Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates

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Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates Hye-Lim Ju a,1 , Jung-Mi Kang a,1 , Sung-Ung Moon b , Young-Yil Bahk c , Pyo-Yun Cho d , Woon-Mok Sohn a , Yun-Kyu Park d , Jae-Won Park e,2 , Tong-Soo Kim d,∗ , Byoung-Kuk Na a,∗∗ a

Department of Parasitology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Republic of Korea Department of Pathology, College of Medicine, Korea University, Seoul 136-705, Republic of Korea c Department of Biotechnology, College of Biomedical and Health Sciences, Konkuk University, Chungju 380-701, Republic of Korea d Department of Parasitology and Inha Research Institute for Medical Sciences, Inha University School of Medicine, Incheon 400-712, Republic of Korea e Department of Microbiology, Graduate School of Medicine, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 10 February 2012 Received in revised form 19 September 2012 Accepted 22 September 2012 Available online xxx Keywords: Plasmodium vivax Duffy binding protein Korea Genetic polymorphism Natural selection

a b s t r a c t Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005–2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Erythrocyte invasion by malaria parasites is an essential step for parasite survival (Chitnis, 2001). Plasmodium vivax triggers erythrocyte invasion through expression of several surface and apical proteins on the merozoite which recognize and bind to receptor proteins on the erythrocyte surface (Barnwell et al., 1989; Adams et al., 1992). P. vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein expressed on the merozoite of P. vivax and plays an essential role in erythrocyte invasion of the parasite through irreversible binding with its corresponding receptor, the Duffy Antigen Receptor for Chemokines (DARC), on the surface

∗ Corresponding author. ∗∗ Corresponding author. Tel.: +82 55 772 8102; fax: +82 55 772 8109. E-mail addresses: [email protected] (T.-S. Kim), [email protected] (B.-K. Na). 1 These authors equally contributed in this study. 2 Deceased.

of erythrocytes (Barnwell et al., 1989; Wertheimer and Barnwell, 1989; Adams et al., 1992; Horuk et al., 1993). Several lines of experimental evidence suggest that PvDBP is an important vaccine candidate antigen. It elicits strong immune responses in naturally infected humans (Michon et al., 1998; Xainli et al., 2002, 2003; Tran et al., 2005), and the antibodies against PvDBP effectively inhibit the interaction of this protein with DARC in vitro and block the invasion of P. vivax into human erythrocytes (Michon et al., 2000; Grimberg et al., 2007; Cerávolo et al., 2008; King et al., 2008; SouzaSilva et al., 2010). Although several recent studies have reported the transmission of P. vivax to a Duffy negative population (Ryan et al., 2006; Dhorda et al., 2011; Mendes et al., 2011), which suggests that P. vivax could have certain alternative invasion pathways, no other invasion pathway has been clearly identified. PvDBP is a high molecular weight protein of 140 kDa and is segmented into 7 different regions (regions I–VII). The critical erythrocyte-binding motif of PvDBP is mapped into a 170 amino-acid stretch of the N-terminal cysteine-rich region (PvDBPII) (Chitnis et al., 1996; Ranjan and Chitnis, 1999). As like many other

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Please cite this article in press as: Ju, H.-L., et al., Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates. Acta Trop. (2012), http://dx.doi.org/10.1016/j.actatropica.2012.09.016

