JOURNAL OF VIROLOC~Y. Oct. 1999, p. 8196-8200 Vol....
JOURNAL OF VIROLOC~Y. Oct. 1999, p. 8196-8200
Vol. 73, No. 10
0022-538X~99/$04.00+0
Copyright 0 1999, American Socicty for Microbiology. Al1 Rights Rescwcd.
Genetic Reassortment of Rift Valley Fever Virus in Nature
A. A. SALL,’ P. M. DE: A. ZANOTTO,’ 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,
Sio Paulo, Brazil’; Institut Pasteur de Madagascar, Antunanarivo, Madagascar’; and Groupe des Bunyavirides,
Institut Pasteur, 75724 Paris Cedex 15, France5
Received 9 April 199YAcceptcd 15 July 1999
Rift Valley fever virus (RW), 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 RW, 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
confu-med 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 emnhasizes 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 Bunyaviridue), which was reported pri-
the genome of members of the Bunyaviridue 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 andior 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 hf 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 seyuelae
been suggested or demonstrated for bunyavirus (8, 12,40) and
(13). Periodic large-scale epidemics, such as the ones in Mau-
hantavirus (11, 14, 27). However, to date, despite some clues
ritania in 1987 and 1998 (7, 43) Madagascar in 1990-1991
derived from unexpectcd 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-I 991
MATERIALS AND METHODS
outbreak (28) revealing that the virus could spread across
considerable distances. possibly beyond Africa. This threat be-
Virus propagation and RIVA extraction. The origina and years of isolation of
cornes more disyuieting 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, 3.5-37). Therefore, understanding the
sets of primers, NS3a-NS2g, MRVlaMRV2g, and Wag-Xg, wcre used to am-
mechanism underlying its dispersa1 and evolution is of para-
pli@ portions of the NSs, G2. and L coding regions. rcspectivcly. Thc NSs coding
mount importance to the control of this disease.
region is located in the S segment, whereas the regions coding for G? and L are
in the M and L segments, respectivcly.
The antisense primera N%ia, MRVla. and
The RVFV genome consists of three negative-sense single-
Wag were used to synthesize thc tirst-strand complementary DNA. Primer sc-
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 phylogenrtic analyses of each segment of the
RVFV genome were performed 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 likclihood and maximum parsimony methods in
G2, which, after cleavage, generate two additional nonstruc-
PAUP (Phylogenetic Analysis Using Parsimony, beta version 4.0: kindly pro-
tural proteins of 78 and 14 kDa. The S segment codes for the
vidcd hy 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 prohabilities among different nucleotides
and the value for the shape pnrameter (alpha) for the gamma distrihuted variable
rates among sites werc empirically determined from the data. Using the optimal
* Corresponding author. Mailing addrcss: Institut Pasteur, Groupe
values of thesc parameters. trççs with similar likelihood were collected and the
des Bunyaviridés, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France.
trec topology stahility around the likclihood maxima was investigated by calcu-
Phonc: 33 1 40 61 31 57. Fax: 33 1 40 61 31 51. E-mail: mhouloy
lating the 50% majority-rule
consçnsus. For an additional comparison,
we used
@pastcur.fr.
a fast tree search algorithm (quartet puzzling) for estimating maximum likeli-
8196
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VOL.13, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
x197
TABLE 1. Origins and years of isolation of RVFV isolates
Genotype”
C o d e
strain
Yr of
Origin
SOUKC
isolation
(SIMIL)
SNS’
Smithburn
1944
Uganda
Entehbe strain
C/C/C
ArUGA-55
Lunyo
1955
Uganda
Mosquito
CKIC
ArCAr-09
Ar B 1976
1969
CAR
Mosquito
CICIC
Cl3
Clone 13
L974
CAR
Human
*Kx
HEGY-77
ZH 54X
1977
QYP~
Human
EIE,/E
MPlZ”
MP12
1977
EgyP
28538 strain
UEIE
ArMAD-79
Ar Mg 811
1979
Madagascar
Mosquito
EI?;E
ArSEN-84
Ar D 3%61
1984
Senegal
Mosquito
E/WiW
AnGUI-
An K 6087
1984
Guinea
Bat
WICIC
ArBUF-84
Ar D 38457
1984
Burkina Faso
Mosquito
W!WIw
HlMAU-87
H D 47502
1987
Mauritania
Human
GWrw
HZMAU-87
H D 47312
1987
Mauritanie
Human
w;wiw
H3MAU-87
H D 47408
1987
Mauritania
Human
WlW/W
H4MAU-87
H D 48255
1987
Mauritania
Human
W/‘?/W
AnMAD-91
An Mg 990
1901
Madagascar
Bovine
cictc
ArSEN-93
Ar D 104769
1993
Sencgal
Mosquito
C/UW
AnSEN-K-93
An D 106417
1993
Senegal
Zcbu
WIWIC
BEGY-93
B EGY 93
1993
J%W
Buffalo
E/E,E
HEGY-93
H EGY 93
1903
WP
Humaii
EIEIE
HKEN-97
384-97.1
1997
Kenya
Human
CICIC
” E, W, and C, Egyptian, Western African. and Central-East Afrirican linragc, respectively; a:, scqucnce too short to hc assigncd a linenge: ?. scquence net determined.
