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See discussions, stats, and author profiles for this publication at: <a href=https://www.researchgate.net/publication/40043328 Detection of a new yellow fever virus lineage within the South American genotype I in Brazil Article in Journal of Medical Virology · November 2009 DOI: 10.1002/jmv.21606 · Source: PubMed CITATIONS 25 READS 80 18 authors , including: Renato Pereira de Souza Instituto Adolfo Lutz 30 PUBLICATIONS 456 CITATIONS SEE PROFILE Maria Anice Sallum University of São Paulo 205 PUBLICATIONS 1,603 CITATIONS SEE PROFILE Peter Gordon Foster Natural History Museum, London 89 PUBLICATIONS 4,383 CITATIONS SEE PROFILE Gizelda Katz Universidad Adolfo Ibáñez 23 PUBLICATIONS 310 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Landscape as regulator of Culicidae diversity and dynamics of Anopheles vectors in rural settlements with malaria cases in the Brazilian Amazon View project All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Maria Anice Sallum Retrieved on: 01 October 2016 " id="pdf-obj-0-2" src="pdf-obj-0-2.jpg">

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/40043328

Article in Journal of Medical Virology · November 2009

DOI: 10.1002/jmv.21606 · Source: PubMed

CITATIONS

25

READS

80

30 PUBLICATIONS 456 CITATIONS SEE PROFILE

205 PUBLICATIONS 1,603 CITATIONS SEE PROFILE

Some of the authors of this publication are also working on these related projects:

  • Landscape as regulator of Culicidae diversity and dynamics of Anopheles vectors in rural settlements with malaria cases in the Brazilian Amazon View project

All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.

Available from: Maria Anice Sallum Retrieved on: 01 October 2016

Journal of Medical Virology 82:175–185 (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 Fabı´ ola 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 Cecı´ lia L.S. dos Santos 1 *

1

Servic¸o de Virologia do Instituto Adolfo Lutz, Sa˜o Paulo, SP, Brazil Department of Zoology, Natural History Museum, London, United Kingdom Departamento de Epidemiologia, Faculdade de Sau´de Pu´blica, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil 4 Divisa˜o de Zoonoses, Centro de Vigilaˆncia Epidemiolo´gica, Sa˜o Paulo, SP, Brazil

2

3

Nucleotide sequences of two regions of the genomes of 11 yellow fever virus (YFV) samples isolated from monkeys or humans with sympto- matic 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 phylo- genetic analysis showed that sequences gener- ated 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 2004–2008 isolates arose about 1985. J. Med. Virol. 82:175–185, 2010.

2009 Wiley-Liss, Inc.

KEY WORDS: yellow fever virus; subclade 1; premembrane and envelope gene junction; 3 0 non-coding region; molecular clock; diver- gence 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, posi- tive sense RNA genome of about 11 kb in length, arranged into a short 5 0 non-coding region (5 0 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 3 0 non-coding region (3 0 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 mosqui- toes 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 Sa˜ o Paulo/SP; Grant sponsor: FAPESP (to C.L.S.S. and M.A.M.S.); Grant number: 05/53973-0.

*Correspondence to: Cecı´lia L.S. dos Santos, Servic¸o de Virologia do Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, CEP 01246/902 Sa˜ o 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)

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de Souza et al.

Amazonas, Amapa´ , Roraima, Rondoˆ 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´ı, 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 re- urbanization 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, Sa˜ o Paulo, and Parana´ . Five of the recent cases were detected between Decem- ber 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, ve genotypes were identied, 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 rst 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

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 300 400 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 3 0 NCR beginning in the NS5 gene stop codon to the 3 0 terminal of the conserved sequence 2 motif (CS2) present in all mosquito-borne aviviruses [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. Repre- sentative sequences from GenBank of the South Amer- ica 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 Brazil- ian 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 uid from C6/36- infected cells using the QIAmp Viral RNA Extraction Kit (Qiagen, Valencia, CA), according to the manufac- turers 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

