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Detection of a new yellow fever virus lineage

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Article in Journal of Medical Virology November 2009
DOI: 10.1002/jmv.21606 Source: PubMed

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Journal of Medical Virology 82:175185 (2010)

Detection of a New Yellow Fever Virus Lineage

Within the South American Genotype I in Brazil
Renato P. de Souza,1 Peter G. Foster,2 Maria Anice M. Sallum,3 Terezinha L.M. Coimbra,1
Adriana Y. Maeda,1 Vivian R. Silveira,1 Eduardo S. Moreno,1 Fernanda G. da Silva,1 Iray M. Rocco,1
Ivani B. Ferreira,1 Akemi Suzuki,1 Fabola M. Oshiro,1 Selma M.C.N. Petrella,1 Luiz E. Pereira,1
Giselda Katz,4 Cilea H. Tengan,4 Melissa M. Siciliano,4 and Ceclia L.S. dos Santos1*
1

Servico de Virologia do Instituto Adolfo Lutz, Sao Paulo, SP, Brazil

Department of Zoology, Natural History Museum, London, United Kingdom
3
Departamento de Epidemiologia, Faculdade de Saude Publica, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
4
Divisao de Zoonoses, Centro de Vigilancia Epidemiologica, Sao Paulo, SP, Brazil
2

Nucleotide sequences of two regions of the

genomes of 11 yellow fever virus (YFV) samples
isolated from monkeys or humans with symptomatic yellow fever (YF) in Brazil in 2000, 2004, and
2008 were determined with the objective of
establishing the genotypes and studying the
genetic variation. Results of the Bayesian phylogenetic analysis showed that sequences generated from strains from 2004 and 2008 formed a
new subclade within the clade 1 of the South
American genotype I. The new subgroup is here
designated as 1E. Sequences of YFV strains
recovered in 2000 belong to the subclade 1D,
which comprises previously characterized YFV
strains from Brazil. Molecular dating analyses
suggested that the new subclade 1E started
diversifying from 1D about 1975 and that the
most recent 20042008 isolates arose about
1985. J. Med. Virol. 82:175185, 2010.
2009 Wiley-Liss, Inc.

KEY WORDS: yellow fever virus; subclade 1;

premembrane and envelope
gene junction; 30 non-coding
region; molecular clock; divergence time

INTRODUCTION
Yellow fever virus (YFV) is the prototype member of
the family Flaviviridae, genus Flavivirus, which
includes approximately 70 human and veterinary
pathogens. Flaviviruses have a single-stranded, positive sense RNA genome of about 11 kb in length,
arranged into a short 50 non-coding region (50 NCR), a
single open-reading frame encoding the structural
proteins capsid (C), premembrane (prM), membrane
(M), envelope (E), the non-structural (NS) proteins NS1,
2009 WILEY-LISS, INC.

NS2a, NS2b, NS3, NS4, NS5, and a 30 non-coding region

(30 NCR) [Chambers et al., 1990].
YFV is the causative agent of yellow fever (YF), an old
disease that caused widespread epidemics in Africa,
North and South Americas, and Europe from the 17th to
the early 20th centuries, and then reemerged in recent
decades in sub-Saharan Africa and tropical South
America [Gubler, 2002, 2004]. In humans, YFV can
cause a broad spectrum of clinical manifestations
ranging from unapparent infection or mild fever to
severe hepatitis and hemorrhagic fever. The mortality
rate of symptomatic patients who develop visceral
disease can vary from 20% to 50% [Monath, 2008].
Despite the availability of an effective vaccine, YF
remains a public health problem in endemic regions of
South America and Africa, where incidence rates can
reach 200,000 infections per year, including 30,000
deaths [WHO, 2003].
YFV is maintained in primitive rainforest cycles,
involving sylvatic mosquitoes and non-human primates.
In the urban cycle the YFV is transmitted among
humans by the mosquito Aedes aegypti Linnaeus. The
sylvatic cycle of YFV includes people who live or work in
the tropical rain forests, and those who visit as tourists.
In Brazil, the sylvatic cycle is associated with mosquitoes of the genera Haemagogus and Sabethes [Monath,
1988].
Circulation of YFV is endemic in both rural and forest
areas in all regions of Brazil, including the states of Acre,
Grant sponsor: Secretaria de Estado da Saude de Sao Paulo/SP;
Grant sponsor: FAPESP (to C.L.S.S. and M.A.M.S.); Grant
number: 05/53973-0.
*Correspondence to: Ceclia L.S. dos Santos, Servico de
Virologia do Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, CEP
01246/902 Sao Paulo, SP, Brazil.
E-mail: simoes.santos@uol.com.br
Accepted 21 June 2009
DOI 10.1002/jmv.21606
Published online in Wiley InterScience
(www.interscience.wiley.com)