h Attenuated strain.
hood trecs from PUZZLE version 3. I (33), which automatically
assigns cstima-
First, some of thc strains in the Central-East Africa and Egypt
tion of support of each interna1 node. Additionally, to investigate the rohuslncss
groups (SNS and ArUGA-55, AnMAD-91 and HKEN-97.
of tree resolution under maximum parsimony. a bootstrapping analysis was donc
with 1,000 random reaamplings
with thc nearcat-neighhor-interchange pertnrha-
HEGY-77 and HEGY-93) are closely related, despite their
tion algorithm within PAUP. For the shortest data set (i.e., the 211.hp L ampli-
dates of isolation, suggesting the existence of an endcmic-
con), a 2% jackknife was used as an alternative resampling
mcthod.
enzootic maintenance cycle of the virus in these areas. Second.
TO determine topological incongruencc among estimates ohtained fwm dif-
no obvious grouping bascd on the host species was observed.
ferent data sets for the same set of tazxü, wc chccked whether the best topology
for each data set is statisticallyworse when rcconstrocted with an alternative data
This could be explained by frequent exchange of viruses be-
set hy using the Kishino and Hasegawa test implemcnted in PAUP 4.0. Addi-
tween different hosts. Third, in cach group, strains isolated
tionally, to test for rcassortment among viruses from different lineages, con-
during epidemic-epizootic and endemic-enzootic periods were
straint trees were constructed; in these, the taxa werc assigned to a given part of
the tree, generating alternative topologies to he tested against the ones obtaincd
found adjacent to each other, suggesting that the virus circu-
from the unconstrained data. Finally, 10 allow thc inspection of the topologies
lates alternatively in enzootic-endemic form through a main-
estimated from different genomic ssgments. the TreeMap program (22) ws 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 numhers. The sequences of the strains listed in
Table 1 and corresponding to the L. Ci2. and N protcin coding regions bave been
distributions of each tree into three clusters. the assignment of
assigned GenBank accession no. AF134782 to AF134801, AF133492 to
some isolates (ArSEN-93. AnSEN-K-93,
ArSEN-84, HlMAU-
AF1345OX, and AF134530 to AF134551, respectively. The sequences of the NSs
87, and AnGUI-84) to one particular group does not remain
protein coding region were deposited in the EMBL datahasc (accession no.
constant within the three phylogenies. Construction of the
Y 12739 to Y12756) as already reported (29).
reconciling parallel trees using the thrce phylogenies empha-
sized that the groupings of the strains in the S, M, and L
RESULTS
segment trees correlate quite well but clearly show some in-
Phylogeny of different RVF’V segments. In a previous papcr,
congruences. For instance, HlMAU-87 and ArSEN-84 possess
phylogenetic analysis carried out with 20 isolates and dcrived
the 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 Ebyptian lineage,
coding region in the S segment showed the existence of three
rcspectively. AnGUI- has thc L and M segments of the Central-
distinct lineages: Ia and Ib in sub-Saharan Africa and II in
East African genotype. whereas 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 809-nucleo-
period in two different places in Senegal, 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 lineage 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 confirmed 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 Ia, Ib, and II, respectively (see Fig. 1).
the L segment belongs to the Western African lineage (Fig. 1).
The support for different nodes on thc 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
the following extreme precautions that were taken to avoid
three phylogenies.
such artifacts. (i) RNA extraction and RT were done sepa-
These phylogenetic analyses raised some general comments.
rately for cach isolate under a laminar-flow containmcnt hood
81%
SALL ET AL.
J. VIROL.
A
6
r H2MAU87
--y
--i ArSEN84 1
/
/
” rHsMA’J87 1
yAnSENk93:
~EST
PFRICA
l
-1
-MPIS
-1
-HEGY93
’
EGYPT
-- BEGY93
5”pll H E G Y 7 7
--.
ArMAD79
l
I AnMAD91
EAST
! [s2’
- ArCAR69
CENTRAL
I
1
EAST
’
CENTRAL
AFRICA
rAnMAD91
AFRICA
fw i---- ,,K,=,,g7
“l
-SNS
---~--- SNS
93
/
r---~~- ArCAR69
- --------ArUGA55
z-‘L-- ArUGA55
-
C
FIG. 1. Maximum likelihood trees for the NSs (A), G2 (B). and L(C) coding
region sequences on thc S, M, and L segmments, rcspectivrly.