Sequence ID

Location/year a

Source

prM/E junction

3 0 NCR

SPH188002

Brazil00B

SP/2000

Human

FJ875515

FJ875521

SPH188057

Brazil00C

SP/2000

Human

FJ875516

FJ875522

SPH258595

Brazil04

AM/2004

Human

FJ875517

FJ875523

SPH287923

Brazil08A

MT/2008

Human

nd

FJ875524

SPH287992

Brazil08B

MS/2008

Human

nd

FJ875525

SPH288116

Brazil08C

GO/2008

Human

nd

FJ875526

SPH288294

Brazil08D

GO/2008

Human

nd

FJ875527

SPAn288183

Brazil08E

SP/2008

Monkey

FJ875518

FJ875528

SPAn288184

Brazil08F

SP/2008

Monkey

FJ875519

FJ875529

SPAn289562

Brazil08G

SP/2008

Monkey

nd

FJ875530

SPAn289568

Brazil08H

SP/2008

Monkey

FJ875520

FJ875531

nd, Not determined. a SP, Sa˜ o Paulo; AM, Amazonas; MT, Mato Grosso; MS, Mato Grosso do Sul; GO, Goia´ s/year of isolation.

(RT)-PCR was performed on puried viral RNA with the SuperScript TM one-step RT-PCR with Platinium R 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 3 0 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 3 0 NCR region that corresponds to the complementary VD8 nucleotide sequence. Thermocycling parameters used for amplication of prM/E junction and 3 0 NCR sequen- ces were those described by Jennings et al. [1994] and Deubel et al. [1997], respectively. The amplied prod- ucts were directly sequenced using the ‘‘ABI PrismR Big DyeM Terminator Cycle Sequencing Ready Reaction Kit’’ (PE Applied Biosystems, Foster City, CA), accord- ing to the manufacturers instructions, in the presence of the primers used in RT-PCR amplication. Sequences were determined using the ABI 377 sequencer (PE Applied Biosystems). Sequences determined in the study are deposited in GenBank under accession numbers FJ875515 FJ875531.

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., Madi- son, WI). This program was also used to determine the end of the NS5 gene and the beginning of the 3 0 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 3 0 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 acces- sion numbers for which prM/E junction and 3 0 NCR

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 Cran- dall, 1998] was employed to choose a model using the Akaike information criterion (AIC). The models sug- gested by Modeltest for prM/E junction and 3 0 NCR were, respectively, GTR þ G þ I and GTR þ G. Bayesian anal- yses were conducted for each of the prM/E junction and 3 0 NCR sequences of YFV separate, and for the con- catenated 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.

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 3 0 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 þ G model for the 3 0 NCR, and the GTR þ I þ 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

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TABLE II. prM/E Junction and 3 0 NCR Sequences Used in This Study

GenBank accession no.

 

Origin/year of

Isolate

Sequence ID

isolation

Source

prM/E junction

3 0 NCR

JSS

Brazil35

Brazil/1935

Human

U52390

BeH111

Brazil54

Brazil/1954

Human

AY540437

AY541335

BeAn23536

Brazil60

Brazil/1960

Cebus spp.

AY540441

AY541338

BeAr44824

Brazil62

Brazil/1962

Haemagogus spp.

AY540443

AY541340

BeH203416

Brazil71

Brazil/1971

Human

AY540449

AY541344

BeAr233436

Brazil73A

Brazil/1973

Haemagogus spp.

AY540452

AY541347

BeH233393

Brazil73B

Brazil/1973

Human

AY540453

AY541348

BeH350698

Brazil78

Brazil/1978

Human

AY541352

BeH379501

Brazil80

Brazil/1980

Human

AY540456

AY541358

BeH413820

Brazil83

Brazil/1983

Human

AY540457

AY566273

BeH 425381

Brazil84A

Brazil/1984

Human

AY540458

BeAr424492

Brazil84B

Brazil/1984

Hg. janthinomys

AY541366

BeH422312

Brazil84C

Brazil/1984

Human

AY541369

BeAr 424083

Brazil84D

Brazil/1984

Hg. albomaculatus

AY540459

BeH511843

Brazil91

Brazil/1991

Human

AY540461

AY541377

BeAr512943

Brazil92A

Brazil/1992

Hg. janthinomys

AY540463

AY541379

BeH512772

Brazil92B

Brazil/1992

Human

AY540465

AY541381

BeAr527198

Brazil94

Brazil/1994

Haemagogus spp.