176

Amazonas, Amapa , Roraima, Rondo nia, Mato Grosso,

Mato Grosso do Sul, Tocantins, Goia s, Distrito Federal,
Maranha o, Para, Minas Gerais, northwestern Sa o
Paulo, and western areas of the states of Parana , Santa
Catarina, and Rio Grande do Sul. The boundaries
between these zones of YFV circulation, which include
part of the states of Piau, Bahia, Espirito Santo, Sa o
Paulo, Parana , Santa Catarina, and Rio Grande do Sul,
are considered to be transitional epizootic areas and are
targets of a continuous surveillance. In Brazil, the only
area considered to be low risk is a narrow region along
the Atlantic coast [Vasconcelos et al., 1997; FUNASA,
1999; CDC, 2009].
The last urban YF epidemics in Brazil occurred in
Sena Madureira, Acre State, in 1942 [Monath, 1988].
The occurrence of a gradual increase in the sylvatic
circulation of YFV beyond traditional boundaries of the
enzootic zone, as those reported in the states of Rio
Grande do Sul [Vasconcelos et al., 2003], and in Minas
Gerais in 2000 and 2001 [Filippis et al., 2002], was
associated with the high mobility of susceptible humans
in those regions where the YF is endemic. As the YFV
circulates in several regions in Brazil, the presence of A.
aegypti was considered to be a real threat for reurbanization of the disease [Travassos da Rosa et al.,
2000].
Since late 2007, several South American countries
have detected episodes of YF in humans and monkeys.
In Brazil, approximately 50 human symptomatic cases
have been registered from December 2007 to early
February 2009, including 29 deaths [Ministry of Health,
Brazil, 2008, 2009]. The YF surveillance program
developed by the Ministry of Health, Brazil, concluded
that these symptomatic cases might have occurred in
forested areas of the states of Goia s, Mato Grosso do Sul,
Distrito Federal, Mato Grosso, Sao Paulo, and Parana .
Five of the recent cases were detected between December 2008 and January 2009 in municipalities of the
northwest of the state of Rio Grande do Sul, near the
border of Argentina [Ministry of Health, Brazil, 2009;
Pan American Health Organization, 2009].
Molecular epidemiological studies revealed that YFV
strains isolated in Africa and South America are
genetically distinct. In Africa, five genotypes were
identified, that is, the west African genotypes I and II,
the east African, the east and central African, and the
Angola genotype [Wang et al., 1996; Mutebi et al., 2001].
In South America two genotypes were registered: the
first being the South American genotype I that includes
strains recovered from Brazil, Panama, Colombia,
Ecuador, Venezuela, and Trinidad, and the second
designated as South America genotype II, including
virus isolated mainly in Peru [Wang et al., 1996; Bryant
and Barrett, 2003].
Recently, Vasconcelos et al. [2004] analyzed the
genetic diversity of multiple YFV strains isolated over
the last 67 years in Brazil. They showed that, except for a
strain from 1983 which belongs to the South America
genotype II, all remaining samples that circulated in
Brazil from 1935 to 2001 clustered together in the South
J. Med. Virol. DOI 10.1002/jmv

de Souza et al.

