.hlJRNAL OF VIROI,OGY, Oct. 1999, p. 8196-8200...
.hlJRNAL OF VIROI,OGY, Oct. 1999, p. 8196-8200
0022-538X/99!$04.00+0
Copyright 0 1999, American Society for Microbiology. Al1 Rights Rcscrved.
Genetic Reassortment of Rift Valley Fever Virus in Nature
A. A. SALL,’ P. M. DE A. ZANOTT0,2 0. K. SENE,’ H. G. ZELLER: J. P. DIGOUTTE,’
Y. THIONGANE 4 AND M. BOULOY’*
.---.----’
Institut Pasteur de Dakar’ and Institut Senegalais de Recherche Agronomique,4 Dakar, Senegal; DIPA-UNIFESP,
Silo Paulo, Brazif; Institut Pasteur de Madagascar, Antananarivo, Madagascar’; and Groupe des Bunyaviride,
Institut Pasteur, 75724 Paris Cedex 15, Fran&
Receivcd 9 April 1999iAccepted 15 July 1999
Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridue family, is an arthropod-borne virus which
emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human
populations. TO assess the genetic variability of RVFV, several isolates from diverse localities of Africa were
investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S),
medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major
lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However,
incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment
occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further
confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of
endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain
forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated
vaccines in areas of endemicity.
Rift Valley fever is a serious emerging arthropod-borne viral
nucleocapsid protein N and the nonstructural protein NSs by
anthropozoonosis caused by a phlebovirus (Rift Valley fever
using an ambisense strategy. Because of its segmented nature,
virus [RVFV], family Bunyaviridae), which was reported pri-
the genome of members of the Bunyaviridae family allows
marily to infect domestic cattle and more recently to cause
RNA segment reassortment (exchange of a whole segment)
massive epidemics in human populations across Africa. Mod-
when cells are coinfected by two closely related viruses of the
ifications in the ecological andlor environmental conditions
same genus or serogroup (reviewed in reference 26). Reassort-
appeared to be responsible for the emergence of the virus (16,
ment between strains of RVFV has been demonstrated exper-
24). Disease in humans exhibits clinical manifestations ranging
imentally in tissue cultures (30) and in mosquitoes that were
from acute febrile illness to severe complications. including
dually infected (38). Naturally occurring reassortants have
hepatitis, encephalitis, hemorragic fever, and ocular sequelae
been suggested or demonstrated for bunyavirus (8, 12,40) and
(13). Periodic large-scale epidemics, such as the ones in Mau-
hantavirus (33, 14, 27). However, to date, despite some clues
ritania in 1987 and 1998 (7, 43). Madagascar in 1990-1991
derived from unexpected groupings in a phylogenetic tree
(17.-19), and Egypt in 1977 and 1993 (2, 16), as well as in East
based on the NSs coding region (29), reassortment among
Africa (Kenya, Somalia, and Tanzania) in 1997-1998, reiterate
natural isolates of RVFV has not been investigated. In this
the potential of this virus as a considerable threat to human
report, we address the question of RVFV genetic reassortment
health, the latter epidemic affecting some 89,000 people and
under natural conditions and its consequences
on the evolution
causing 500 deaths (1). Analysis of one strain isolated from a
and epidemiology of the disease.
fatal human case during this epidemic showed a close related-
ness with a strain isolated in Madagascar during the 1990-1991
outbreak (28), revealing that the virus could spread across
MATEHIALS
A N D M E T H O D S
considerable distances, possibly beyond Africa. This threat be-
Virus propagatinn and RNA extraction. The origins and ycars of isolation of
cornes more disquieting when we consider that numerous mos-
RVFV isolates are shown in Table 1. Propagation of viruses and cytoplasmic
RNA extraction from infected cells were done as previously described (29).
quito species around the world are competent laboratory vec-
Reverse transcription (RT)-PCR and sequencing procedures. Three different
tors for RVFV (9, 35-37). Therefore, understanding the
sets of primcrs, NS3a-NS2g, MRVla-MRV2g, and Wag-Xg. were used to am-
mechanism underlying its dispersa1 and evolution is of para-
plify portions of the NSs, G2, and L coding regions. respectively. The NSs coding
mount importance to the control of this disease.
region is located in the S segment, whcreas the regions coding for G2 and L are
The RVFV genome consists of three negative-sense single-
in the M and L segments, rcspectively. The antisense primers NS3a. MRVLa, and
Wag were used to synthcsize thc first-strand complementary DNA. Primer se-
stranded RNA segments designated L (large), M (medium),
quences, protocols for cDNA synthesis, PCR amplification, and direct sequenc-
and S (small) (for reviews, see references
10 and 31). The L
ing of PCR products were described previously (20, 21, 29, 34).
segment codes for the L viral polymerase. The M segment
Phylogenetic
reconstruction. The phylogenetic analyses of each segment of the
RVFV genome were performcd with data sets for the NSs, G2, or L protein
codes for the precursor to the envelope glycoproteins, Gl and
coding region by using maximum likelihood and maximum parsimony methods in
G2, which, after cleavage, generate two additional nonstruc-
PAUP (Phylogenetic Analysis Using Parsimony, brta version 4.0; kindly pro-
tural proteins of 78 and 14 kDa. The S segment codes for the
vided by David L. Swofford, Smithsonian Institution, Washington, D.C.). For a
more realistic tree reconstruction and branch length estimates for each data set,
the optimal values for the transition probahilities among different nucleotides
and the value for the shape parameter (alpha) for the gamma distributed variable
* Corrcsponding author. Mailing address: Institut Pasteur. Groupe
rates among sites were empirically determined from the data. Using the optimal
values of these parameters. trecs with similar likelihood were collected and the
des Bunyaviridés, 25, rue du Dr. Roux, 75724 Paris Ccdcx 15, France.
tree topology stability around the likelihood maxima was investigated hy calcu-
Phonc: 33 1 40 61 31 57. Fax: 33 1 40 61 31 51. E-mail: mbouloy
lating the 50% majority-rule consensus. For an additional comparison, we used
@pasteur.fr.
