Georges, A.

Gonzalez, Jean-Paul

Johnson, E.D.

Horizon / Pleins textes

i 
536 
Filovi& activity among selected ethnic groups inhabiting the tropical forest of 
eq u at o ri a I Africa 
E. D. Johnson1, J. P. Gonzalez2 and Alain Georges3 ‘United States A m y  Medical Research Institute of Infectious 
Diseases, Frederick, Maryland, USA;  2Institut Français de Recherche Scientifique pour le Développement en Coopération 
(ORSTOM), Paris, France; 3Institut Pasteur, Bangui, Central African Republic 
Abstract 
Seroepidemiological surveys were conducted to determine the frequency and distribution of filovirus activ- 
ity among selected ethnic groups inhabiting the tropical forests of the Central African Republic. 427 serum 
specimens were collected from hunter-gatherers and subsistence farmers living in forest environs in the Lo- 
baye District south of the river Lobaye and west of the river Oubangui. Striking serological evidence for 
filovirus activity was found in both populations. Ebola virus appears to be the most active filovirus; 17.6% 
(75/427) of the Lobaye survey population were seropositive for Ebola virus reactive antibody while 1.2% 
(5/427) were seroreactive with Marburg viral antigens. Ethnic background appeared to be an important risk 
factor influencing filovirus exposure in the forest communities. The filovirus antibody prevalence among 
21-40 years old male Aka Pygmy hunter-gatherers was significantly (P=0.03) 3 times higher (37.5%) than 
that in similarly aged male Monzombo and Mbati subsistence farmers (13.2%). Continued epidemiological 
investigations are needed to define ethnic-related events influencing human filovirus activity in the Congo 
basin of equatorial Africa. 
Introduction 
The filoviruses Ebola and Marburg viruses have 
caused sporadic but widespread epidemics of fatal hae- 
morrhagic disease in sub-Saharan Africa. These highly 
pathogenic viruses circulate undetected, presumably as 
enzootic infections, until conditions change favouring 
their expression and recognition as severe human pa- 
thogens (SIEGERT, 1970; SMITH, 1978). 
A concerted effort was begun in 1984 to define risk 
factors for infections and the natural threat posed by the 
filoviruses in central Africa. Over the 4 year period 
19841987 cross-sectional and prospective epidemiologi- 
cal and clinical surveillance studies were conducted in 
which serosurvey samples and clinical specimens from 
fever cases of unknown aetiology were assessed for evi- 
dence of haemorrhagic fever virus activity. Our early 
findings, suggesting frequent filovirus exposures in se- 
lected central African populations, have been presented 
elsewhere (GONZALEZ et al., 1982, 1989; MEUNIER et 
al., 1987). 
This paper reports the frequency of filovirus activity, 
as measured by the prevalence of virus reactive antibody, 
among selected ethnic groups, Aka Pygmies or hunter- 
gatherers and Monzombo and Mbati villagers or subsist- 
ence farmers, inhabiting the Lobaye District forest envi- 
rons of the Central African Republic. Potential 
epidemiological and ecological factors which may in- 
fluence endemic virus activity are also discussed. 
Materials and Methods 
Survey populations 
During the 1987 dry season, formt villages in the 
Mongoumba region of the Lobaye District were serosur- 
veyed. Medical clinics were held in the Monzombo and 
Mbati villages Molabaye, Yabongo, Ikoumba, Saboulou, 
and in Gouga and Aka forest camps to assess the general 
health of the local forest populations. Physical injuries, 
burns, minor myalgic and arthritic disorders, bacterial, 
fungal and parasitic infections were treated. Village and 
forest residents were informed of the purpose of the sero- 
survey; volunteers were interviewed, serosurveyed, and 
issued a registration card containing the individual’s 
name and identification number used to label corre- 
sponding specimens. An interview form was completed 
documenting the individual‘s name, identification num- 
ber, estimated age, sex, village of residence, length of 
residence, ethnic group, occupation, father’s name, 
mother’s name, and husband’s or wife’s name. 
Sample collection 
Venous blood was drawn aseptically from the ante- 
cubital fossa following accepted standards governing pro- positive for virus reactive 
cedures to be used with human subjects. The blood was 
allowed to clot overnight at 4°C. The serum was separ- 
ated by centrifugation, dispensed into cryotubes and im- 
mediately frozen in liquid nitrogen for transport to the 
laboratory. 
Immunofluorescent antibody test 
An indirect immunofluorescent anribody test (IFAT) 
using monovalent and polyvalent spot slides of acetone- 
fixed virus and sham-infected cells waspsed throughout 
the study to detect and measure haemorrhagic fever virus 
(HFV) reactive antibody (JOHNSON, E. D. et al., 1993). 
Monovalent slides were prepared using uninfected cells 
mked 1O:l with cells infected with a single HFV. Poly- 
valent slides were produced using a mixture of equivalent 
numbers of sham-infected cells and cells infected with 
each of 6 African haemorrhagic fever viruses (AHFV) 