America genotype I. There are two major subclades

within the clade composed of South American genotype
I. One subclade contains strains from Para isolated from
1954 until 1968, and the second major subclade is
formed by strains that circulated from 1969 until 2001.
The hypothesis that YFV evolved in Africa prior to its
introduction in South America was reinforced by dating
studies recently published by Bryant et al. [2007]. This
study suggested that the currently circulating strains of
YFV arose in Africa within the last 1,500 years and
emerged in the Americas about 300400 years ago as a
consequence of the slave trade. After being introduced,
the virus spread westward, and still persists in forest
cycles [Bryant et al., 2007].
Strains isolated from the 2008 YF outbreak were
analyzed for the present study. We sequenced a 670 bp
fragment of the prM/E junction, and an approximately
300 bp of the 30 NCR beginning in the NS5 gene stop
codon to the 30 terminal of the conserved sequence 2
motif (CS2) present in all mosquito-borne flaviviruses
[Chambers et al., 1990]. To these we added strains
isolated by the Virology Laboratory of the Instituto
Adolfo Lutz in 2000 from human symptomatic infections
which occurred in Sa o Paulo state, and one sample from
a patient infected in Amazonas state in 2004. Representative sequences from GenBank of the South America genotypes I and II, including previously published
strains from Brazil, as well as sequences of YFV from
west, central, and east Africa, were included in the study
for comparison with the sequences of the recent Brazilian isolates. The main objectives of the study are: (1) to
examine the genotypes of YFV strains that circulated in
the 2008 epidemics in Brazil; (2) to establish genetic
relationships among Brazilian YFV strains, and (3) to
estimate time of both emergence and divergence of the
YFV subtypes.
MATERIALS AND METHODS
Viruses
Eight strains from human or monkeys infected with
YFV in 2008 plus three strains from human infection
detected in 2000 and 2004 in Brazil were characterized
in the study (Table I). Viral isolation from clinical
samples of infected patients or necropsy tissues of
monkeys that have died with symptoms of YF was made
by inoculation onto monolayer cultures of clone C6/36
cells of Aedes albopictus and into suckling mouse brains.
RNA Isolation, RT-PCR, and Nucleotide
Sequencing
Viral RNA was extracted either from the serum of
viremic patients or supernatant fluid from C6/36infected cells using the QIAmp Viral RNA Extraction
Kit (Qiagen, Valencia, CA), according to the manufacturers instructions. For viral RNA extraction from
brain and liver of suckling mouse infected with necropsy
tissue of monkeys, the QIAmp Blood Viral RNA
Extraction Kit was used instead. Reverse transcription

Genetic Diversity of Yellow Fever Virus

177

TABLE I. Origin of Brazilian Yellow Fever Virus Investigated in This Study

GenBank accession number
Strain
SPH188002
SPH188057
SPH258595
SPH287923
SPH287992
SPH288116
SPH288294
SPAn288183
SPAn288184
SPAn289562
SPAn289568

Sequence ID

Location/yeara

Source

prM/E junction

30 NCR

Brazil00B
Brazil00C
Brazil04
Brazil08A
Brazil08B
Brazil08C
Brazil08D
Brazil08E
Brazil08F
Brazil08G
Brazil08H

SP/2000
SP/2000
AM/2004
MT/2008
MS/2008
GO/2008
GO/2008
SP/2008
SP/2008
SP/2008
SP/2008

Human
Human
Human
Human
Human
Human
Human
Monkey
Monkey
Monkey
Monkey

FJ875515
FJ875516
FJ875517
nd
nd
nd
nd
FJ875518
FJ875519
nd
FJ875520

FJ875521
FJ875522
FJ875523
FJ875524
FJ875525
FJ875526
FJ875527
FJ875528
FJ875529
FJ875530
FJ875531

nd, Not determined.

a
SP, Sao Paulo; AM, Amazonas; MT, Mato Grosso; MS, Mato Grosso do Sul; GO, Goia s/year of isolation.

(RT)-PCR was performed on purified viral RNA with the

SuperScriptTM one-step RT-PCR with PlatiniumR Taq
System (Invitrogen/Life Technologies, Carlbad, CA).
One set of studies employed primers described by
Jennings et al. [1994] designed to amplify a fragment
of 670-bp spanning the prM/E junction genomic region of
YFV. The second set of studies focused the 30 NCR region.
Flavivirus universal primers EMF1 and VD8 described
by Pierre et al. [1994] were used to amplify a fragment of
YFV genome comprising the distal part of the NS5 gene
to the end of the CS2 element located in the 30 NCR
region that corresponds to the complementary VD8
nucleotide sequence. Thermocycling parameters used
for amplification of prM/E junction and 30 NCR sequences were those described by Jennings et al. [1994] and
Deubel et al. [1997], respectively. The amplified products were directly sequenced using the ABI PrismR Big
DyeM Terminator Cycle Sequencing Ready Reaction
Kit (PE Applied Biosystems, Foster City, CA), according to the manufacturers instructions, in the presence of
the primers used in RT-PCR amplification. Sequences
were determined using the ABI 377 sequencer (PE
Applied Biosystems). Sequences determined in the
study are deposited in GenBank under accession
numbers FJ875515FJ875531.

