fourth passage in
Vero cells of a strain from Sudan (BON) (titer,
106 . 5 TCIDso/ml) were inoculated into suckling
mice. For comparison, a 1979 strain (MAL) from
Sudan (titer, 105 .5 TCIDso) was also inoculated into
suckling mice. Litters of Swiss ler mice were inoculated intracerebrally with 0.02 ml of the BON
strain at dilutions of 10- 1 through 10-5 • The MAL
strain was inoculated undiluted, and the MAY
strain was inoculated at dilutions of 10- 1 through
10-8 • The LD so values for each strain were
calculated by the method of Karber [15].
Virus neutralization tests. We examined the
ability of homologous and heterologous human
serum to neutralize the lethal effect of the MAY
strain in suckling mice. Dilutions of 10- 1 to 10-5 of
the MAY virus pool (titer, 106 •7 TCIDso/ml) were
made, and I-ml aliquots were incubated at 37 C
with an equal volume of human convalescentphase plasma. The Zaire plasma (no. 096029) has
an IFA titer of 1:256 and was from a patient who
underwent plasmapheresis after the epidemic of
EHF in Zaire in 1976. The Sudan plasma (no.
015176) has an IFA titer of 1:2,048 and was from
a patient who recovered from EHF in Sudan in
1979 (the same patient from whom strain MAL
was isolated). Aliquots of virus were also mixed
3
See footnote 2.
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the Nzara area of Sudan resulted in 620/0 mortality.2
Collection of human specimens. Blood, serum,
or biopsy specimens for virus isolation collected
during the Zaire epidemic in 1976 and the Sudan
epidemic in 1979 were taken during the acutefebrile phase of the illness or shortly after death.
The specimens were stored in either dry ice or liquid nitrogen for delivery to the Maximum Containment Laborat'ory at the Centers for Disease
Control (CDC), Atlanta. Material from two
acutely ill patients was available from the Zaire
epidemic for virus isolation [2]. During the Sudan
epidemic in 1979, serum was collected from eight
acutely ill patients, and biopsy specimens were collected from two of those eight. A single specimen
was available from an isolated case of EHF in
northwestern Zaire, which occurred in 1977 [l 0].
Virus isolation. For primary virus isolation,
all specimens were inoculated in Vero-76 cells, a
cell line that is maintained at the CDC. The cells
were grown in Eagle's minimal essential medium
containing 1 unit of penicillin/ml, 0.5 /-lg of streptomycin/ml, and 0.2 /-lg of amphotericin B/ml
with 100/0 fetal calf serum. The cells were maintained in minimal essential medium with 2% fetal
calf serum. Serum specimens were inoculated undiluted and at dilutions of 10-0 .6 , 10-1, 10-2, and 10-3
into two screw-cap tubes per dilution. These were
observed for 14 days for CPE. After seven days of
incubation, cell scrapings were removed and tested
by indirect fluorescent antibody (IFA) testing [11]
for the presence of viral antigen. After 14 days of
incubation, cells were again tested for viral antigen, and the culture fluids (undiluted and diluted
to 10-2 ) were inoculated into fresh cell culture
tubes. These were also held for 14 days and tested
as before. The eight specimens from Sudan were
also inoculated at the same dilution in a human
lung carcinoma cell line (SW-13), which is maintained at the CDC. Finally, the eight specimens
from Sudan were inoculated into 300-g male Hartley guinea pigs in a final effort to recover virus.
Virus presence was confirmed by IFA testing in
cell cultures. For recovery of virus from guinea
pigs, the liver and spleens were harvested at day 10
after inoculation, and 0.1 ml of tissue suspensions
in minimal essential medium (0.1 g/ml) was inoc-
265
266
McCormick et af.
Table 1. Determination of LDso values in suckling
mice of two strains of Ebola virus isolated in Zaire and
Sudan.
Virus dilution
10- 1
10-2
10-3
10-4
10- 5
10-6
Zaire (MAY)
0/8
0/8
0/9
0/8
4/8
8/8
Sudan (BON)
7/7
6/6
8/8
7/7
8/8
ND
NOTE. Data are no. of survivors/no. of mice inoculated.
ND = not done.