a fast tree search algorithm (quartet puzzling) for estimating maximum likeli-
8196
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VOL. 73, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
8197
TABLE 1. Origins and years of isolation of RVFV isolatcs
Grnotype”
S1rain
Yr of
isolati«n
(SIMIL)
SNS’
Smithburn
1 9 4 4
Uganda
Entcbbe strain
CICIC
ArUGA-55
Lunyo
1 9 5 3
Uganda
Mosquito
c/cic
ArCAr-69
Ar El 1976
1 9 6 9
CAR
Mosquilo
CICIC
Cl3
Clone 13
1974
CAR
Human
*K7c
HEGY-77
ZH 54X
1 9 7 7
Eiwt
Human
EiE,‘E
MPIZh
MP12
1 9 7 7
Egwt
ZHS48 strain
E/E/E
ArMAD-79
Ar Mg 811
1 9 7 9
Madagascar
Mosquito
E/?/E
ArSEN-84
Ar D 38661
1 9 8 4
Scnegal
Mosquito
EI’W~W
AnGUI-
An K 6087
1984
Guinca
Bat
WICIC
ArBUF-84
Ar D 38457
1 9 8 4
Burkina Faso
Mosquito
w,ww
HlMAU-87
H D 47502
1 9 8 7
Mauritania
Human
ciww
H2MAU-87
H D 47311
1987
Mauritania
Human
w/w/w
H3MAU-87
H D 47408
1 9 8 7
Mauritania
Human
w,wnv
H4MAU-87
H D 48255
1 9 8 7
Mauritania
Human
w,~?iw
AnMAD-91
An Mg 990
1991
Madagascar
Bovine
CICIC
ArSEN-93
Ar D 104769
1993
Senegal
Mosquito
CiGW
AnSEN-K-93
An D 106417
1 9 9 3
Scncgal
Zebu
Wlw!C
BEGY-93
B EGY 93
1 9 9 3
Emt
Buffalo
EiEiE
HEGY-93
H EGY 93
1 9 9 3
EgYPt
Human
EIEE
HKEN-97
384-97.1
1 9 9 7
Kenya
Human
C/C/C
” E, W. and C, Egyptian, Western African. and Central-East African lineagc, respcctively: :j:, scyuencc too short to he a.rsigned a lincage; ‘?, sequence n»t dctermined.
’ Attenuated strain.
hood trees from PUZZLE version 3.1 (33), which automatically
assigns estima-
First, somc of the strains in the Central-East Africa and Egypt
tion of support of each interna1 node. Additionally. to investigatc thc robustness
groups (SNS and ArUGA-55, AnMAD-91 and HKEN-97.
of tree resolution under maximum parsimony. a hootstrapping analysis was done
with 1,000 random resamplings with the ncarcst-neighbor-interchange perturba-
HEGY-77 and HEGY-93) are closely related, despite their
tion algorithm within PAUP. For the ahortest data set (i.c., the 211~bp L ampli-
dates of isolation. suggesting the existence of an endemic-
con), a 2% jackknife wx used as an alternative resampling method.
enzootic maintenance cycle of the virus in these areas. Second,
To determine topological incongruence among estimatcs obtained from dif-
no obvious grouping based on thc host species was observed.
ferent data sets for the same set of taxa, wve chcckcd whether the hest topology
for each data set is statistically worse when rcconstructcd with an alternative data
This could be explained by frequent exchange of viruses bc-
set by using the Kishino and Hasegawa les1 implemcnhxl in PAUP 4.0. Addi-
twecn different hosts. Third, in each group, strains isolated
tionally, t« test for reassortment among viruacs from dilTcrent lineages. con-
during epidemic-cpizootic and endemic-enzootic periods were
straint trees wcre constructcd; in these, the taxa wcrc assigned to a given part of
found adjacent to each other, suggesting that the virus circu-
the tree, generating alternative topologies to he tested against the ones ohtaincd
from the unconstrained data. Finally, to allow the inspection of the topologies
lates alternatively in enzootic-endemic form through a main-
estimated from different genomic segments, thc TrecMap program (22) was uscd
tenance cycle or epidemic-epizootic mode, both cycles provid-
to construct parallel phylogenies.
ing each other with viral strains. However. despite similar
Nucleotide sequence accession numbers. The sequcnces of thc strains listcd in
Table 1 and corresponding to the L, G2, and N protein coding regions bave hecn
distributions of each tree into three clusters, the assignment of
assigned GenBank accession n». AF134782 10 AF134XOl. AF134492 to
some isolates (ArSEN-93, AnSEN-K-93, ArSEN-84, HIMAU-
AF134508, and AF134530 t« AF134551, rcspectively. The sequcncçs of thc NSs
87, and AnGUI-84) to onc particular group does not remain
protein coding region werc depositcd in the EMHL database (accession no.
constant within the three phylogenies. Construction of the
Y12739 to Y12756) as already reported (29).
reconciling parallel trees using the three phylogenies empha-
sized that the groupings of the strains in the S, M. and L
RESULTS
segment trecs correlate quite well but clearly show somc in-
Phylogeny of different RVFV segments. In a previous papcr,
congruenccs. For instance, HlMAU-87 and ArSEN-84 possess
phylogenetic analysis carried out with 20 isolates and derived
thc L and M segments of the West African genotype and the S
from the 669-nucleotide-long sequences of the NSs protein
segment from the Central-East African or Egyptian lineage,
coding region in the S segment showcd the existence of three
respectively. AnGUI- has the L and M segments of the Central-
distinct lineages: la and Ib in sub-Saharan Africa and II in
East African genotype, whercas the S segment belongs to the
Egypt (29). Additional sequencing of the M and L segments
West African lineage. Finally, two strains isolated during the same
was performed after RT-PCR amplification of an 80%nucleo-
period in two different places in Scnegal, Barkedji (North) and
tide-long DNA fragment located within the region coding for
Kolda (Casamance, South), ArSEN-93 and AnSEN-K93, have a
the G2 protein and a 212-nucleotide-long
DNA fragment lo-
different distribution within clusters: AnSEN-K93 was located
cated in the L protein. Phylogenetic analyses of these se-
within the Western African lincage with regards to its S and M
quences using the maximum likelihood and maximum parsi-
segments and within the Central-East African lineage with re-
mony methods confirmcd the distribution pattern within the
gards to its L segment. Reciprocally, in the case of ArSEN-93, its
three lineages, which were renamed Central-East Africa, West
S and M segments belong to the Central-East African lineage and
Africa, and Egypt for la, Ib, and II, respectively (see Fig. 1).
the L segment belongs to the Western African Iineage (Fig. 1).
The support for different nodes on the tree was evaluated by
The possibility that incongruences might be due to cross
various methods (i.e., bootstrap, jackknife, or maximum like-
contamination among isolates seems highly improbably given
lihood topology consensus) and showed strong support for the
thc following extreme prccautions that were taken to avoid
three phylogenies.
such artifacts. (i) RNA extraction and RT were done sepa-
These phylogenetic analyses raised some general comments.
ratcly for çach isolate undcr a laminar-flow containment hood
8198
SALI, ET AL.