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plasmodial proteins, PvDBP also shows a high degree of genetic polymorphism and the most polymorphic residues are clustered in PvDBPII (Cole-Tobian et al., 2002; Cole-Tobian and King, 2003). The cysteine and some hydrophobic amino acid residues in PvDBPII are well conserved within and between P. vivax populations from different geographic regions, but the remaining amino acid residues are highly polymorphic (Ampudia et al., 1996; Xainli et al., 2000; Kho et al., 2001; Cole-Tobian and King, 2003; Sousa et al., 2006; Gosi et al., 2008; Babaeekho et al., 2009; Sousa et al., 2010; Ju et al., 2012). Although it seems likely that these polymorphisms do not affect the capacity to bind DARC (Xainli et al., 2000; VanBuskirk et al., 2004a), some of them may alter immune recognition of PvDBP (Tsuboi et al., 1994; Michon et al., 2000; Cole-Tobian and King, 2003; VanBuskirk et al., 2004b). It has been suggested that PvDBPII undergoes positive natural pressure, which may induce allelic variation as a mechanism for immune evasion (Sousa et al., 2010; Premaratne et al., 2011; Ju et al., 2012). Since antigenic variation and the associated strain specific immunity present major limitation in successful design of effective vaccine, characterization of the nature and genetic polymorphism in PvDBPII among P. vivax isolates from different geographic regions is important for the rational design of vaccines against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection of PvDBPII in 70 P. vivax Korean isolates collected between 2005 and 2010. Our results suggest that a limited level of genetic polymorphism of PvDBPII is found among the filed isolates of P. vivax in Korea and the protein region is under natural selection. 2. Methods 2.1. Blood samples and DNA preparation The 70 blood samples used in this study were collected from patients who were infected with P. vivax in Korea between 2005 and 2010 (2005–2008, n = 10 for each year; 2009 and 2010, n = 15 for each year). All the patients inhabited in malaria endemic areas, Ilsan, Kimpo or Yonchon, and have not been abroad at least in recent 2 years when their blood samples were collected. P. vivax infection was confirmed by microscopic examination of thin and thick blood smears and polymerase chain reaction (Moon et al., 2009). Blood collections were made with full informed consent of the patients and following institutional ethical guidelines that were reviewed and approved by the Ethics committee of Gachon University of Medicine and Science or Inha University School of Medicine. Genomic DNA was isolated from 200 ␮l of whole blood using a QIAamp Blood kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. 2.2. Amplification and sequencing analysis of PvDBPII The PvDBPII region was amplified by the polymerase chain reaction (PCR) using the specific primers, PvDBPII 5 -ACCACGATCTCTAGTGCTATTATA-3 and PvDBPII R: F: 5 -ATTTGTCACAACTTCCTGAGTATT-3 (Ju et al., 2012). The amplification reaction was performed using the following thermal cycling conditions: 94 ◦ C for 5 min, 30 cycles at 94 ◦ C for 1 min, 50 ◦ C for 1 min, and 72 ◦ C for 1 min, followed by a 72 ◦ C extension for 10 min. Ex Taq DNA polymerase (Takara, Otsu, Japan) was used in all PCR reactions to reduce possible nucleotide mis-incorporation. The PCR product was analyzed on a 1.2% agarose gel, gel-purified, and ligated into the T&A cloning vector (Real Biotech Cooperation, Banqiaa City, Taiwan). Each ligation mixture was transformed into Escherichia coli DH5␣ competent cells, and positive clones were selected with colony PCR for the presence of plasmid with the appropriate insert. The nucleotide sequences of cloned insert

were analyzed by automatic sequencing. To verify the sequences, at least 2 clones from each isolate were sequenced in both directions. The nucleotide sequences reported here have been deposited in the GenBank database under the accession numbers JN989472–JN989484. 2.3. Sequence and phylogenetic analyses Nucleotide and deduced amino acid sequences of the cloned PvDBPII were analyzed using the EditSeq program and Clustal in the Megalign program, a multiple alignment program of the DNASTAR package (DNASTAR, Madison, WI, USA). The phylogeny tree was constructed using the neighbor-joining method with MEGA4 version 4.0 (Tamura et al., 2007). Bootstrap proportions were used to assess the robustness of the tree with 1000 bootstrap replications. 2.4. DNA sequence polymorphism analysis DNA sequence polymorphism analysis was performed on 70 Korean PvDBPII sequences. The number of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (), and average number of pairwise nucleotide differences within the population (K) were estimated using the DnaSP ver. 5.10.00 (Librado and Rozas, 2009). The  was also calculated on a sliding window of 100 bases, with a step size of 25 bp to estimate the stepwise diversity across PvDBPII. The rates of synonymous (Ks ) and non-synonymous (Kn ) substitutions were estimated and compared by the Z-test (P < 0.05) in the MEGA4 program (Tamura et al., 2007) using the Nei and Gojobori’s method (Nei and Gojobori, 1986) with the Jukes and Cantor correction. Tajima’s D test (Tajima, 1989) was performed with DnaSP ver. 5.10.00 to test the neutral theory of evolution. 2.5. Analysis of polymorphism associated with B- and T-cell epitopes and MHC binding regions To assess the possibility that diversity in PvDBPII within the Korean P. vivax isolates may have been associated with the host’s immune pressure, we examined the genetic diversity in identified or predicted B- and T-cell epitopes and MHC binding regions in PvDBPII (Sousa et al., 2010). Polymorphism of each region was analyzed by DnaSP ver. 5.10.00 (Librado and Rozas, 2009), as described above. 2.6. Recombination parameters and linkage disequilibrium The recombination parameter (R), which included the effective population size and probability of recombination between adjacent nucleotides per generation, and the minimum number of recombination events (Rm ) were measured using DnaSP ver. 5.10.00 (Librado and Rozas, 2009). Linkage disequilibrium (LD) between different polymorphic sites was computed in terms of the R2 index using DnaSP ver. 5.10.00 (Librado and Rozas, 2009). 3. Results 3.1. Genetic polymorphisms and amino acid changes Nucleotide sequence analysis of PvDBPII among 70 P. vivax Korean isolates revealed that there was no size variation among the sequences and that 17 single nucleotide polymorphisms (SNP) were found compared to the reference sequence of Sal I (DQ156512). Five of these 17 SNP (14 non-synonymous and 3 synonymous mutations) occurred at the first base of the codon, 7 at the second base, and 5 at the third base of the codon, resulting in amino acid changes through the PvDBPII among Korean isolates. Most