listed below. 
The Mayinga strain (Zaire isolate) and Boniface strain 
(Sudan isolate) of Ebola virus (EBOV), the Musoki strain 
(Kenya isolate) of Marburg virus (MBGV), the Josiah 
strain (Sierra Leone isolate) of Lassa virus, the 10200 
strain (Uganda isolate) of Crimean-Congo haemorrhagic 
fever virus (CCHFV), and the ZH501 strain (Egypt iso- 
late) of Rift Valley fever virus (RVFV) were used to pre- 
pare serological reagents (JOHNSON, B. K. et al., 1982; 
JOHNSON, E. D. et al., 1993). Infectious HFV prepara- 
tions were handled within the containment facilities de- 
signed and equipped for maximum biological contain- 
ment (level 3 and 4) at the United States Army Medical 
Research Institute of Infectious Diseases. 
The IFAT was standardized using convalescent 
plasma or immune serum from confirmed HFV infec- 
tions and goat anti-human y globulin conjugated with 
fluorescein isothiocyanate. Each test was performed 
double-blind. Questionable IFAT reactions were re- 
examined by a reviewer and technician andlor an outside 
observer. The tests were repeated when a consensus 
could not be reached concerning inconclusive reactions. 
The few sera which produced inconclusive reactions on 
retesting were considered non-specific IFAT reactors. 
The survey specimens were screened for virus-reactive 
antibody at 1: 16 dilution on polyvalent slides. Virus spe- 
cificity was determined by rescreening the IFAT sero- 
reactive specimens on monovalent slides. The level of se- 
roreactivity was determined by titrating the IFAT 
reactions on monovalent slides using two-fold serial dilu- 
tions. Antibody wefe recorded as the reciprocal of 
the last clearly posiuve dilution. Specimens that reacted 
with infected but not uninfected monovalent cell prep- 
arations at 1:128 or greater dilution were considered 
high antibody titre, 1:128, was selected as the cut-off 
point between seropositives and seronegatives to reduce 
the likelihood of including false positives (JOHNSON, E. 
D. etal., 1993). 
Statistical analysis 
The significance of differences between HFV antibody 
prevalence rates was determined by x2 analysis (SNEDE- 
COR & COCHRAN, 1969). 
Results 
African haemorrhagic fever virus antibody prevalence 
Evidence of AHVF activity was found in the Lobaye 
study population; 18.7% (801427) of the population were 
seropositive for HFV, with titres 2128. The filoviruses 
appeared to have been the most active AHFV in 1987: 
17.5% (751427) of inhabitants were filovirus seropositive; 
0.7% (31427) were RVFV seropositive; and 0.5% (2/427) 
were positive for Lassa virus antibody. Serological evi- 
dence for human CCHFV activity was not found. 
Filovirus antibody prevalence 
The majority of filovirus seroreactive specimens from 
Lobaye District (94.6%, 71/75) reacted with the EBOV 
strains and not with MBGV (GONZALEZ et al., 1983). 
Sixty-one per cent (45/74) of the EBOV seropositive 
samples reacted with both EBOV-Zaire (EBOV-Z) and 
EBOV-Sudan (EBOV-S) viral antigens and were con- 
sidered positive for EBOV group specific antibody. 
Broad 'family' reactions were observed in 4.1% (3/74) of 
the seropositive samples which reacted with both EBOV 
and MBVG antigens. Filovirus strain-specific responses 
(EBOY-Z, EBOV-S, or MBGV) were also observed; 
8.0% (6/75), 30.7% (23175) and 1.3% (1175) of the total 
number positive were EBOV-Z, EBOV-S or MBGV se- 
roreactive, respectively. 
Table. Sex distribution of filovirus antibody seropositivity 
among hunter-gäeerers and farmers in Lobaye District, 
Central African Republic 
No. seropositive" 
Total Male Female 
Hunter-gatherers 31/127 (24.4) 13/56 (23.2) 18/71 (25.3) 
Farmers 421300 (14.4) 181169 (10.7) 24/131 (18.3) 
"Number seropositive/total number tested (with percentages in 
parentheses). 
Risk factors forfiloviws infections 
Ethnic background appeared to be an important factor 
influencing filovirus antibody activity among the forest 
communities (Table). The filovirus antibody prevalence 
in hunter-gatherers was significantly higher (P=O.O11) 
than that among farmers. The antibody prevalence in 
both male and female forest dwellers was higher than the 
level found in corresponding male and female village 
residents; however, only the male difference was statisti- 
cally significant (P=0.025). 
Ethnic background remained an important factor 
when sex- and age-specific filovirus reactive antibody 
prevalence rates were compared (Figure). The antibody 
prevalence appeared to be 2 or 3 times higher among 
male hunter-gatherers than in similarly aged male 
farmers. However, the difference was statistically signifi- 
cant (P=0.03) in only the 21-40 years old male age 
group (Pygmies, 37.5%; farmers 13.2%). Differences be- 
tween female Pygmy and farmer age-specific antibody 
prevalences were less striking. The antibody prevalence 
was higher among the 21-40 year old female hunter- 
gatherers (30.8%) than among similarly aged farmers 
(18.6%), but the difference was not statistically signifi- 
cant (P=0.22). 
Discussion 
Our results indicate that filovirus infections are com- 
MALES 2 7 ~  I FEMALES 
I 
I 
I 
I 
25.0 ' 