J. VIROL.
B
A
-
r H2MAU87
--~
ri ArSEN84j
l
--+AnSENk93
/
H3MAU87
I-
~
- H4MAU87
WEST
AFRICA
~EST
FFRICA
ArBUF84
l
j
AnGU184 1
i
.-
-MP12
+iEGY93
1
I
EGYPT
I
EGYPT
-’
BEGY93
‘Bpi HEGY77
1
i
I AnMAD91
~ ArMAD79
1
j
EAST
/
CENTRAL
EAST
’
AFRICA
CENTRAL
1
;& r_“S;,,
AFRICA
k%’
.-- -.-- SNS
ArUGA55
-
C
FIG. 1. Maximum likelihood trees for the NSs (A), Ci2 (B), and L(C) coding
region sequences on the S, M, and L segments, respectively. Values helow the
H2MAU87
diffcrent nodes indicate their rohustness by 5% jacknife (indicated in boldface
-7
and italic type), maximum likelihood quartet puzzliny (indicated in hrackets).
and hootstrapping (indicated in roman type) methods. Putative reassortant
strains are hoxed.
- - - H3MAU87
I
----H4MAU87
;WEST
IAFRICA
- ~ A r B U F 8 4
c-il
sequenced several times and led to identical sequences, many
hArSEN93:
of them being processed in laboratories at least 4,000 km apart
i
-
(Paris, France, and Dakar, Senegal).
r--ArMAD79
Given that trees for different genes are clearly incongruent
in their topologies, it could be assumed that these incongru-
pl/BEGY93
,EGYPT
ences are due to genetic exchange. Regarding the segmentation
jHEGY77
of the RVFV genome, the most probable device to explain the
r HEGY93
exchange of genetic material is RNA segment reassortment.
‘MPI2
Tests for topological structure as indicative of reassortment.
Two approaches
were used to evaluate the incongruences ob-
---1
I
AnSENk93 >:
served among specific isolates of different trees. First, we con-
I
-------HKEN97
I
structed constrained trees and tested their topologies by using
SS!
likelihood against the unconstrained trees shown in Fig. 1. The
~91 AnMAD91
j
EAST
;-----
CENTRAL
likelihood value for the unconstrained tree resulting from the
- - - - - - iAnGU1B;il
AFR,(.A
use of the NSs sequences was better than the one obtained for
the constrained tree where the ArSEN-93 and HlMAU-87
-i!‘lÀrRccp369
isolates were forced into the West African group (-1nL =
-ArUGA55
1,774.76, difference of 1nL = 57.21, P < 0.001). Similarly, the
S N S
values obtained for the G2 and L sequence phylogenies (for
G2, difference of 1nL = 42.95, P = 0.0002; for L region, dif-
5 % jackknife IWO mplhticms
[% ML quartet puzzlinp]
ference of 1nL = 9.74, P = 0.06), in which ArSEN-93 and
60% MR bootsVapi000 replicatiom
AnGUI- or AnSEN-K-93 and AnGUI-84, respectively, were
forced into the West African group, led to the same conclu-
sion. Second, we used the Kishino and Hasegawa test on the
which was decontaminated between each manipulation. (ii)
difference of the likelihoods for trees obtained for taxa com-
PCR mix preparation, PCRs, and electrophoresis were per-
mon to both G2 and NSs data sets. The maximum likelihood
formed in separate rooms. (iii) For cach isolate, PCR products
tree for G2 (-1nL = 1,684.40) had a lower likelihood (differ-
derived from independent RNA extraction and RT-PCR were
ence of 1nL = 30.35, P = 0.0603) under the NSs data set than
VOL. 73, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
8199
the NSs tree did (-1nL = 1,654.06), and the NSs trec (-1nL =
meric viruses werc viable and their host did not exert a nega-
1,842.69) had a lower likelihood (difference of lnI, = 51.99,
tive sclcction against them. We do not know whether the
P < 0.0001) under the G2 data set than the G2 tree did
strains identified as reassortants werc host restricted, but they
(-1nL = 1,790.69). Although the difference of likelihood (InL
were isolated from different hosts, including humans, mosqui-
difference = 30.35) for the G2 tree under the NSs data set may
toes, bats (An GUI-84), and zebu (AnSEN-K93). With regard
not be statistically different under the 95% confidence level,
to cocirculation of different strains which cari potentially un-
likelihood tests using either constrained trees or the Kishino
dergo reassortment, Senegal is a very instructive example as an
and Hasegawa test support the notion that the incongruences
area where at least two different lineages circulated at the same
observed among trees are due not to poor phylogcnetic topol-
time in 1993. Since RVFV is an arbovirus, reassortment among
ogy inferences but rather to genetic exchange.
strains cari occur in a dually infected mosquito or vertebrate
In light of the recent report of recombination in hantavirus
host. Reassortment was demonstrated experimentally in ham-
evolution (32), it could not be excluded that the incongruences
sters as well as in mosquitoes that were dually infected with two
in the RVFV tree topologies could be due to intramolecular
RVFV natural isolates (38). It seems likely that vertebrate
recombination. Although such events appear, SO far? to be very
hosts cari be naturally infected with different strains of RVFV
rare among negative-strand RNA viruses (26), we considered
since a human isolate from the Republic of Central Africa was
this possibility and analyzed the NSs, G2, and L data with the
shown to be composcd of a heterogenous population of viral
split decomposition method, by using the program Splits ver-
clones with ditlerent biological properties (21). Mosquitoes cari
sion 1.0 (3), but we did not find any clear-tut indication of
bc dually infected by interrupted fceding or feeding on a dually
recombination, i.e., networked evolution (data not shown),
infected vertebrafe host. Both mechanisms have been demon-
within the fragments sequenced. Thus, reassortment events gen-
strated cxperimentally for RVFV with a natural mosquito vec-
erating combination between L, M, and S segments of RVFV
tor, Cukxpipierw, suggesting that under natural conditions, the
natural isolates appear to be the most likely explanation.
process is probably the same. There exists a third theoretical
possibility for a mosquito to be dually infected: a female in-
DISCUSSION
fected transovarially or venereally with one strain could be-
corne superinfected during a bloodmeal with another strain.
This study indicated that 5 of the 20 (25%) isolates, ArSEN-
However, due to homologous interference,
vertically infected
93, AnSEN-K-93, ArSEN-84, HlMAU-87, and AnGUI-84, ap-
mosquitocs may be refractory to superinfection with the same
pear to result from a reassortmcnt event. This percentage may
virus spccies. Therefore, both mosquitoes and vertebrate hosts
be underestimated since the method used herein to identify
may act as a site for RVFV reassortment in nature but their
reassortants took into account only reassortment events be-
relative contribution in the natural process of reassortment
tween the three major lineages (Egypt, West Africa, and Cen-
remains to be dctermincd.