Values helw the
H2MAU87
different nodes indicate their robuatness by 5% jacknife (indicated in boldface
--/
and italic type). maximum likelihood quartet puzzling (indicated in brackets).
_ HIMAlJ87 j
and bnotstrapping (indicated in roman type) methods. Putative tussortant
---- H3MAU87
strains are hoxed.
---H4MAU87
WEST
AFRICA
!---- ArBUF84
i-- fArSEN84
sequenced several times and led to identical sequences, many
!LArsEN93;
/
of them being processed in laboratories at least 4,000 km apart
(Paris, France, and Dakar, Senegal).
7 ArMAD79
-1
Given that trees for different genes are clearly incongruent
.-- j
;,-jBEGY93
in their topologies, it could be assumed that these incongru-
E !
EGYPT
ences are due to genetic exchange. Regarding the segmentation
,
j H E G Y 7 7
of the RVFV genome, the most probable device to explain the
1
kHEGY93
/
I
exchange of genetic material is RNA segment reassortment.
I
‘MPi2
Tests for topological structure as indicative of reassortment.
Ti
[96] ,--.---
Two approaches
werc used to evaluate the incongruences ob-
‘AnSEiVk93 ~
scrved among specific isolates of different trees. First, we con-
1
Cr-_-r---------HKEN97
structed constrained trees and tested their topologies by using
EAST
likelihood against the unconstrained trees shown in Fig. 1. The
likelihood value for the unconstrained tree resulting from the
_-.-~-- - - - ‘AT;suJ84
71 CENTRAL
i--d 1 AFRICA
use of the NSs sequences was bctter than the one obtained for
95 c-c13
the constrained tree where the ArSEN-93 and HlMAU-87
~
%fl iArRCA69
1
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
I
values obtained for the G2 and L sequence phylogenies (for
G2, difference of 1nL = 42.95, P = 0.0002; for L region, dif-
S%Jackknife IOC4 replicailons
1% ML quaflet puzli&
ference of 1nL = 9.74, P = 0.06) in which ArSEN-93 and
54% MR bootstrapi0W replicatms
AnGU1-84 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 each 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 InL = 30.35, P = 0.0603) under thc 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 tree (--hlL =
meric viruses were viable and their host did not exert a nega-
1,842.69) had a lower likelihood (difference of 1nL = 51.99,
tive selection against them. We do not know whether the
P < 0.0001) under the G2 data set than the Ci2 tree did
strains identified as reassortants were host restricted, but they
(-InL = 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
tocs, 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 wherc at least two different lineages circulated at the same
observed among trees are due not to poor phylogenetic 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 mosquitocs that were dually infected with two
in the RVFV tree topologies could be duc to intramolecular
RVFV natural isolates (38). It secms 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 composed of a heterogenous population of viral
split decomposition method, by using the program Splits ver-
clones with different biological propcrties (21). Mosquitoes cari
sion 1.0 (3), but we did not find any clear-tut indication of
be dually infected by intcrrupted feeding or feeding on a dually
recombination, i.e., networked evolution (data not shown),
infected vertebrate host. Both mechanisms have been demon-
within the fragments sequcnced. Thus, reassortment events gen-
strated experimentally for RVFV with a natural mosquito vec-
erating combination between L, M, and S segments of RVFV
tor, Culexpipiens, suggesting that under natural conditions, the
natural isolates appear to be the most Iikely 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-
mosquitoes may be refractory to superinfection with the same
pear to result from a reassortment event. This percentagc may
virus species. Therefore, both mosquitoes and vertebrate hosto
be underestimated since the method used herein tu 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 determined.
tral-East Africa). Interestingly, a11 the strains identilied as re-
The occurrence of reassortment among natural isolates from
assortants, ArSEN-93, AnSEN-K-93,
ArSEN-84, HlMAU-87,
West Africa and Central-East Africa or, less frequently. Egypl
and AnGUI-84, were isolated from West Africa (Senegal,
suggests two possibilities: (i) one strain was transported to s
Guinea, and Mauritania) and involved reassortment with
place where another strain was circulating; or (ii) the virus bac
strains of the Central-East African or Egyptian lineage (Ar-
to make its way through the rain forest and to adapt to thc
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 cxistencc of a sylvatic cycle would have to be dem-
SEN-84, AnGUI-84, and HlMAU-87) and M plus S segments
onstrated, the circulation of RVFV in the rain forest has beer
(AnSEN-K-93 and ArSEN-93) (Table 1). In dual infection of
revealed by the high antibody prevalence in wild animals anc
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 oi’
tions with a larger sample of RVFV field isolates would be
Perinet in Madagascar (15). In addition, data recently publishec
needed to indicate whether generation and selection of reas-
by Pretorius et al. (25) indicatcd that rodents were involved as 2
sortants in nature are random or not.