sequences of YFV were obtained for phylogenetic

analysis are listed in Table II. Sequence alignment
was performed manually using the BioEdit software
[Hall, 1999].
The program MrBayes [Huelsenbeck and Ronquist,
2001] was used in the Bayesian analysis without the
clock model. Modeltest version 3.7 [Posada and Crandall, 1998] was employed to choose a model using the
Akaike information criterion (AIC). The models suggested by Modeltest for prM/E junction and 30 NCR were,
respectively, GTR G I and GTR G. Bayesian analyses were conducted for each of the prM/E junction and
30 NCR sequences of YFV separate, and for the concatenated data set. In the concatenated analysis, we
used only the strains for which there were sequence
information for both genes, which includes 30 samples
from Latin America and 8 strains from Africa.
For each alignment, two separate MCMC runs were
made, each with four chains in the Metropolis-coupled
MCMC. Runs were done for 2,000,000 generations,
sampling every 1,000. A burn-in of half of the samples
was used. The post-burn-in samples were compared
between the two runs, and showed good parameter and
topology convergence, as shown by PSRF values near
unity, and by low values (<0.01) of the average standard
deviation of split frequencies.

Data Analysis
Nucleotide sequence of prM/E junction fragment was
translated into the amino acid sequence by using the
program Edit Seq (Lasergene, DNASTAR, Inc., Madison, WI). This program was also used to determine the
end of the NS5 gene and the beginning of the 30 NCR in
the nucleotide sequence obtained with primers set VD8/
EMF1. The portion of the NS5 gene was eliminated and
further analysis was undertaken using the region of the
30 NCR of about 300-bp starting with the stop codon to
the end of the CS2 motif.
Phylogenetic Analysis
The sources, geographic origins, and GenBank accession numbers for which prM/E junction and 30 NCR

Molecular Dating of Recent Divergences of YFV

The program Beast v1.5a1 [Drummond and Rambaut,
2007] was used to estimate the divergence pattern of the
prM/E junction and 30 NCR sequences (results not
shown) using a relaxed clock model [Drummond et al.,
2006]. Each alignment was analyzed with the nucleotide
substitution model suggested by ModelTest, the
GTR G model for the 30 NCR, and the GTR I G for
the prM/E junction region. Each analysis was run twice,
each for 20 million generations, each with a burn-in of 2
million generations. Convergence was assessed using
the estimated sample size (ESS) values of the numerical
parameters, which were all over 100. Post-burn-in
samples from the two skyline runs were pooled for the
J. Med. Virol. DOI 10.1002/jmv

178

de Souza et al.
0

TABLE II. prM/E Junction and 3 NCR Sequences Used in This Study
GenBank accession no.
Isolate

Sequence ID

JSS
BeH111
BeAn23536
BeAr44824
BeH203416
BeAr233436
BeH233393
BeH350698
BeH379501
BeH413820
BeH 425381
BeAr424492
BeH422312
BeAr 424083
BeH511843
BeAr512943
BeH512772
BeAr527198
BeH 535010
BeAr 544276
Tennessee
BeAn604552
BeH603325
BeH605427
BeAr614320
BeAR628124
BeAr630768
BeAr631464
BeAr645693
V528A
Trinidad79
CAREC 788379
1345
OBD 5041
614819
1368
1899/81
1914
Peru95(153)
ARV 0548
03-5350-98
IQT 5591
OBS7687
OBS8026
35720
Asibi
M 90-5
69056
IB AR 45244
H117505
Uga
ggA 709-4-A2
STA-LSF-4-4143
Serie 227
Senegal65
SH 1446
Ar B 8883

Brazil35
Brazil54
Brazil60
Brazil62
Brazil71
Brazil73A
Brazil73B
Brazil78
Brazil80
Brazil83
Brazil84A
Brazil84B
Brazil84C
Brazil84D
Brazil91
Brazil92A
Brazil92B
Brazil94
Brazil95
Brazil96A
Brazil96B
Brazil98A
Brazil98B
Brazil98C
Brazil99
Brazil00A
Brazil01A
Brazil01B
Brazil01C
Colombia79
Trinidad79A
Trinidad79B
Ecuador81
Ecuador97
Panama74
Peru77
Peru81A
Peru81B
Peru95A
Peru95B
Peru98A
Peru98B
Bolivia99A
Bolivia99B
Venezuela98
Ghana27
Sudan40
Nigeria46
Nigeria69
Nigeria87
Uganda48A
Uganda48B
Zaire58
Ethiopia61
Senegal65A
Senegal65B
CAR77A