tral-East Africa). Interestingly, all the strains identified as re-
The occurrence of reassortmcnt among natural isolates from
assortants, ArSEN-93, AnSEN-K-93,
ArSEN-84, HI MAU-87,
West Africa and Central-East Africa or, less frequently, Egypt
and AnGUI-84, were isolated from West Africa (Scncgal,
suggests two possibilities: (i) one strain was transported to a
Guinea, and Mauritania) and involved reassortment with
place where another strain was circulating; or (ii) the virus had
strains of the Central-East African or Egyptian lineage (Ar-
to make its way through the rain forest and to adapt to the
SEN-84) (Table 1). In addition, these reassortants have re-
different fauna and ecotopes, establishing a sylvatic cycle. Al-
tained combinations of homologous L plus M segments (Ar-
though the existence of a sylvatic cycle would have to be dem-
SEN-84, AnGUI-84, and HlMAU-87) and M plus S segments
onstrated, thc circulation of RVFV in the rain forest has heen
(AnSEN-K-93 and ArSEN-93) (Table 1). In dual infection of
revealed by thc high antibody prevalcnce in wild animals and
BHK-21 cells with La Crosse and Snowshoe hare viruses, some
pygmy populations in Central African Republic (6) and mul-
segment associations appeared to be preferred (39). Investiga-
tiple strain isolations from mosquitoes in the primary forest of
tions with a larger sample of RVFV field isolates would be
Perinct in Madagascar (15). In addition, data recently published
needed to indicate whether generation and selection of reas-
by Pretorius et al. (25) indicatcd that rodents were involved as a
sortants in nature are random or not.
potential reservoir of RVFV in South Africa. It is likely that
Replication errors such as base substitution and deletions or
different strains of RVFV circulate in the rain forest area among
insertions are the most common mechanism of RNA virus
wild mosquitoes (16) and vcrtcbratcs like monkeys (23) and bats
evolution. However, major changes of viral genotype may in-
(5), depending on the dynamic of the interaction among hosts and
volve exchanges of RNA segments (genetic shift), as exempli-
virus populations. Therefore, the search and characterization of a
fied by influenza virus (42). Comparison of RVFV isolates
sylvatic cycle of RVFV are of utmost importance because this
sampled from different geographical localities showed that
would lead to better understanding and control of the epidemi-
evolution of this virus in nature not only appears to be due to
ology of Rift Valley fever and its potential cmergence.
point mutations, the percentage of base substitution varying
Thc existence of reassortment as a mechanism of evolution
from 0 to 9.6% in the S segment (29), but also may occur by
raises the question of thc cpidemic potential of these viruses
genetic exchange. Indeed, the apparent contradictions in the
and their pathogenicity. Isolation of a potential reassortant
groupings of the different segments within the three distinct lin-
from a fatal case during the 1987 Mauritanian epidemic leaves
eages could be explained by reassortment and strongly suggest
this question open, even though othcr strains which were not
that this evolution mechanism is a common trait in RVFV natural
identified as reassortants were isolated from fatal cases. A
history. Arboviruses apparently evolve approximately lO-fold
similar question concerning the outbreak of Sin Nombre virus
more slowly than RNA viruses that have only a single vertebrate
was raised (1 1), but in spite of thc evidence of reassortment
host (4,41). T~US, the evolution of RVFV driven by reassortment
among Sin Nombre virus isolates (II, 14, 27). there was no
and by point mutations may depend on the host number.
indication that thc emcrgcncc of hantavirus pulmonary syn-
Reassortment under natural conditions implies the existence
drome was due to a novel chimeric virus. On the other hand.
of at least two different strains present at the same time, in the
reassortment appeared to be involved in the triggering of an
same area, and infecting the same host. One additional requi-
influenza virus pandemic (42).
site for finding reassortants is that, once generated, these chi-
In conclusion, the high rate of reassortment among RVFV
8200
SALL ET AL.
J. VIROL.
isolatcs raises interesting issues on vaccination with live atten-
17. Morvan, J., P. E. Rollin, S. Laventure, 1. Rakotoarivony,
and J. Roux. 1992b.
uated vaccines during epidemics when virulent strains are cir-
Rift Valley fever epizootic in the central highlands of Madagascar. Res.
culating. Since reassortants containing one attenuated and two
Viral. 143:407-415.
18. Morvan, J., P. E. Rollin, and J. Roux. 1992a. La lièvre de la vallée du Rift à
virulent segments werc shown to be attenuated (30) rcassort-
Madagascar en 1991. Enquête scro-épidémiologique chez ICS bovins. Rev.
ment between the wild and vaccine strains would generate
Elev. Med. Vet. Pays Trop. 45121-127.
attcnuated viruses with protective effects against the discase,
19. Morvan, J., J. F. Saluzzo, D. Fontenille, P. E. Rollin, and P. Coulanges. 1991.
provided attenuation markers were present in each genomic
Rift Valley fever on the east toast of Madagascar. Res. Viral. 142:475-4X2.
20. Muller, R., 0. Poch, M. Delarue, D. H. L. Bishop,
and M. Bouloy. 1994. Rift
segment of the live attenuated strain. Therefore, attenuated
Valley fever virus L segment: correction of the sequence and possible func-
vaccine strains with several attenuation markers present in
tional role of newlv identificd regions conserved in RNA-dependent poly-
cach segment would be recommended for control of RVFV.
merases. J. Gen. Vhol. 75:134-i-1352.
This safety strategy also stands a better chance to minimize the
21. Muller, R., J. F. Saluzzo, N. Lapez, T. Dreier, M. Turell, J. Smith, and M.
Bouloy. 1995. Characterization of clone 13. a naturally attenuated avirulent
probability of reversion toward virulence.
isolate of Rift Vallev fever virus, which is altered in thc small segment. Am. J.
Trop. Med. Hyg. 53:405-411.
ACKNOWLEDGMENTS
22. Page, R. D. M. 1995. Parallel phylogenies: reconstructing the history of
host-parasite
assemblages. Cladistics 10:155-173.
WC :arc gratcful to A. Billecocq, B. Lc Gucnno. and C. PrChaud for
23. Pelissier,
A., and R. Rousselot. 1954. Enquéte sérologique sur l’incidence des
fruitful discussicms, to M. Diallo for critically rcadiug thc manuscript,
virus neurotropcs chez quelques singes de l’Afrique équatoriale française.
and to R. Swancpoel for praviding us with the 1997 Kenyan strain. We
Bull. Soc. Pathol. Exot. 4222X-230.
24. Peters, C. J., and K. J. Linthicum. 1994. Rift Vallev fever. p. 125-138. In
are intlchtcd to ORSTOM and thc WHO collaborating ccntcr for
B. G. W. and J. H. Steele (ed.), Handhook of zoom& section B. Viral, 2nd
arbovirus and hcmorrhagic fcver virus rcscarch (CRORA). whose
ed. CRC Press, Boca Raton, Fla.
collabcrrativc work allowed us to obtain most of thc RVFV isolatcs
25. Pretorius, A., M. J. Oelofsen,
M. S. Smith, and E. van der Ryst. 1997. Rift
analyzcd in this study. Thc excellent tcchnical cxpcrtise of P. Vialat is
Valley fever virus: a scroepidemiologic study of small terrestrial vertebrates
acknowledgcd.
in South Africa. Am. J. Trop. Med. Hyg. 57:693-69X.