potential reservoir of RVFV in South Africa. It is likely thal
Replication errors such as base substitution and deletions or
different strains of RVFV circulate in the rain forest area amons,
insertions are the most common mechanism of RNA virus
wild mosquitoes (16) and vertcbrates like monkeys (23) and bat!,
evolution. However, major changes of viral genotype may in-
(5), depending on the dynamic of the interaction among hosts anc.
valve exchanges of RNA segments (genetic shift), as exempli-
virus populations. Therefore, the search and characterization of 2.
fied by influenza virus (42). Comparison of RVFV isolates
sylvatic cycle of RVFV are of utmost importance because this
sampled from different geographical localities showcd 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 emergence.
point mutations, the percentage of base substitution varying
Thc existence of reassortmcnt as a mechanism of evolutior.
from 0 to 9.6% in the S segment (29), but also may occur by
raiscs the question of the epidemic potential of these viruses
genetic exchange. Indeed, the apparent contradictions in the
and their pathogenicity. Isolation of a potential reassortam.
groupings of the different segments within the three distinct lin-
from a fatal case during thc 1987 Mauritanian epidemic leaves
eages could be explained by reassortment and strongly suggest
this question open, even though other strains which were no’:
that this evolution mechanism is a common trait in RVFV natural
identitied as reassortants wcre isolated from fatal cases. A
history. Arboviruses apparently evolve approximately 1 0-fold
similar question concerning thc outbreak of Sin Nombre virus
more slowly than RNA viruses that have only a single vertebrate
was raised (1 l), but in spite of the evidence of reassortmen:
host (4,41). Thus, the evolution of RVFV driven by reassortment
among Sin Nombre virus isolates (1 1, 14, 27), there was no
and by point mutations may depend on the host numbcr.
indication that the emergence 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 hvo different strains present at the same timc, in the
reassortmcnt appeared to be involved in the triggering of an
same area, and infecting the samc host. One additional requi-
influenza virus pandemic (42).
site for finding reassortants is that, once generated, thesc chi-
In conclusion, the high rate of reassortment among RVF\\’
8200
SALL ET AL.
J. VIKOL.
isolates 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 fcver epizootic in the central highlands of Madagascar. Res.
culating. Since reassortants containing one attenuated and two
Viral. 143i407315..
18.
virulent segments were shown to be attenuated (30) reassort-
Morvan, J., P. E. Rollin, and J. Roux. lY92a. La lièvre de la vallée du Rift à
Madagascar en 1991. Enquête séro-épidémiologique chez les bovins, Rev.
ment bctween the wild and vaccine strains would generate
Elev. Med. Vet. Pays Trop. 45:121-127.
attenuated viruses with protective effects against the disease,
1 9 . Morvan, J., J. F. Saluzzo, D. Fontenille, P. E. Rollin. and P. Coulanges. 1991.
provided attenuation markers werc present in each genomic
Rift Valley fever on the east toast of Madagascar. Res. Viral. 142:475-482.
segment of the live attenuated strain. Therefore, attenuated
20. Muller, R., 0. Poch, M. Delarue, D. H. L. Bishop, and M. Bouloy. 1994. Rift
Vallcy fever virus L segment: correction of the sequence and possible func-
vaccine strains with several attenuation markers present in
tional role of newly identified regions conserved in RNA-dcpendent poly-
each segment would be recommended for control of RVFV.
merases. J. Gen. Viral. 75:134S-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.
probability of reversion toward virulence.
Bouloy. 1995. Characterization of clone 13, a naturally attenuated avirulent
isolate of Rift Valley fever virus, which is altered in the small segment. Am. J.
Trop. Med. Hyg. 53:405~11.
ACKNOWLEDGMENTS
22. Page, R. D. M. 1995. Parallel phylogenies: reconstructing the history of
host-parasite assemblages. Cladistics 10:155-173.
We arc grateful to A. Billccocq, B. Le Guenno. and C. PrEhaud for
23. Pelissier, A., and R. Rousselot. 1954. Enquête sérologique sur l’incidence des
fruitful discussions, to M. Diallo for critically reading the manuscript,
virus neurotropes chez quelques singes de l’Afrique équatoriale fmnçaise.
and to R. Swanepocl for providing us with the 1997 Kenyan strain. W
Bull. Soc. Pathol. Exot. 47:22X-230.