Ar B 9005

CAR77B

85-82H
IvoryC1999

IvoryCoast82
IvoryCoast99

J. Med. Virol. DOI 10.1002/jmv

Origin/year of
isolation
Brazil/1935
Brazil/1954
Brazil/1960
Brazil/1962
Brazil/1971
Brazil/1973
Brazil/1973
Brazil/1978
Brazil/1980
Brazil/1983
Brazil/1984
Brazil/1984
Brazil/1984
Brazil/1984
Brazil/1991
Brazil/1992
Brazil/1992
Brazil/1994
Brazil/1995
Brazil/1996
Brazil/1996
Brazil/1998
Brazil/1998
Brazil/1998
Brazil/1999
Brazil/2000
Brazil/2001
Brazil/2001
Brazil/2001
Colombia/1979
Trinidad/1979
Trinidad/1979
Ecuador/1981
Ecuador/1997
Panama/1974
Peru/1977
Peru/1981
Peru/1981
Peru/1995
Peru/1995
Peru/1998
Peru/1998
Bolivia/1999
Bolivia/1999
Venezuela/1998
Ghana/1927
Sudan/1940
Nigeria/1946
Nigeria/1969
Nigeria/1987
Uganda48
Uganda/1948
Zaire/1958
Ethiopia/1961
Senegal/1965
Senegal/1965
Central African
Republic/1977
Central African
Republic/1977
Ivory Coast/1982
Ivory Coast/1999

Source

prM/E junction

30 NCR

Human
Human
Cebus spp.
Haemagogus spp.
Human
Haemagogus spp.
Human
Human
Human
Human
Human
Hg. janthinomys
Human
Hg. albomaculatus
Human
Hg. janthinomys
Human
Haemagogus spp.
Human
Hg. janthinomys
Human
Alouatta belzebul
Human
Human
Haemagogus spp.
Hg. janthinomys
Hg. janthinomys
Sa. chloropterus
Haemagogus sp.
Human
Hg. spegazzini
Haemagogus
Human
Human
Human
Human
Human
Sentinel mice
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Mosquito
Human
Human
Aedes africanus
Human
Human
Human
Human
Ae. africanus

AY540437
AY540441
AY540443
AY540449
AY540452
AY540453

AY540456
AY540457
AY540458

AY540459
AY540461
AY540463
AY540465
AY540469
AY540471
AY540472
AY540473

AY540436

AY540475

DQ872412
AY540477
AY540478
AY540479
AY161928

AY161933

AY161946
AY161948
AY161950
AY540431
AY540434
AY540489
AY640589
AF369692
U52403
AF369677
AF369684

AF369694
AF369697
AF369674

AF369687
U52392

U52390
AY541335
AY541338
AY541340
AY541344
AY541347
AY541348
AY541352
AY541358
AY566273

AY541366
AY541369

AY541377
AY541379
AY541381
AY541387

AY541394
AY541397
AY541398
AY541399
AY541328
AY541332
AY541333
AY541334
AY541403
U52420

AY541405
AY541406
AY541412
AY541414
U52411

U52407
AY541430
AY541432
AY541434
AY541326
AY541327
AY541443
AY326411
AY326414
U52403

AY541410
U52423

AY541445
AY541407
U52414

U52393

U52395

U52396

U54798
AY603338

U54798

Ae. africanus
Human
Human

Genetic Diversity of Yellow Fever Virus

final results, from which the maximum clade credibility

(MCC) tree was made.
RESULTS
Sequence Variation
prM/E junction fragment. A fragment of 670 bp,
which includes the last 108 nucleotides of prM proteincoding gene, the entire 225 nucleotides of M proteincoding gene, and the first 337 nucleotides of E proteincoding gene, was determined for six new strains of this
study. The nucleotide and amino acid sequences of the
prM/E junction fragment determined for the six new
strains amplified in this study were compared with the
prototype Asibe strain, showing a paired identity
ranging from 85.8% to 100% (nucleotide) and 97.8% to
100% (amino acid) (MEGALIGN, Lasergene, DNASTAR, Inc.).
30 NCR. A fragment of about 300 bp, beginning with
the NS5 gene stop codon to the end of the CS2 motif, was
determined for the 11 new YFV strains of this study.
Alignment of these novel 30 NCR sequences with the
corresponding 30 NCR sequence of the prototype Asibe
strain revealed the extreme heterogeneity of this region
of YFV genome, as previously reported by others [Wang
et al., 1996; Mutebi et al., 2004; Bryant et al., 2005]. The
most important feature was a deletion of 65 bases
located in domain I of the 30 NCR (not shown), which
comprises the first and second repeated hairpin motifs
identified in YFV isolated in African continent [Wang
et al., 1996; Bryant et al., 2005].
Phylogenetic Analysis
The alignment of the prM/E junction sequences
included 50 sequences, with 670 nucleotides; that of
the 30 NCR sequences included 60 sequences with 391
nucleotides. In the prM/E junction alignment, of the 670
nucleotides, 412 were constant and 258 were variable, of
which 210 were parsimony informative. In the 30 NCR
alignment, of the 391 nucleotides, 249 were constants
and 142 variables, with 120 parsimony informative.
Topologies generated in Bayesian analyses without the
molecular clock model of either prM/E junction or 30 NCR
sequences were nearly identical (Figs. 1 and 2).
A new and strongly supported subclade was found
within clade 1 of the South American genotype I,
composed of YF strains obtained from humans or
monkeys infected in 2004 and 2008 in Brazil, and a
sequence from 1998 from Venezuela. Bayesian posterior
probability for the split leading to the group is 1.00 when
employing either the 30 NCR sequence data or the prM/E
junction sequence data. The new subclade is here
designated subclade 1E, when following the classification proposed by Vasconcelos et al. [2004]. YFV strains
from the state of Sa o Paulo in 2000 belong to the
subclade 1D, which also comprises YFV strains from
Para state isolated in 19981999, and from the states of
Tocantins, Goia s, Bahia, and Minas Gerais isolated in
20002001. YFV from Para isolated in 1971, 1987, and