P.M.A.Z. was funded by a PQ Scholarship from CNPc4 and a
26. Pringle, C. R. 1996. Genetics and genome segment reassortment, p. 1X9-226.
PRONEXIPADCT grant cndowcd hy the Brazilian Governmcnt.
In R. M. Elliott (cd.), The viruscs. The Bunyaviridac. Plenum Press, New
York, N.Y.
27. Rodriguez, L. L., J. H. Owens, C. J. Peters, and S. T. Nichol. 1998. Genetic
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0022-538X/99!$04.00+0
Copyright 0 1999, American Society for Microbiology. Al1 Rights Rcscrved.
Genetic Reassortment of Rift Valley Fever Virus in Nature
A. A. SALL,’ P. M. DE A. ZANOTT0,2 0. K. SENE,’ H. G. ZELLER: J. P. DIGOUTTE,’
Y. THIONGANE 4 AND M. BOULOY’*
.---.----’
Institut Pasteur de Dakar’ and Institut Senegalais de Recherche Agronomique,4 Dakar, Senegal; DIPA-UNIFESP,
Silo Paulo, Brazif; Institut Pasteur de Madagascar, Antananarivo, Madagascar’; and Groupe des Bunyaviride,
Institut Pasteur, 75724 Paris Cedex 15, Fran&
Receivcd 9 April 1999iAccepted 15 July 1999
Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridue family, is an arthropod-borne virus which
emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human
populations. TO assess the genetic variability of RVFV, several isolates from diverse localities of Africa were
investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S),
medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major
lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However,
incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment
occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further
confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of
endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain
forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated
vaccines in areas of endemicity.
Rift Valley fever is a serious emerging arthropod-borne viral
nucleocapsid protein N and the nonstructural protein NSs by
anthropozoonosis caused by a phlebovirus (Rift Valley fever
using an ambisense strategy. Because of its segmented nature,
virus [RVFV], family Bunyaviridae), which was reported pri-
the genome of members of the Bunyaviridae family allows
marily to infect domestic cattle and more recently to cause
RNA segment reassortment (exchange of a whole segment)
massive epidemics in human populations across Africa. Mod-
when cells are coinfected by two closely related viruses of the
ifications in the ecological andlor environmental conditions
same genus or serogroup (reviewed in reference 26). Reassort-
appeared to be responsible for the emergence of the virus (16,
ment between strains of RVFV has been demonstrated exper-
24). Disease in humans exhibits clinical manifestations ranging
imentally in tissue cultures (30) and in mosquitoes that were
from acute febrile illness to severe complications. including
dually infected (38). Naturally occurring reassortants have
hepatitis, encephalitis, hemorragic fever, and ocular sequelae
been suggested or demonstrated for bunyavirus (8, 12,40) and
(13). Periodic large-scale epidemics, such as the ones in Mau-
hantavirus (33, 14, 27). However, to date, despite some clues
ritania in 1987 and 1998 (7, 43). Madagascar in 1990-1991
derived from unexpected groupings in a phylogenetic tree
(17.-19), and Egypt in 1977 and 1993 (2, 16), as well as in East
based on the NSs coding region (29), reassortment among
Africa (Kenya, Somalia, and Tanzania) in 1997-1998, reiterate
natural isolates of RVFV has not been investigated. In this
the potential of this virus as a considerable threat to human
report, we address the question of RVFV genetic reassortment
health, the latter epidemic affecting some 89,000 people and
under natural conditions and its consequences
on the evolution
causing 500 deaths (1). Analysis of one strain isolated from a
and epidemiology of the disease.
fatal human case during this epidemic showed a close related-
ness with a strain isolated in Madagascar during the 1990-1991
outbreak (28), revealing that the virus could spread across
MATEHIALS
A N D M E T H O D S
considerable distances, possibly beyond Africa. This threat be-
Virus propagatinn and RNA extraction. The origins and ycars of isolation of
cornes more disquieting when we consider that numerous mos-
RVFV isolates are shown in Table 1. Propagation of viruses and cytoplasmic
RNA extraction from infected cells were done as previously described (29).
quito species around the world are competent laboratory vec-
Reverse transcription (RT)-PCR and sequencing procedures. Three different
tors for RVFV (9, 35-37). Therefore, understanding the
sets of primcrs, NS3a-NS2g, MRVla-MRV2g, and Wag-Xg. were used to am-
mechanism underlying its dispersa1 and evolution is of para-
plify portions of the NSs, G2, and L coding regions. respectively. The NSs coding
mount importance to the control of this disease.
region is located in the S segment, whcreas the regions coding for G2 and L are
The RVFV genome consists of three negative-sense single-
in the M and L segments, rcspectively. The antisense primers NS3a. MRVLa, and
Wag were used to synthcsize thc first-strand complementary DNA. Primer se-
stranded RNA segments designated L (large), M (medium),
quences, protocols for cDNA synthesis, PCR amplification, and direct sequenc-
and S (small) (for reviews, see references
10 and 31). The L
ing of PCR products were described previously (20, 21, 29, 34).
segment codes for the L viral polymerase. The M segment
Phylogenetic
reconstruction. The phylogenetic analyses of each segment of the
RVFV genome were performcd with data sets for the NSs, G2, or L protein
codes for the precursor to the envelope glycoproteins, Gl and
coding region by using maximum likelihood and maximum parsimony methods in
G2, which, after cleavage, generate two additional nonstruc-
PAUP (Phylogenetic Analysis Using Parsimony, brta version 4.0; kindly pro-
tural proteins of 78 and 14 kDa. The S segment codes for the
vided by David L. Swofford, Smithsonian Institution, Washington, D.C.). For a
more realistic tree reconstruction and branch length estimates for each data set,
the optimal values for the transition probahilities among different nucleotides
and the value for the shape parameter (alpha) for the gamma distributed variable
* Corrcsponding author. Mailing address: Institut Pasteur. Groupe
rates among sites were empirically determined from the data. Using the optimal
values of these parameters. trecs with similar likelihood were collected and the
des Bunyaviridés, 25, rue du Dr. Roux, 75724 Paris Ccdcx 15, France.
tree topology stability around the likelihood maxima was investigated hy calcu-
Phonc: 33 1 40 61 31 57. Fax: 33 1 40 61 31 51. E-mail: mbouloy
lating the 50% majority-rule consensus. For an additional comparison, we used
@pasteur.fr.