C
24. Peters, C. J., and K. J. Linthicum. 1904. Rift Valley fever, p. 125-138. In
arc indcbtcd to ORSTOM and the WHO collaborating ccntcr for
B. G. W. and J. H. Ste& (cd.), Handbook of zoonoscs. section B. Viral, 2nd
nrhovirus and hcmorrhagic fevcr virus rcscarch (CRORA), whose
ed. CRC Press, Boca Raton, Fla.
collahorative work allowed us to obtain most of the RVFV isolatcs
25. Pretorius, A., M. J. Oelofsen, M. S. Smith, and E. van der Ryst. 1997. Rift
analyzed in this study. The excellent technical expertise of P. Vialat is
Valley fever virus: a seroepidemiologic study of small terrestrial vertebrates
acknowlcdgcd.
in South Africa. Am. J. Trop. Med. Hyg. 57:693-6Y8.
P.M.A.Z. was fundcd hy a PQ Scholarship from CNPq and a
26. Pringle, C. R. 1996. Genetics and genome segment reassortment, p, 1X9-226.
PRONEXIPADCT grant endowed by the Brazilian Government.
In R. M. Elliott (ed.). The viruses. The Bunyaviridae. Plenum Press. New
York, N.Y.
27. Rodrignez, L. L., J. H. Owens, C. J. Peters, and S. T. Nichol. 1998. Genetic
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Vol. 73, No. 10
0022-538X~99/$04.00+0
Copyright 0 1999, American Socicty for Microbiology. Al1 Rights Rescwcd.
Genetic Reassortment of Rift Valley Fever Virus in Nature
A. A. SALL,’ P. M. DE: A. ZANOTTO,’ 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,
Sio Paulo, Brazil’; Institut Pasteur de Madagascar, Antunanarivo, Madagascar’; and Groupe des Bunyavirides,
Institut Pasteur, 75724 Paris Cedex 15, France5
Received 9 April 199YAcceptcd 15 July 1999
Rift Valley fever virus (RW), 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 RW, 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
confu-med 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 emnhasizes 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 Bunyaviridue), which was reported pri-
the genome of members of the Bunyaviridue 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 andior 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 hf 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 seyuelae
been suggested or demonstrated for bunyavirus (8, 12,40) and
(13). Periodic large-scale epidemics, such as the ones in Mau-
hantavirus (11, 14, 27). However, to date, despite some clues
ritania in 1987 and 1998 (7, 43) Madagascar in 1990-1991
derived from unexpectcd 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-I 991
MATERIALS AND METHODS
outbreak (28) revealing that the virus could spread across
considerable distances. possibly beyond Africa. This threat be-
Virus propagation and RIVA extraction. The origina and years of isolation of
cornes more disyuieting 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, 3.5-37). Therefore, understanding the
sets of primers, NS3a-NS2g, MRVlaMRV2g, and Wag-Xg, wcre used to am-
mechanism underlying its dispersa1 and evolution is of para-
pli@ portions of the NSs, G2. and L coding regions. rcspectivcly. Thc NSs coding
mount importance to the control of this disease.
region is located in the S segment, whereas the regions coding for G? and L are
in the M and L segments, respectivcly.
The antisense primera N%ia, MRVla. and
The RVFV genome consists of three negative-sense single-
Wag were used to synthesize thc tirst-strand complementary DNA. Primer sc-
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 phylogenrtic analyses of each segment of the
RVFV genome were performed 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 likclihood and maximum parsimony methods in
G2, which, after cleavage, generate two additional nonstruc-
PAUP (Phylogenetic Analysis Using Parsimony, beta version 4.0: kindly pro-
tural proteins of 78 and 14 kDa. The S segment codes for the
vidcd hy 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 prohabilities among different nucleotides
and the value for the shape pnrameter (alpha) for the gamma distrihuted variable
rates among sites werc empirically determined from the data. Using the optimal
* Corresponding author. Mailing addrcss: Institut Pasteur, Groupe
values of thesc parameters. trççs with similar likelihood were collected and the
des Bunyaviridés, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France.
trec topology stahility around the likclihood maxima was investigated by calcu-
Phonc: 33 1 40 61 31 57. Fax: 33 1 40 61 31 51. E-mail: mhouloy
lating the 50% majority-rule
consçnsus. For an additional comparison,
we used
@pastcur.fr.