179

1998, a 1980 strain from Maranha o, and a 1994 strain

from Minas Gerais were placed in the subclade 1C in the
analyses of the both prM/E junction and 30 NCR data
sets. The strains from the northern and central states,
including Brazil94 and Brazil96A (Rondonia), Brazil96B (Para), Brazil84A (Amapa ), Brazil73B, Brazil79
(Goia s), and Brazil92A (Mato Grosso do Sul), formed the
subclade 1B. The subclade 1A contained the strains
isolated from the northern and central states Brazil72,
Brazil73 (Goias), Brazil84B (Para), Brazil91 (Roraima),
and Brazil92A and Brazil92B (Mato Grosso do Sul). The
phylogenetic positions of the prM/E junction sequences
of the strains Brazil84A (Amapa ), Panama74, and
Ecuador 81, as well as the prM/E junction and 30 NCR
sequences of the strain Colombia79, were not resolved.
When using the prM/E junction fragments, the
strains identified as Brazil60, Brazil54, and Brazil62
clustered together in the Old Para group proposed by
Vasconcelos et al. [2004]. However, in the topology
generated in the analysis employing sequences of
30 NCR, relationships among sequences Brasil54, Brasil62, and Brasil60 were not resolved. Similarly, the
phylogenetic position of Brasil35 remains unresolved.
YFV strains from Trinidad, Peru, Bolivia, Ecuador,
and a single strain isolated from Brazil in 1983 were
classified in the South American genotype II. Finally,
the phylogenetic analyses of the prM/E junction and
30 NCR sequences of the African strains included in the
study identified two distinct major groups, which
contain viruses from the west (Nigeria, Ivory Coast,
Senegal, and Ghana), and east (Sudan, Ethiopia,
Zaire, Uganda, and Central African Republic) of the
continent.
In the Bayesian analyses using the concatenated prM/
E junction and 30 NCR sequences, the groups and
subgroups are better supported than in the separate
data set analyses (not shown). Posterior probability for
the new subclade 1E is 1.0, and Venezuela98 strain is
clustered into this subgroup as sister to Brazil04,
Brazil08E, Brazil08F, Brazil08H strains. In the concatenated data Brazilian strains Brazil08E, Brazil08F,
and Brazil08H clustered together in a strongly supported clade sister to Brazil04.
Molecular Dating of Divergences of YFV Strains
Bayesian molecular dating of YFV divergence times
was conducted employing the prM/E junction data set
only, which had smaller variance than the 30 NCR.
Sequences were analyzed using BEAST, using the
uncorrelated log-normal relaxed clock model [Drummond and Rambaut, 2007]. Three different coalescent
demographic models were tested: Bayesian skyline,
constant population size, and exponential population
growth. The marginal likelihood as given by the
harmonic mean estimator did not differ much between
the three models (not shown), and having no compelling
evidence to choose one demographic model over another
the Bayesian skyline demographic model was used. The
estimated divergence time among YFV of the west
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de Souza et al.

Fig. 1. Bayesian analysis of the prM/E junction sequences. This analysis used the GTR G I model, as
suggested by Modeltest. Duplicate sequences were collapsed before analysis (as shown by compound leaf
names). Details of the MCMC are given in the Materials and Methods Section. The consensus of post-burnin trees is shown, with nodes with less than 70% support collapsed. Numbers above the nodes represent
posterior probabilities of those branches. Black squares indicate the new sequences generated for this
study.