a fast tree search algorithm (quartet puzzling) for estimating maximum likeli-
8196
-
/
VOL. 73, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
8197
TABLE 1. Origins and years of isolation of RVFV isolatcs
Grnotype”
S1rain
Yr of
isolati«n
(SIMIL)
SNS’
Smithburn
1 9 4 4
Uganda
Entcbbe strain
CICIC
ArUGA-55
Lunyo
1 9 5 3
Uganda
Mosquito
c/cic
ArCAr-69
Ar El 1976
1 9 6 9
CAR
Mosquilo
CICIC
Cl3
Clone 13
1974
CAR
Human
*K7c
HEGY-77
ZH 54X
1 9 7 7
Eiwt
Human
EiE,‘E
MPIZh
MP12
1 9 7 7
Egwt
ZHS48 strain
E/E/E
ArMAD-79
Ar Mg 811
1 9 7 9
Madagascar
Mosquito
E/?/E
ArSEN-84
Ar D 38661
1 9 8 4
Scnegal
Mosquito
EI’W~W
AnGUI-
An K 6087
1984
Guinca
Bat
WICIC
ArBUF-84
Ar D 38457
1 9 8 4
Burkina Faso
Mosquito
w,ww
HlMAU-87
H D 47502
1 9 8 7
Mauritania
Human
ciww
H2MAU-87
H D 47311
1987
Mauritania
Human
w/w/w
H3MAU-87
H D 47408
1 9 8 7
Mauritania
Human
w,wnv
H4MAU-87
H D 48255
1 9 8 7
Mauritania
Human
w,~?iw
AnMAD-91
An Mg 990
1991
Madagascar
Bovine
CICIC
ArSEN-93
Ar D 104769
1993
Senegal
Mosquito
CiGW
AnSEN-K-93
An D 106417
1 9 9 3
Scncgal
Zebu
Wlw!C
BEGY-93
B EGY 93
1 9 9 3
Emt
Buffalo
EiEiE
HEGY-93
H EGY 93
1 9 9 3
EgYPt
Human
EIEE
HKEN-97
384-97.1
1 9 9 7
Kenya
Human
C/C/C
” E, W. and C, Egyptian, Western African. and Central-East African lineagc, respcctively: :j:, scyuencc too short to he a.rsigned a lincage; ‘?, sequence n»t dctermined.
’ Attenuated strain.
hood trees from PUZZLE version 3.1 (33), which automatically
assigns estima-
First, somc of the strains in the Central-East Africa and Egypt
tion of support of each interna1 node. Additionally. to investigatc thc robustness
groups (SNS and ArUGA-55, AnMAD-91 and HKEN-97.
of tree resolution under maximum parsimony. a hootstrapping analysis was done
with 1,000 random resamplings with the ncarcst-neighbor-interchange perturba-
HEGY-77 and HEGY-93) are closely related, despite their
tion algorithm within PAUP. For the ahortest data set (i.c., the 211~bp L ampli-
dates of isolation. suggesting the existence of an endemic-
con), a 2% jackknife wx used as an alternative resampling method.
enzootic maintenance cycle of the virus in these areas. Second,
To determine topological incongruence among estimatcs obtained from dif-
no obvious grouping based on thc host species was observed.
ferent data sets for the same set of taxa, wve chcckcd whether the hest topology
for each data set is statistically worse when rcconstructcd with an alternative data
This could be explained by frequent exchange of viruses bc-
set by using the Kishino and Hasegawa les1 implemcnhxl in PAUP 4.0. Addi-
twecn different hosts. Third, in each group, strains isolated
tionally, t« test for reassortment among viruacs from dilTcrent lineages. con-
during epidemic-cpizootic and endemic-enzootic periods were
straint trees wcre constructcd; in these, the taxa wcrc assigned to a given part of
found adjacent to each other, suggesting that the virus circu-
the tree, generating alternative topologies to he tested against the ones ohtaincd
from the unconstrained data. Finally, to allow the inspection of the topologies
lates alternatively in enzootic-endemic form through a main-
estimated from different genomic segments, thc TrecMap program (22) was uscd
tenance cycle or epidemic-epizootic mode, both cycles provid-
to construct parallel phylogenies.
ing each other with viral strains. However. despite similar
Nucleotide sequence accession numbers. The sequcnces of thc strains listcd in
Table 1 and corresponding to the L, G2, and N protein coding regions bave hecn
distributions of each tree into three clusters, the assignment of
assigned GenBank accession n». AF134782 10 AF134XOl. AF134492 to
some isolates (ArSEN-93, AnSEN-K-93, ArSEN-84, HIMAU-
AF134508, and AF134530 t« AF134551, rcspectively. The sequcncçs of thc NSs
87, and AnGUI-84) to onc particular group does not remain
protein coding region werc depositcd in the EMHL database (accession no.
constant within the three phylogenies. Construction of the
Y12739 to Y12756) as already reported (29).
reconciling parallel trees using the three phylogenies empha-
sized that the groupings of the strains in the S, M. and L
RESULTS
segment trecs correlate quite well but clearly show somc in-
Phylogeny of different RVFV segments. In a previous papcr,
congruenccs. For instance, HlMAU-87 and ArSEN-84 possess
phylogenetic analysis carried out with 20 isolates and derived
thc L and M segments of the West African genotype and the S
from the 669-nucleotide-long sequences of the NSs protein
segment from the Central-East African or Egyptian lineage,
coding region in the S segment showcd the existence of three
respectively. AnGUI- has the L and M segments of the Central-
distinct lineages: la and Ib in sub-Saharan Africa and II in
East African genotype, whercas the S segment belongs to the
Egypt (29). Additional sequencing of the M and L segments
West African lineage. Finally, two strains isolated during the same
was performed after RT-PCR amplification of an 80%nucleo-
period in two different places in Scnegal, Barkedji (North) and
tide-long DNA fragment located within the region coding for
Kolda (Casamance, South), ArSEN-93 and AnSEN-K93, have a
the G2 protein and a 212-nucleotide-long
DNA fragment lo-
different distribution within clusters: AnSEN-K93 was located
cated in the L protein. Phylogenetic analyses of these se-
within the Western African lincage with regards to its S and M
quences using the maximum likelihood and maximum parsi-
segments and within the Central-East African lineage with re-
mony methods confirmcd the distribution pattern within the
gards to its L segment. Reciprocally, in the case of ArSEN-93, its
three lineages, which were renamed Central-East Africa, West
S and M segments belong to the Central-East African lineage and
Africa, and Egypt for la, Ib, and II, respectively (see Fig. 1).
the L segment belongs to the Western African Iineage (Fig. 1).
The support for different nodes on the tree was evaluated by
The possibility that incongruences might be due to cross
various methods (i.e., bootstrap, jackknife, or maximum like-
contamination among isolates seems highly improbably given
lihood topology consensus) and showed strong support for the
thc following extreme prccautions that were taken to avoid
three phylogenies.
such artifacts. (i) RNA extraction and RT were done sepa-
These phylogenetic analyses raised some general comments.
ratcly for çach isolate undcr a laminar-flow containment hood
8198
SALI, ET AL.