a fast tree search algorithm (quartet puzzling) for estimating maximum likeli-
8196
Y’
-
-
-
VOL.13, 1999
RIFT VALLEY FEVER VIRUS GENETIC EXCHANGE
x197
TABLE 1. Origins and years of isolation of RVFV isolates
Genotype”
C o d e
strain
Yr of
Origin
SOUKC
isolation
(SIMIL)
SNS’
Smithburn
1944
Uganda
Entehbe strain
C/C/C
ArUGA-55
Lunyo
1955
Uganda
Mosquito
CKIC
ArCAr-09
Ar B 1976
1969
CAR
Mosquito
CICIC
Cl3
Clone 13
L974
CAR
Human
*Kx
HEGY-77
ZH 54X
1977
QYP~
Human
EIE,/E
MPlZ”
MP12
1977
EgyP
28538 strain
UEIE
ArMAD-79
Ar Mg 811
1979
Madagascar
Mosquito
EI?;E
ArSEN-84
Ar D 3%61
1984
Senegal
Mosquito
E/WiW
AnGUI-
An K 6087
1984
Guinea
Bat
WICIC
ArBUF-84
Ar D 38457
1984
Burkina Faso
Mosquito
W!WIw
HlMAU-87
H D 47502
1987
Mauritania
Human
GWrw
HZMAU-87
H D 47312
1987
Mauritanie
Human
w;wiw
H3MAU-87
H D 47408
1987
Mauritania
Human
WlW/W
H4MAU-87
H D 48255
1987
Mauritania
Human
W/‘?/W
AnMAD-91
An Mg 990
1901
Madagascar
Bovine
cictc
ArSEN-93
Ar D 104769
1993
Sencgal
Mosquito
C/UW
AnSEN-K-93
An D 106417
1993
Senegal
Zcbu
WIWIC
BEGY-93
B EGY 93
1993
J%W
Buffalo
E/E,E
HEGY-93
H EGY 93
1903
WP
Humaii
EIEIE
HKEN-97
384-97.1
1997
Kenya
Human
CICIC
” E, W, and C, Egyptian, Western African. and Central-East Afrirican linragc, respectively; a:, scqucnce too short to hc assigncd a linenge: ?. scquence net determined.
h Attenuated strain.
hood trecs from PUZZLE version 3. I (33), which automatically
assigns cstima-
First, some of thc strains in the Central-East Africa and Egypt
tion of support of each interna1 node. Additionally, to investigate the rohuslncss
groups (SNS and ArUGA-55, AnMAD-91 and HKEN-97.
of tree resolution under maximum parsimony. a bootstrapping analysis was donc
with 1,000 random reaamplings
with thc nearcat-neighhor-interchange pertnrha-
HEGY-77 and HEGY-93) are closely related, despite their
tion algorithm within PAUP. For the shortest data set (i.e., the 211.hp L ampli-
dates of isolation, suggesting the existence of an endcmic-
con), a 2% jackknife was used as an alternative resampling
mcthod.
enzootic maintenance cycle of the virus in these areas. Second.
TO determine topological incongruencc among estimates ohtained fwm dif-
no obvious grouping bascd on the host species was observed.
ferent data sets for the same set of tazxü, wc chccked whether the best topology
for each data set is statisticallyworse when rcconstrocted with an alternative data
This could be explained by frequent exchange of viruses be-
set hy using the Kishino and Hasegawa test implemcnted in PAUP 4.0. Addi-
tween different hosts. Third, in cach group, strains isolated
tionally, to test for rcassortment among viruses from different lineages, con-
during epidemic-epizootic and endemic-enzootic periods were
straint trees were constructed; in these, the taxa werc assigned to a given part of
the tree, generating alternative topologies to he tested against the ones obtaincd
found adjacent to each other, suggesting that the virus circu-
from the unconstrained data. Finally, 10 allow thc inspection of the topologies
lates alternatively in enzootic-endemic form through a main-
estimated from different genomic ssgments. the TreeMap program (22) ws 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 numhers. The sequences of the strains listed in
Table 1 and corresponding to the L. Ci2. and N protcin coding regions bave been
distributions of each tree into three clusters. the assignment of
assigned GenBank accession no. AF134782 to AF134801, AF133492 to
some isolates (ArSEN-93. AnSEN-K-93,
ArSEN-84, HlMAU-
AF1345OX, and AF134530 to AF134551, respectively. The sequences of the NSs
87, and AnGUI-84) to one particular group does not remain
protein coding region were deposited in the EMBL datahasc (accession no.
constant within the three phylogenies. Construction of the
Y 12739 to Y12756) as already reported (29).
reconciling parallel trees using the thrce phylogenies empha-
sized that the groupings of the strains in the S, M, and L
RESULTS
segment trees correlate quite well but clearly show some in-
Phylogeny of different RVF’V segments. In a previous papcr,
congruences. For instance, HlMAU-87 and ArSEN-84 possess
phylogenetic analysis carried out with 20 isolates and dcrived
the 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 Ebyptian lineage,
coding region in the S segment showed the existence of three
rcspectively. AnGUI- has thc L and M segments of the Central-
distinct lineages: Ia and Ib in sub-Saharan Africa and II in
East African genotype. whereas 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 809-nucleo-
period in two different places in Senegal, 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 lineage 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 confirmed 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 Ia, Ib, and II, respectively (see Fig. 1).
the L segment belongs to the Western African lineage (Fig. 1).