J. Med. Virol. DOI 10.1002/jmv

Genetic Diversity of Yellow Fever Virus

181

Fig. 2. Bayesian analysis of the 30 NCR sequences. Conditions were as described in Figure 1, using the
GTR G model. Numbers above the nodes represent posterior probabilities of those branches. Black
squares indicate the new sequences generated for this study.

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de Souza et al.

African genotype and the strains from South American

genotypes I and II was at approximately the year 1640
(95% highest posterior density (HPD) of 14171852)
(not shown).
Further analysis was then performed to focus on the
divergence times of the South American genotype I
strains. For this, sequences of African YFV were omitted
and a data set of prM/E junction sequences (37 taxa) was
reanalyzed in BEAST. The results of this analysis did
not differ much in comparison with those obtained with
all strains. As shown in the estimated MCC tree of
Figure 3, the mean time of origin of the two South
American genotypes was about 1867 (95% HPD of 1773
1943). Considering the Brazilian YFV strains, our
analyses suggested that the Old Para clade arose in
about 1904 (95% HPD of 18511943), while the genetic
diversity of the clade 1 that contains isolates recovered
from 1969 to 2008 was of more recent origin, with the
subgroups 1A to 1E diverging beginning in about 1950
(95% HPD of 19271965). The strain Venezuela98
appears to have been the first registered member of
the new subclade 1E. We estimate that subclade 1E
began to diverge from the other recent Brazilian isolates
about 1975 (95% HPD of 19631985). Within subclade
1E, the Brazilian strains Brazil04, and Brazil08E, F,
and H diverged from the single Venezuelan strain about
1985 (95% HPD of 19801989; Table III).
DISCUSSION
Based on sequences derived from YFV strains recovered from the last 80 years, previous phylogenetic
analyses have shown that the YFV is genetically stable
[Chang et al., 1995; Wang et al., 1996; Mutebi et al.,
2001; Vasconcelos et al., 2004; Bryant et al., 2007]; this
genetic stability was considered to be indicative that
YFV evolves slowly [Barret and Higgs, 2007]. However,
results for Brazilian isolates in the present study
suggest that YFV strains circulating in the country are
diversifying.
Vasconcelos et al. [2004] found that Brazilian YFV
strains isolated from 1969 until 2001 belonged to
South American genotype I, except for one strain
isolated from Rondo nia state in 1983, which belongs
to genotype II. In the epidemics registered in 2008,
nucleotide sequences obtained from YFV from human
or howler monkeys (Alouatta caraya) infected in
distinct geographic regions (Mato Grosso, Sa o Paulo,
and Goia s) clustered together in a strongly supported
new group, here designated subclade 1E, within clade
1 of South American genotype I. The sequences
obtained in 2008 clustered together with other
sequences generated from human symptomatic infections in Amazonas state, Brazil, in 2004, and was also
closely related to the strain Venezuela98 from the
state of Amazon, Venezuela [Bryant et al., 2005].
Results of dating studies carried out in the present
study indicate that this particular virus lineage
evolved in the northern Amazon in the last 33 years
and was first detected in the Brazilian Amazon in
J. Med. Virol. DOI 10.1002/jmv

2004. The most recent 20042008 isolates diverged

about 23 years ago.
Furthermore, our results show that the viral lineages
causing the historic epidemics tend to disappear, and
samples from more recent epidemics belong to new
lineages. In this sense, virus isolates that caused the
2008 epidemics belong to a new lineage that replaced the
former lineages, which then become less frequent or
undetectable. This event corroborates a previous observation by Vasconcelos et al. [2004], when it was noted
that viruses from the Old Para clade had not been
detected since 1968.
Results of several studies showed that the Amazon
region concentrates more sporadic sylvatic cases than
other regions outside the Amazon, and consequently the
epidemics occurred mostly within areas in the endemic
region [Vasconcelos et al., 2001b, 2004]. Since mild and
asymptomatic cases are likely underestimated, we can
speculate that asymptomatic infections can encourage
virus dispersion. Moreover, human activities such as
deforestation and land use can favor contact among
infected semi-domestic and sylvatic mosquitoes, monkeys, and unvaccinated man, causing outbreaks in
zones of virus emergence. In facilitating the involvement of either urban or semi-domestic species of
mosquito in the dynamics of YFV transmission, mans
activities can encourage virus evolution. Similarly,
Bertolotti et al. [2007, 2008] discussed that anthropogenic factors may have affected West Nile Virus (WNV)
evolution in USA. Moreover, these authors suggested
that WNV may evolve at different rates in a cyclic
pattern influenced by the intensity of transmission and
amplification events associated with urbanization and
landscape characteristics affecting both host and vector
populations. YFV and other flaviviruses may show
similar influences, and thus could evolve at faster rates
during epidemics.
It may be that each sporadic case of sylvatic YF, or
each small outbreak occurring throughout an endemic
area, represents an exposure to a different strain of the
virus. Consequently, these virus strains can disperse
and infect non-human primates, mosquitoes, and
humans in areas in which the circulation of the virus
is not endemic. The symptomatic infections registered in
2008 may have been caused by virus strains that
emerged in the Amazon region and thus dispersed
throughout central Brazil. The process of virus dispersion from endemic to non-endemic areas may involve
distinct mosquito species, monkeys, and vaccinated and
unvaccinated humans, causing an increase in the
genetic divergence of the YFV. The introduction of new
virus strains seems to be a continuous, overlapping
process that have been causing low-level virus transmission, and a progressive genetic divergence. Moreover, the intensive human migration and heavy
deforestation caused by the use of land may drive
changes in mosquito species composition, alteration in
mosquito behavior, and an increase in non-human
primate dispersion [Vasconcelos et al., 2001a; Vasconcelos, 2002; Camargo-Neves et al., 2005].