Please cite this article in press as: Ju, H.-L., et al., Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates. Acta Trop. (2012), http://dx.doi.org/10.1016/j.actatropica.2012.09.016

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B

299 320 371 381 384 390 396 417 420 424 437 474 493 503

A Sal I

V KK FD RN NWLWV N I

Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4 Haplotype 5 Haplotype 6 Haplotype 7 Haplotype 8 Haplotype 9 Haplotype 10 Haplotype 11 Haplotype 12 Haplotype 13

V V I V V V V V V V V V V

K K K K K K K I K K K K K

K K K K K K E E E E K E E

L F F F F F F F F F F F F

G G G G G G G G G G D G G

H H H H H H H H H H R H H

N N N N S N N N N N N N N

NW NW NW N R NW NW KW KW KW KW KW KW KW

I I I I I I I I I I I I I

WV WV WV WV WV WV RA R V R V R V R V R V WV

N N N N N N N N S N N N N

I I I I I I K K K K K I K

3

Total 2 29 1 3 2 2 3 2 2 15 7 1 1

V299I K320I K371E F381L D384G R390H N396S N417K W420R L424I W437R V474A N493S I503K 0

25

50

75

100

Frequency (%)

Fig. 1. Sequence polymorphism of PvDBPII in P. vivax Korean isolates. (A) The changes in amino acid sequences. Polymorphic amino acid residues are listed for each haplotype. Amino acid residues identical to those of the reference sequence, Sal I (DQ156512), are marked in yellow. The dimorphic amino acid changes are marked in blue. Total number of sequences for each haplotype is listed in right panel. (B) Frequencies of amino acid changes found in PvDBPII among Korean isolates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of the non-synonymous polymorphisms were non-conservative, resulting in changes in the physico-chemical family of the respective amino acid. An analysis of the deduced amino acid sequences classified them into 13 different haplotypes (haplotypes 1–13) with amino acid changes at 14 positions, in which all were dimorphic (Fig. 1A). Seven of the 14 non-synonymous changes were previously identified, whereas the other 7 changes (V299I, K320I, F381L, N396S, W420R, V474A, and N493S) were unique in Korean isolates, which were not hitherto reported. No mutation was observed at position 306 among all Korean isolates and all the cysteine residues were well conserved. Haplotype 2 was predominant (n = 29, 41.4%) among the Korean isolates. Haplotypes 2 and 6 encoded the same amino acid sequences, but they differed in nucleotide sequences: haplotype 6 had a synonymous mutation on D461 (GAT to GAC). High frequencies of variant amino acids (>50%), compared to the Sal I sequence, were found for D384G (63/70, 90.0%), R390H (63/70, 90.0%), and L424I (70/70, 100.0%) (Fig. 1B). A comparison of the most common amino acid changes in PvDBPII among presently studied P. vivax populations revealed that Korean isolates showed a different pattern compared to isolates from other geographical regions (Table 1). R308S, E385K, and K386N were not identified among Korean isolates, but the frequencies of D384G, H390R, and L424I in Korean isolates were higher than those in P. vivax isolates