AY540469

AY541387

BeH 535010

Brazil95

Brazil/1995

Human

AY540471

BeAr 544276

Brazil96A

Brazil/1996

Hg. janthinomys

AY540472

Tennessee

Brazil96B

Brazil/1996

Human

AY540473

BeAn604552

Brazil98A

Brazil/1998

Alouatta belzebul

AY541394

BeH603325

Brazil98B

Brazil/1998

Human

AY541397

BeH605427

Brazil98C

Brazil/1998

Human

AY541398

BeAr614320

Brazil99

Brazil/1999

Haemagogus spp.

AY541399

BeAR628124

Brazil00A

Brazil/2000

Hg. janthinomys

AY540436

AY541328

BeAr630768

Brazil01A

Brazil/2001

Hg. janthinomys

AY541332

BeAr631464

Brazil01B

Brazil/2001

Sa. chloropterus

AY541333

BeAr645693

Brazil01C

Brazil/2001

Haemagogus sp.

AY541334

V528A

Colombia79

Colombia/1979

Human

AY540475

AY541403

Trinidad79

Trinidad79A

Trinidad/1979

Hg. spegazzini

U52420

CAREC 788379

Trinidad79B

Trinidad/1979

Haemagogus

DQ872412

1345

Ecuador81

Ecuador/1981

Human

AY540477

AY541405

OBD 5041

Ecuador97

Ecuador/1997

Human

AY540478

AY541406

614819

Panama74

Panama/1974

Human

AY540479

AY541412

1368

Peru77

Peru/1977

Human

AY161928

AY541414

1899/81

Peru81A

Peru/1981

Human

U52411

1914

Peru81B

Peru/1981

Sentinel mice

AY161933

Peru95(153)

Peru95A

Peru/1995

Human

U52407

ARV 0548

Peru95B

Peru/1995

Human

AY161946

AY541430

03-5350-98

Peru98A

Peru/1998

Human

AY161948

AY541432

IQT 5591

Peru98B

Peru/1998

Human

AY161950

AY541434

OBS7687

Bolivia99A

Bolivia/1999

Human

AY540431

AY541326

OBS8026

Bolivia99B

Bolivia/1999

Human

AY540434

AY541327

35720

Venezuela98

Venezuela/1998

Human

AY540489

AY541443

Asibi

Ghana27

Ghana/1927

Human

AY640589

AY326411

M 90-5

Sudan40

Sudan/1940

Human

AF369692

AY326414

69056

Nigeria46

Nigeria/1946

Human

U52403

U52403

IB AR 45244

Nigeria69

Nigeria/1969

Mosquito

AF369677

H117505

Nigeria87

Nigeria/1987

Human

AF369684

AY541410

Uga

Uganda48A

Uganda48

Human

U52423

ggA 709-4-A2

Uganda48B

Uganda/1948

Aedes africanus

AF369694

STA-LSF-4-4143

Zaire58

Zaire/1958

Human

AF369697

AY541445

Serie 227

Ethiopia61

Ethiopia/1961

Human

AF369674

AY541407

Senegal65

Senegal65A

Senegal/1965

Human

U52414

SH 1446

Senegal65B

Senegal/1965

Human

AF369687

Ar B 8883

CAR77A

Central African

Ae. africanus

U52392

U52393

 

Republic/1977

Ar B 9005

CAR77B

Central African

Ae. africanus

U52395

U52396

 

Republic/1977

85-82H

IvoryCoast82

Ivory Coast/1982

Human

U54798

U54798

IvoryC1999

IvoryCoast99

Ivory Coast/1999

Human

AY603338

Genetic Diversity of Yellow Fever Virus

179

nal 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 protein- coding gene, the entire 225 nucleotides of M protein- coding gene, and the rst 337 nucleotides of E protein- coding 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 amplied 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, DNAS- TAR, Inc.). 3 0 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 3 0 NCR sequences with the corresponding 3 0 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 3 0 NCR (not shown), which comprises the rst and second repeated hairpin motifs identied 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 3 0 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 3 0 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 3 0 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 3 0 NCR sequence data or the prM/E junction sequence data. The new subclade is here designated subclade 1E, when following the classica- tion 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 1998 1999, and from the states of Tocantins, Goia´ s, Bahia, and Minas Gerais isolated in 2000 2001. YFV from Para isolated in 1971, 1987, and