Results
Isolates of strains of Ebola virus from Zaire were
readily obtained after primary inoculation of cell
cultures. In contrast, using exactly the same cell
line and technique, the strains from Sudan were
uniformly difficult to isolate, requiring multiple
passages in Vero cells or SW-13 cells or inoculation into guinea pigs before virus could be
detected. Two strains were isolated in Vero cells,
three were detected in SW-13 cells, and one was
recovered only by inoculation into guinea pigs. In
all, only six isolates were obtained from specimens
from eight acutely ill Sudanese patients with proven EHF.
To determine whether the differences in human
mortality and the difficulty in primary isolation
observed between strains from the two countries
were manifested in other biologic properties, we
measured the LOso of several strains for suckling
mice (table 1). Using an inoculum with a TCIOso of
106 . 7 /ml, the LOso of the MAY strain was
10-6 . 7 /ml, or approximately one infectious particle. In contrast, there were no deaths when mice
were inoculated with >1Os TCIOso of either of the
Sudan strains (BON or MAL). All strains were of
nearly identical passage history, and none had
been passaged in mice.
We were unable to show any neutralizing effect
of Zaire virus with human plasma having positive
IFA test results against either Sudan or Zaire
strains of Ebola virus.
The observed differences in mortality during the
Zaire and Sudan epidemics of 1976 were not
clearly understood. Host and ecological variables,
differences, in case definitions, and differential
mortality following parenteral versus contact
transmission of the viruses were all suggested as
potentially important [1, 2]. All parenteral infections in Zaire were lethal [2]. The case definition
in the 1979 Sudan outbreak, however, was the
same as that in Zaire in 1976. Contact infections in
Zaire resulted in 620/0 mortality, suggestive if not
conclusive evidence for inherently greater human
virulence of Zaire strains.
The relative facility of isolation of the Zaire
strains compared to the Sudan strains also indicates a fundamental difference in strains from the
respective countries. The eight Zaire strains isolated in 1976 were recovered without blind passage, and the initial titrations were on the order of
106 TCIOso [2]. In contrast, the Sudan strains were
very difficult to isolate in 1976, requiring passage
into guinea pigs [1]. Sudan strains did not initially
produce CPE in cell cultures, whereas the Zaire
strains produce marked CPE. Ellis et al. noted
larger numbers of RNA-free particles in cell cultures infected with Sudan strains as compared
with Zaire strains [16]. Small RNA species which
might characterize defective-interfering particles
were not found when concentrated virus preparations of Zaire and Sudan isolates were examined
[8]. Also, the failure to detect virions lethal for
mice in a high-titer Sudan strain is evidence
against the hypothesis of defective-interfering particles.
Further evidence was provided by experimental
work in rhesus monkeys, which were uniformly
killed by the ECK strain (Zaire) but which nearly
completely survived infection with the BON strain
(Sudan) [12]. Pathologic lesions in monkeys infected with the ECK strain were more extensive
compared to infections with the BON strain [16,
17]. Our expansion of previously reported data
[18] and comparative assessment of Ebola virus
geographic strain differences in virulence for
suckling mice also support the conclusion that the
Zaire strains are biologically more virulent than
those from Sudan for a variety of mammals. Also,
the establishment of infections that were lethal to
guinea pigs proved much more difficult for the
BON strain compared to the ECK strain [12].
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with 1% bovine serum albumin in phosphatebuffered saline, which was used as the diluent for
the virus. The virus-antibody mixtures were incubated at 37 C for 1 hr, and the mice were then
inoculated with 0.02 ml of the mixtures.
Discussion
Ebola Virus from Zaire and Sudan
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We were unable, as others before us [19], to
demonstrate neutralization of Ebola virus. Indeed, efforts to study immunologic cross-protection between the two strains have so far ended in
contradiction. Guinea pigs were protected against
lethal challenge with the ECK strain by prior infection and immunization with the BON (Sudan)
strain. Such was not the case for monkeys [12].
Nevertheless, the consistent biologic difference in
strains of Ebola virus from distinct geographic
regions leads us to conclude that the epidemics of
EHF in 1976 were remarkably coincident but completely independent events. More fundamental
quantitative properties of the viruses presented
elsewhere [5-7] help to reinforce this conclusion.
267