J. VIROL.
B
A
-
r H2MAU87
--~
ri ArSEN84j
l
--+AnSENk93
/
H3MAU87
I-
~
- H4MAU87
WEST
AFRICA
~EST
FFRICA
ArBUF84
l
j
AnGU184 1
i
.-
-MP12
+iEGY93
1
I
EGYPT
I
EGYPT
-’
BEGY93
‘Bpi HEGY77
1
i
I AnMAD91
~ ArMAD79
1
j
EAST
/
CENTRAL
EAST
’
AFRICA
CENTRAL
1
;& r_“S;,,
AFRICA
k%’
.-- -.-- SNS
ArUGA55
-
C
FIG. 1. Maximum likelihood trees for the NSs (A), Ci2 (B), and L(C) coding
region sequences on the S, M, and L segments, respectively. Values helow the
H2MAU87
diffcrent nodes indicate their rohustness by 5% jacknife (indicated in boldface
-7
and italic type), maximum likelihood quartet puzzliny (indicated in hrackets).
and hootstrapping (indicated in roman type) methods. Putative reassortant
strains are hoxed.
- - - H3MAU87
I
----H4MAU87
;WEST
IAFRICA
- ~ A r B U F 8 4
c-il
sequenced several times and led to identical sequences, many
hArSEN93:
of them being processed in laboratories at least 4,000 km apart
i
-
(Paris, France, and Dakar, Senegal).
r--ArMAD79
Given that trees for different genes are clearly incongruent
in their topologies, it could be assumed that these incongru-
pl/BEGY93
,EGYPT
ences are due to genetic exchange. Regarding the segmentation
jHEGY77
of the RVFV genome, the most probable device to explain the
r HEGY93
exchange of genetic material is RNA segment reassortment.
‘MPI2
Tests for topological structure as indicative of reassortment.
Two approaches
were used to evaluate the incongruences ob-
---1
I
AnSENk93 >:
served among specific isolates of different trees. First, we con-
I
-------HKEN97
I
structed constrained trees and tested their topologies by using
SS!
likelihood against the unconstrained trees shown in Fig. 1. The
~91 AnMAD91
j
EAST
;-----
CENTRAL
likelihood value for the unconstrained tree resulting from the
- - - - - - iAnGU1B;il
AFR,(.A
use of the NSs sequences was better than the one obtained for
the constrained tree where the ArSEN-93 and HlMAU-87
-i!‘lÀrRccp369
isolates were forced into the West African group (-1nL =
-ArUGA55
1,774.76, difference of 1nL = 57.21, P < 0.001). Similarly, the
S N S
values obtained for the G2 and L sequence phylogenies (for
G2, difference of 1nL = 42.95, P = 0.0002; for L region, dif-
5 % jackknife IWO mplhticms
[% ML quartet puzzlinp]
ference of 1nL = 9.74, P = 0.06), in which ArSEN-93 and
60% MR bootsVapi000 replicatiom
AnGUI- or AnSEN-K-93 and AnGUI-84, respectively, were
forced into the West African group, led to the same conclu-
sion. Second, we used the Kishino and Hasegawa test on the
which was decontaminated between each manipulation. (ii)
difference of the likelihoods for trees obtained for taxa com-
PCR mix preparation, PCRs, and electrophoresis were per-
mon to both G2 and NSs data sets. The maximum likelihood
formed in separate rooms. (iii) For cach isolate, PCR products
tree for G2 (-1nL = 1,684.40) had a lower likelihood (differ-
derived from independent RNA extraction and RT-PCR were
ence of 1nL = 30.35, P = 0.0603) under the NSs data set than
VOL. 73, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
8199
the NSs tree did (-1nL = 1,654.06), and the NSs trec (-1nL =
meric viruses werc viable and their host did not exert a nega-
1,842.69) had a lower likelihood (difference of lnI, = 51.99,
tive sclcction against them. We do not know whether the
P < 0.0001) under the G2 data set than the G2 tree did
strains identified as reassortants werc host restricted, but they
(-1nL = 1,790.69). Although the difference of likelihood (InL
were isolated from different hosts, including humans, mosqui-
difference = 30.35) for the G2 tree under the NSs data set may
toes, bats (An GUI-84), and zebu (AnSEN-K93). With regard
not be statistically different under the 95% confidence level,
to cocirculation of different strains which cari potentially un-
likelihood tests using either constrained trees or the Kishino
dergo reassortment, Senegal is a very instructive example as an
and Hasegawa test support the notion that the incongruences
area where at least two different lineages circulated at the same
observed among trees are due not to poor phylogcnetic topol-
time in 1993. Since RVFV is an arbovirus, reassortment among
ogy inferences but rather to genetic exchange.
strains cari occur in a dually infected mosquito or vertebrate
In light of the recent report of recombination in hantavirus
host. Reassortment was demonstrated experimentally in ham-
evolution (32), it could not be excluded that the incongruences
sters as well as in mosquitoes that were dually infected with two
in the RVFV tree topologies could be due to intramolecular
RVFV natural isolates (38). It seems likely that vertebrate
recombination. Although such events appear, SO far? to be very
hosts cari be naturally infected with different strains of RVFV
rare among negative-strand RNA viruses (26), we considered
since a human isolate from the Republic of Central Africa was
this possibility and analyzed the NSs, G2, and L data with the
shown to be composcd of a heterogenous population of viral
split decomposition method, by using the program Splits ver-
clones with ditlerent biological properties (21). Mosquitoes cari
sion 1.0 (3), but we did not find any clear-tut indication of
bc dually infected by interrupted fceding or feeding on a dually
recombination, i.e., networked evolution (data not shown),
infected vertebrafe host. Both mechanisms have been demon-
within the fragments sequenced. Thus, reassortment events gen-
strated cxperimentally for RVFV with a natural mosquito vec-
erating combination between L, M, and S segments of RVFV
tor, Cukxpipierw, suggesting that under natural conditions, the
natural isolates appear to be the most likely explanation.
process is probably the same. There exists a third theoretical
possibility for a mosquito to be dually infected: a female in-
DISCUSSION
fected transovarially or venereally with one strain could be-
corne superinfected during a bloodmeal with another strain.