The support for different nodes on thc 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
the following extreme precautions that were taken to avoid
three phylogenies.
such artifacts. (i) RNA extraction and RT were done sepa-
These phylogenetic analyses raised some general comments.
rately for cach isolate under a laminar-flow containmcnt hood
81%
SALL ET AL.
J. VIROL.
A
6
r H2MAU87
--y
--i ArSEN84 1
/
/
” rHsMA’J87 1
yAnSENk93:
~EST
PFRICA
l
-1
-MPIS
-1
-HEGY93
’
EGYPT
-- BEGY93
5”pll H E G Y 7 7
--.
ArMAD79
l
I AnMAD91
EAST
! [s2’
- ArCAR69
CENTRAL
I
1
EAST
’
CENTRAL
AFRICA
rAnMAD91
AFRICA
fw i---- ,,K,=,,g7
“l
-SNS
---~--- SNS
93
/
r---~~- ArCAR69
- --------ArUGA55
z-‘L-- ArUGA55
-
C
FIG. 1. Maximum likelihood trees for the NSs (A), G2 (B). and L(C) coding
region sequences on thc S, M, and L segmments, rcspectivrly.
Values helw the
H2MAU87
different nodes indicate their robuatness by 5% jacknife (indicated in boldface
--/
and italic type). maximum likelihood quartet puzzling (indicated in brackets).
_ HIMAlJ87 j
and bnotstrapping (indicated in roman type) methods. Putative tussortant
---- H3MAU87
strains are hoxed.
---H4MAU87
WEST
AFRICA
!---- ArBUF84
i-- fArSEN84
sequenced several times and led to identical sequences, many
!LArsEN93;
/
of them being processed in laboratories at least 4,000 km apart
(Paris, France, and Dakar, Senegal).
7 ArMAD79
-1
Given that trees for different genes are clearly incongruent
.-- j
;,-jBEGY93
in their topologies, it could be assumed that these incongru-
E !
EGYPT
ences are due to genetic exchange. Regarding the segmentation
,
j H E G Y 7 7
of the RVFV genome, the most probable device to explain the
1
kHEGY93
/
I
exchange of genetic material is RNA segment reassortment.
I
‘MPi2
Tests for topological structure as indicative of reassortment.
Ti
[96] ,--.---
Two approaches
werc used to evaluate the incongruences ob-
‘AnSEiVk93 ~
scrved among specific isolates of different trees. First, we con-
1
Cr-_-r---------HKEN97
structed constrained trees and tested their topologies by using
EAST
likelihood against the unconstrained trees shown in Fig. 1. The
likelihood value for the unconstrained tree resulting from the
_-.-~-- - - - ‘AT;suJ84
71 CENTRAL
i--d 1 AFRICA
use of the NSs sequences was bctter than the one obtained for
95 c-c13
the constrained tree where the ArSEN-93 and HlMAU-87
~
%fl iArRCA69
1
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
I
values obtained for the G2 and L sequence phylogenies (for
G2, difference of 1nL = 42.95, P = 0.0002; for L region, dif-
S%Jackknife IOC4 replicailons
1% ML quaflet puzli&
ference of 1nL = 9.74, P = 0.06) in which ArSEN-93 and
54% MR bootstrapi0W replicatms
AnGU1-84 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 each 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 InL = 30.35, P = 0.0603) under thc 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 tree (--hlL =
meric viruses were viable and their host did not exert a nega-
1,842.69) had a lower likelihood (difference of 1nL = 51.99,
tive selection against them. We do not know whether the
P < 0.0001) under the G2 data set than the Ci2 tree did
strains identified as reassortants were host restricted, but they
(-InL = 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
tocs, 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 wherc at least two different lineages circulated at the same
observed among trees are due not to poor phylogenetic 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 mosquitocs that were dually infected with two
in the RVFV tree topologies could be duc to intramolecular
RVFV natural isolates (38). It secms 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 composed of a heterogenous population of viral
split decomposition method, by using the program Splits ver-
clones with different biological propcrties (21). Mosquitoes cari
sion 1.0 (3), but we did not find any clear-tut indication of
be dually infected by intcrrupted feeding or feeding on a dually
recombination, i.e., networked evolution (data not shown),
infected vertebrate host. Both mechanisms have been demon-
within the fragments sequcnced. Thus, reassortment events gen-
strated experimentally for RVFV with a natural mosquito vec-
erating combination between L, M, and S segments of RVFV
tor, Culexpipiens, suggesting that under natural conditions, the
natural isolates appear to be the most Iikely 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-
mosquitoes may be refractory to superinfection with the same
pear to result from a reassortment event. This percentagc may
virus species. Therefore, both mosquitoes and vertebrate hosto
be underestimated since the method used herein tu 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 determined.