Genetic Diversity of Yellow Fever Virus

183

Fig. 3. Maximum clade credibility (MCC) tree from a Bayesian analysis using a molecular clock model for
the prM/E junction. Numbers above the branches show the posterior probability of those branches.
Numbers to the right of nodes are the estimated dates of divergence. The gray bars represent the 95%
confidence region for those estimates. (The confidence interval for the root is not shown because it is very
big; instead the confidence interval is shown in parentheses.) For the genotype nomenclature, we adopted
the classification of Vasconcelos et al. [2004]. Numbers inside the circles represent the nodes for which
molecular divergence times were estimated as indicated in Table III.

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de Souza et al.
TABLE III. Estimated Molecular Divergence Times for YFV of the Clades Old Para and Subclades of Clade I of the
South American Genotype I

Nodea
1
2
3
4
5
6
7
8
9

Dateb

Clade/subclades
Old Para, clade 1
Old Para
Clade 1
1A (1C, 1D, 1E)
1C (1D, 1E)
Brazil84 (1D, 1E)
1D, 1E
Venezuela98 (Brazil04 Brazil08E, Brazil08F, Brazil08H)
Brazil04 (Brazil08E, Brazil08F, Brazil08H)

1904 (18511953)
1935 (19151950)
1950 (19271965)
1954 (19351967)
1957 (19411969)
1969 (19511981)
1975 (1963-1985)
1985 (19801989)
1997 (19882003)

As indicated in Figure 3.
Estimates of the median time of the most recent common ancestor under relaxed molecular clock using the GTR I G model. 95% HPD
confidence intervals are shown between parentheses.

Another factor that may be affecting the YFV

divergence is the high pressure imposed by the vaccination program. It has been suggested that differences
observed in demographic behavior of Japanese Encephalitis in the last 85 years could be associated with
vaccine that caused a damping effect on the virus
population growth, but also because the constant
population size demographic model changed to be a
low rate of exponential growth in response to the
evolutionary pressure [Twiddy et al., 2003]. We
hypothesize that the virus circulating in endemic areas
during inter-epidemics periods show low genetic divergence causing sporadic cases mainly because the
majority of people are vaccinated and the number of
susceptible non-humans is low. This would occur
throughout the endemic zone; however, it would be
particularly relevant in the Amazon region, where
vaccine coverage is between 80% and 95%, in contrast
to the lower coverage found outside the endemic zone.
The human population in the Amazon region, with high
vaccine coverage, represents a barrier for the virus
spreading and would keep a low level of divergence in
the area. However, when the virus disperses to other
areas with lower vaccine coverage each isolated case or
epidemic becomes an opportunity for divergence and an
increase in genetic heterogeneity. The YF cases detected
in 2008 occurred associated with gallery forests situated
along rivers within Brazilian cerrado eco-region, involving a different fauna of monkeys and Aedini and
Sabethini mosquitoes than those present in the Amazon
region. It is imperative to understand the factors leading
to a faster divergence of the YFV genome and also the
involvement of non-sylvatic mosquito species in the
dynamics of the transmission of YFV in areas outside
the Amazon region.
ACKNOWLEDGMENTS
We thank Edward C. Holmes for helpful hints in the
BEAST analysis and Dr. Celso F.H. Granato for
donating the Brazil04 (SPH258595) sample.
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