; 20.5 m 
. I 
537 
Age Groups (Years) 
District, Central African Republic. 
to occur without significant overt disease or increased 
mortality, suggesting that African filoviruses may be less 
pathogenic in their natural environment than expected, 
or that filovirus infections are unrecognized, being con- 
fused with some common local illness or culturally 
defined disease occurring in the Lobaye forest. 
The surveyed population of 427 forest inhabitants con- 
sisted of 400 members of 3 ethnic groups, 127 Aka, 219 
Monzombo, and 54 Mbati, and 27 individuals whose eth- 
nic background was not determined. These groups share 
the forest resources south of the Lobaye river and west of 
1988; BAHUCHET et a¿. ,1990). 
The difference in high titre filovirus antibody pre- 
valences between adult male and female Aka forest dwel- 
lers strongly suggests that filovirus exposure in the Lo- 
baye forest is influenced by factors associated with the 
hunter-gatherer subsistence strategy. The previously re- 
ported findings that the filovirus antibody prevalence is 
higher among Pygmy hunter-gatherers than Bantu sub- 
sistence farmers of the Lombie region of Cameroon sup- 
port this interpretation (BAUREE & BERGMANN, 1983). 
Monzombo and Mbati females maintain multiple forest 
538 
garden plantations throughout the year, while males tend 
to be fishermen, often spending long periods in fishing 
camps along the river (BAHUCHET, 1988). 
Collectively, our findings suggest the filoviruses are 
encountered during their daily occupation by a distinct 
subpopulation of male and female hunter-gatherers and 
female farmers inhabiting the eastern Lobaye. The puta- 
tive risk factors may be related to the collection and 
handling of meat. The forest provides the bulk of the 
animal protein consumed by Lobaye residents. Large 
mammals like antelope and bush-pigs are netted or 
trapped during the dry season by Aka hunting bands 
consisting of both males and females while smaller ani- 
mals such as monkeys and fruit bats are stalked by soli- 
tary male hunters during the August and September 
rainy season. Females prepare and distribute the best 
meat among the hunting band and often use the remain- 
der to barter with village women (HART, 1978). 
The consumption of Cercopithecus monkey meat may 
involve important risk factors for filovirus infection. 
Feral monkeys from diverse African habitats have been 
shown to be seropositive for filovirus antibody (JOHN- 
SON, B. K. et al., 1982; MATHIOT et al.,  1990); seronega- 
tive monkeys have been shown to be susceptible to labor- 
atory filovirus infection but resistant to fatal disease (P. 
B. Jahrling, personal communication); wild-caught mon- 
keys have been associated with two large filovirus out- 
breaks (HENDERSON et al., 1971; JARHLING et al., 1990); 
and the August 1976 EBOV haemorrhagic fever index 
case in Zaire had purchased fresh monkey meat 10 d be- 
fore becoming fatally ill (JOHNSON, K. M., 1978). Mon- 
keys, however, may not play a central role in the natural 
filovirus cycle; they may live in close association with the 
primary maintenance reservoir and only occasionally 
become infected. Filovirus transmission, therefore, may 
occur only in human populations whose subsistence 
strategy places them in frequent and prolonged contact 
with freshly killed animals. 
An association between filovirus antibody and contact 
with feral monkeys among forest inhabitants may be 
unique to distinct forest groups. The antibody pre- 
valence found in 1987 Lobaye hunter-gatherers (311127) 
was roughly 3 times higher than that observed in 1984 
serosurveys of Pygmies living in the Sangha district 
(9/121) of south-western Central African Republic 
(P=O.Ol) (E. D. Johnson & J. P. Gonzalez, unpublished 
observations). A difference in filovirus antibody pre- 
valences was also observed between Lodorodorf-Bipindi 
and Lombie Pygmies of Cameroon (BAUREE & BERG- 
MANN, 1983). These differences may be the consequence 
of variations in environment or culture. Subsistence 
methods and, presumably, the importance of monkey 
meat vary among Pygmy groups of central Africa; some 
hunter-gatherer bands have given up their nomadic 
forest life style to become forest edge cultivators (HEW- 
LETT et al., 1986; BAHUCHET, 1990; BAHUCHET ct al., 
Nevertheless, the hypothetical association between 
filovirus reactive antibody and feral monkeys should be 
explored by epidemiological and anthropological investi- 
gations, which may resolve the paradox of high antibody 
prevalences for the highly pathogenic filoviruses without 
notable disease; the prevalence of filovirus antibody may 
be high among survivors of a common disease associated 
with contact with feral monkeys in the tropical forest of 
equatorial Africa. 
,+I<! 
1990). 
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Received 28 September 1992; revised 2 F e b r u a y  1993; 
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Filovirus activity among selected ethnic groups inhabiting the tropical forest of equatorial Africa

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