tral-East Africa). Interestingly, a11 the strains identilied as re-
The occurrence of reassortment among natural isolates from
assortants, ArSEN-93, AnSEN-K-93,
ArSEN-84, HlMAU-87,
West Africa and Central-East Africa or, less frequently. Egypl
and AnGUI-84, were isolated from West Africa (Senegal,
suggests two possibilities: (i) one strain was transported to s
Guinea, and Mauritania) and involved reassortment with
place where another strain was circulating; or (ii) the virus bac
strains of the Central-East African or Egyptian lineage (Ar-
to make its way through the rain forest and to adapt to thc
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 cxistencc of a sylvatic cycle would have to be dem-
SEN-84, AnGUI-84, and HlMAU-87) and M plus S segments
onstrated, the circulation of RVFV in the rain forest has beer
(AnSEN-K-93 and ArSEN-93) (Table 1). In dual infection of
revealed by the high antibody prevalence in wild animals anc
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 oi’
tions with a larger sample of RVFV field isolates would be
Perinet in Madagascar (15). In addition, data recently publishec
needed to indicate whether generation and selection of reas-
by Pretorius et al. (25) indicatcd that rodents were involved as 2
sortants in nature are random or not.
potential reservoir of RVFV in South Africa. It is likely thal
Replication errors such as base substitution and deletions or
different strains of RVFV circulate in the rain forest area amons,
insertions are the most common mechanism of RNA virus
wild mosquitoes (16) and vertcbrates like monkeys (23) and bat!,
evolution. However, major changes of viral genotype may in-
(5), depending on the dynamic of the interaction among hosts anc.
valve exchanges of RNA segments (genetic shift), as exempli-
virus populations. Therefore, the search and characterization of 2.
fied by influenza virus (42). Comparison of RVFV isolates
sylvatic cycle of RVFV are of utmost importance because this
sampled from different geographical localities showcd 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 emergence.
point mutations, the percentage of base substitution varying
Thc existence of reassortmcnt as a mechanism of evolutior.
from 0 to 9.6% in the S segment (29), but also may occur by
raiscs the question of the epidemic potential of these viruses
genetic exchange. Indeed, the apparent contradictions in the
and their pathogenicity. Isolation of a potential reassortam.
groupings of the different segments within the three distinct lin-
from a fatal case during thc 1987 Mauritanian epidemic leaves
eages could be explained by reassortment and strongly suggest
this question open, even though other strains which were no’:
that this evolution mechanism is a common trait in RVFV natural
identitied as reassortants wcre isolated from fatal cases. A
history. Arboviruses apparently evolve approximately 1 0-fold
similar question concerning thc outbreak of Sin Nombre virus
more slowly than RNA viruses that have only a single vertebrate
was raised (1 l), but in spite of the evidence of reassortmen:
host (4,41). Thus, the evolution of RVFV driven by reassortment
among Sin Nombre virus isolates (1 1, 14, 27), there was no
and by point mutations may depend on the host numbcr.
indication that the emergence 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 hvo different strains present at the same timc, in the
reassortmcnt appeared to be involved in the triggering of an
same area, and infecting the samc host. One additional requi-
influenza virus pandemic (42).
site for finding reassortants is that, once generated, thesc chi-
In conclusion, the high rate of reassortment among RVF\\’
8200
SALL ET AL.
J. VIKOL.
isolates 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 fcver epizootic in the central highlands of Madagascar. Res.
culating. Since reassortants containing one attenuated and two
Viral. 143i407315..
18.
virulent segments were shown to be attenuated (30) reassort-
Morvan, J., P. E. Rollin, and J. Roux. lY92a. La lièvre de la vallée du Rift à
Madagascar en 1991. Enquête séro-épidémiologique chez les bovins, Rev.
ment bctween the wild and vaccine strains would generate
Elev. Med. Vet. Pays Trop. 45:121-127.
attenuated viruses with protective effects against the disease,
1 9 . Morvan, J., J. F. Saluzzo, D. Fontenille, P. E. Rollin. and P. Coulanges. 1991.
provided attenuation markers werc present in each genomic
Rift Valley fever on the east toast of Madagascar. Res. Viral. 142:475-482.
segment of the live attenuated strain. Therefore, attenuated
20. Muller, R., 0. Poch, M. Delarue, D. H. L. Bishop, and M. Bouloy. 1994. Rift
Vallcy fever virus L segment: correction of the sequence and possible func-
vaccine strains with several attenuation markers present in
tional role of newly identified regions conserved in RNA-dcpendent poly-
each segment would be recommended for control of RVFV.
merases. J. Gen. Viral. 75:134S-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.
probability of reversion toward virulence.
Bouloy. 1995. Characterization of clone 13, a naturally attenuated avirulent
isolate of Rift Valley fever virus, which is altered in the small segment. Am. J.
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