This study indicated that 5 of the 20 (25%) isolates, ArSEN-
However, due to homologous interference,
vertically infected
93, AnSEN-K-93, ArSEN-84, HlMAU-87, and AnGUI-84, ap-
mosquitocs may be refractory to superinfection with the same
pear to result from a reassortmcnt event. This percentage may
virus spccies. Therefore, both mosquitoes and vertebrate hosts
be underestimated since the method used herein to identify
may act as a site for RVFV reassortment in nature but their
reassortants took into account only reassortment events be-
relative contribution in the natural process of reassortment
tween the three major lineages (Egypt, West Africa, and Cen-
remains to be dctermincd.
tral-East Africa). Interestingly, all the strains identified as re-
The occurrence of reassortmcnt among natural isolates from
assortants, ArSEN-93, AnSEN-K-93,
ArSEN-84, HI MAU-87,
West Africa and Central-East Africa or, less frequently, Egypt
and AnGUI-84, were isolated from West Africa (Scncgal,
suggests two possibilities: (i) one strain was transported to a
Guinea, and Mauritania) and involved reassortment with
place where another strain was circulating; or (ii) the virus had
strains of the Central-East African or Egyptian lineage (Ar-
to make its way through the rain forest and to adapt to the
SEN-84) (Table 1). In addition, these reassortants have re-
different fauna and ecotopes, establishing a sylvatic cycle. Al-
tained combinations of homologous L plus M segments (Ar-
though the existence of a sylvatic cycle would have to be dem-
SEN-84, AnGUI-84, and HlMAU-87) and M plus S segments
onstrated, thc circulation of RVFV in the rain forest has heen
(AnSEN-K-93 and ArSEN-93) (Table 1). In dual infection of
revealed by thc high antibody prevalcnce in wild animals and
BHK-21 cells with La Crosse and Snowshoe hare viruses, some
pygmy populations in Central African Republic (6) and mul-
segment associations appeared to be preferred (39). Investiga-
tiple strain isolations from mosquitoes in the primary forest of
tions with a larger sample of RVFV field isolates would be
Perinct in Madagascar (15). In addition, data recently published
needed to indicate whether generation and selection of reas-
by Pretorius et al. (25) indicatcd that rodents were involved as a
sortants in nature are random or not.
potential reservoir of RVFV in South Africa. It is likely that
Replication errors such as base substitution and deletions or
different strains of RVFV circulate in the rain forest area among
insertions are the most common mechanism of RNA virus
wild mosquitoes (16) and vcrtcbratcs like monkeys (23) and bats
evolution. However, major changes of viral genotype may in-
(5), depending on the dynamic of the interaction among hosts and
volve exchanges of RNA segments (genetic shift), as exempli-
virus populations. Therefore, the search and characterization of a
fied by influenza virus (42). Comparison of RVFV isolates
sylvatic cycle of RVFV are of utmost importance because this
sampled from different geographical localities showed that
would lead to better understanding and control of the epidemi-
evolution of this virus in nature not only appears to be due to
ology of Rift Valley fever and its potential cmergence.
point mutations, the percentage of base substitution varying
Thc existence of reassortment as a mechanism of evolution
from 0 to 9.6% in the S segment (29), but also may occur by
raises the question of thc cpidemic potential of these viruses
genetic exchange. Indeed, the apparent contradictions in the
and their pathogenicity. Isolation of a potential reassortant
groupings of the different segments within the three distinct lin-
from a fatal case during the 1987 Mauritanian epidemic leaves
eages could be explained by reassortment and strongly suggest
this question open, even though othcr strains which were not
that this evolution mechanism is a common trait in RVFV natural
identified as reassortants were isolated from fatal cases. A
history. Arboviruses apparently evolve approximately lO-fold
similar question concerning the outbreak of Sin Nombre virus
more slowly than RNA viruses that have only a single vertebrate
was raised (1 1), but in spite of thc evidence of reassortment
host (4,41). T~US, the evolution of RVFV driven by reassortment
among Sin Nombre virus isolates (II, 14, 27). there was no
and by point mutations may depend on the host number.
indication that thc emcrgcncc of hantavirus pulmonary syn-
Reassortment under natural conditions implies the existence
drome was due to a novel chimeric virus. On the other hand.
of at least two different strains present at the same time, in the
reassortment appeared to be involved in the triggering of an
same area, and infecting the same host. One additional requi-
influenza virus pandemic (42).
site for finding reassortants is that, once generated, these chi-
In conclusion, the high rate of reassortment among RVFV
8200
SALL ET AL.
J. VIROL.
isolatcs raises interesting issues on vaccination with live atten-
17. Morvan, J., P. E. Rollin, S. Laventure, 1. Rakotoarivony,
and J. Roux. 1992b.
uated vaccines during epidemics when virulent strains are cir-
Rift Valley fever epizootic in the central highlands of Madagascar. Res.
culating. Since reassortants containing one attenuated and two
Viral. 143:407-415.
18. Morvan, J., P. E. Rollin, and J. Roux. 1992a. La lièvre de la vallée du Rift à
virulent segments werc shown to be attenuated (30) rcassort-
Madagascar en 1991. Enquête scro-épidémiologique chez ICS bovins. Rev.
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Elev. Med. Vet. Pays Trop. 45121-127.
attcnuated viruses with protective effects against the discase,
19. Morvan, J., J. F. Saluzzo, D. Fontenille, P. E. Rollin, and P. Coulanges. 1991.
provided attenuation markers were present in each genomic
Rift Valley fever on the east toast of Madagascar. Res. Viral. 142:475-4X2.
20. Muller, R., 0. Poch, M. Delarue, D. H. L. Bishop,
and M. Bouloy. 1994. Rift
segment of the live attenuated strain. Therefore, attenuated
Valley fever virus L segment: correction of the sequence and possible func-
vaccine strains with several attenuation markers present in
tional role of newlv identificd regions conserved in RNA-dependent poly-
cach segment would be recommended for control of RVFV.
merases. J. Gen. Vhol. 75:134-i-1352.
This safety strategy also stands a better chance to minimize the
21. Muller, R., J. F. Saluzzo, N. Lapez, T. Dreier, M. Turell, J. Smith, and M.
Bouloy. 1995. Characterization of clone 13. a naturally attenuated avirulent
probability of reversion toward virulence.
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ACKNOWLEDGMENTS
22. Page, R. D. M. 1995. Parallel phylogenies: reconstructing the history of
host-parasite
assemblages. Cladistics 10:155-173.
WC :arc gratcful to A. Billecocq, B. Lc Gucnno. and C. PrChaud for
23. Pelissier,
A., and R. Rousselot. 1954. Enquéte sérologique sur l’incidence des
fruitful discussicms, to M. Diallo for critically rcadiug thc manuscript,
virus neurotropcs chez quelques singes de l’Afrique équatoriale française.
and to R. Swancpoel for praviding us with the 1997 Kenyan strain. We
Bull. Soc. Pathol. Exot. 4222X-230.
24. Peters, C. J., and K. J. Linthicum. 1994. Rift Vallev fever. p. 125-138. In
are intlchtcd to ORSTOM and thc WHO collaborating ccntcr for
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arbovirus and hcmorrhagic fcver virus rcscarch (CRORA). whose
ed. CRC Press, Boca Raton, Fla.
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