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 3 0 NCR data sets. The strains from the northern and central states, including Brazil94 and Brazil96A (Rondonia), Bra- zil96B (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 (Goia´ s), 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 3 0 NCR sequences of the strain Colombia79, were not resolved. When using the prM/E junction fragments, the strains identied 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 3 0 NCR, relationships among sequences Brasil54, Bra- sil62, 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 classied in the South American genotype II. Finally, the phylogenetic analyses of the prM/E junction and 3 0 NCR sequences of the African strains included in the study identied 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 3 0 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 con- catenated data Brazilian strains Brazil08E, Brazil08F, and Brazil08H clustered together in a strongly sup- ported 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 3 0 NCR. Sequences were analyzed using BEAST, using the uncorrelated log-normal relaxed clock model [Drum- mond 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

180 de Souza et al.
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de Souza et al.

Fig. 1. Bayesian analysis of the prM/E junction sequences. This analysis used the GTR þ G þ 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-burn- in 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.

Genetic Diversity of Yellow Fever Virus

181

Genetic Diversity of Yellow Fever Virus 181 Fig. 2. Bayesian analysis of the 3 NCR sequences.

Fig. 2. Bayesian analysis of the 3 0 NCR sequences. Conditions were as described in Figure 1, using the GTR þ 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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African genotype and the strains from South American genotypes I and II was at approximately the year 1640 (95% highest posterior density (HPD) of 1417 1852) (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 1851 1943), 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 1927 1965). The strain Venezuela98 appears to have been the rst 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 1963 1985). Within subclade 1E, the Brazilian strains Brazil04, and Brazil08E, F, and H diverged from the single Venezuelan strain about 1985 (95% HPD of 1980 1989; Table III).

DISCUSSION

Based on sequences derived from YFV strains recov- ered 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 Rondoˆ 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 infec- tions 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 rst detected in the Brazilian Amazon in

2004. The most recent 2004 2008 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 obser- vation 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, mon- keys, and unvaccinated man, causing outbreaks in zones of virus emergence. In facilitating the involve- ment 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 anthropo- genic 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 inuenced by the intensity of transmission and amplication events associated with urbanization and landscape characteristics affecting both host and vector populations. YFV and other aviviruses may show similar inuences, 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 disper- sion 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 trans- mission, and a progressive genetic divergence. More- over, 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; Vascon- celos, 2002; Camargo-Neves et al., 2005].

Genetic Diversity of Yellow Fever Virus

183

Genetic Diversity of Yellow Fever Virus 183 Fig. 3. Maximum clade credibility (MCC) tree from a

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% condence region for those estimates. (The condence interval for the root is not shown because it is very big; instead the condence interval is shown in parentheses.) For the genotype nomenclature, we adopted the classication 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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TABLE III. Estimated Molecular Divergence Times for YFV of the Clades Old Para and Subclades of Clade I of the South American Genotype I

Node a

Clade/subclades

Date b

  • 1 Old

Para, clade 1

1904

(1851 1953)

  • 2 Old

Para

1935

(1915 1950)

  • 3 Clade 1

 

1950

(1927 1965)

  • 4 1D, 1E)

1A

(1C,

1954

(1935 1967)

  • 5 1C (1D, 1E)

1957

(1941 1969)

  • 6 Brazil84 (1D, 1E)

1969

(1951 1981)

  • 7 1D, 1E

1975 (1963-1985)

  • 8 Venezuela98 (Brazil04 Brazil08E, Brazil08F, Brazil08H)

1985

(1980 1989)

  • 9 Brazil04 (Brazil08E, Brazil08F, Brazil08H)

1997

(1988 2003)

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

Another factor that may be affecting the YFV divergence is the high pressure imposed by the vacci- nation program. It has been suggested that differences observed in demographic behavior of Japanese Ence- phalitis 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 diver- gence 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, involv- ing 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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