from other geographical regions. Interestingly, the frequencies of the amino acid changes found in 70 Korean isolates analyzed here were significantly different from those of previous studies that analyzed genetic diversity of PvDBPII from Korean P. vivax isolates collected at early re-emergence period (Table 1). A BLAST search used the GenBank database was done to compare the Korean P. vivax haplotypes with the previously identified PvDBPII sequences and showed that 11 of 13 (except haplotypes 1 and 8) Korean PvDBPII haplotypes were novel. Phylogenetic analysis revealed that the 13 Korean PvDBPII haplotypes were clustered into 3 different clades, SK-1, SK-2, and SK-3 (Fig. 2). SK-3, which includes haplotypes 1–6, was the most prevalent (39/70, 55.7%). 3.2. Nucleotide diversity and natural selection of PvDBPII DNA sequence analyses were conducted to determine nucleotide diversity and genetic differentiation of PvDBPII among the Korean P. vivax isolates. The average number of pairwise nucleotide differences (K) for the 963 bp of the PvDBPII region was 2.87826 (Table 2). The overall haplotype diversity (Hd) and nucleotide diversity () for all 70 sequences were 0.775 ± 0.040 and 0.00299 ± 0.00018, respectively (Table 2). The  values for isolates collected in each year were analyzed separately, and

Table 1 Comparison of most common variant amino acids in PvDBPII among P. vivax populations. Frequency

Korea Myanmara Thailandb Iranc Sri Lankad Papua New Guineae Colombiae Brazile South Koreae

R308S

K371E

D384G

E385K

K386N

H390R

N417K

L424I

W437R

I503K

0 22.2 26.7 6.6 13.0 69.0 0 7.3 0

34.3 22.2 20.0 17.3 34.0 11.5 17.6 26.0 46.7

90.0 85.2 76.7 61.3 94.0 34.5 41.2 18.7 46.7

0 33.3 46.6 6.7 20.0 9.7 17.6 20.3 6.7

0 33.3 40.0 6.6 20.0 9.7 17.6 22.8 6.7

90.0 63.0 56.6 41.3 66.0 50.4 94.1 50.4 53.3

44.3 38.9 36.6 44.0 36.0 33.6 41.2 39.8 93.3

100 83.3 86.7 50.6 49.0 68.1 41.2 48.0 100

42.9 61.1 63.3 45.3 37.0 32.7 11.8 48.8 100

42.9 77.8 56.7 70.6 55.0 42.5 5.9 43.1 100

The first letter represents the amino acid in that position in Sal I sequence, and the other letter represents the substituted amino acid. a Ju et al. (2012). b Gosi et al. (2008). c Babaeekho et al. (2009). d Premaratne et al. (2011). e Sousa et al. (2011).

Please cite this article in press as: Ju, H.-L., et al., Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates. Acta Trop. (2012), http://dx.doi.org/10.1016/j.actatropica.2012.09.016

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0.00071 0.00299 ± 0.00018 14 70

2

14

16

2.87826

13

0.775 ± 0.040

0.00352

Ks Kn  ± SD Hd ± SD H K Total no. of mutations Parsimony informative sites Singleton variable sites Segregating sites (S) Total no. of isolates

Table 2 Estimates of DNA sequence polymorphism and tests of neutrality at PvDBPII among Korean isolates.

Fig. 2. Phylogenetic analysis. The phylogenetic tree for the 13 haplotypes of PvDBPII was constructed with a neighbor-joining method using the MEGA4 program. Numbers on the branches indicate bootstrap proportions (1000 replicates).

−0.39146 (P > 0.1)

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Tajima’s D

4

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ARTICLE IN PRESS K, average number of pairwise nucleotide differences; H, number of haplotypes; Hd, haplotype diversity; , observed average pairwise nucleotide diversity; Kn , rate of non-synonymous mutations; Ks , rate of synonymous mutations. a B, B-cell epitope; T, T-cell epitope; None, no epitope identified.

−1.06734 (P > 0.1) 0 0 0 −0.84077 (P > 0.1) −0.62049 (P > 0.1) −0.70113 (P > 0.1) 0 0 0.67187 (P > 0.1) 0 1.72580 (P > 0.1) 1.74802 (P > 0.05) −0.74135 (P > 0.05) 0 0 0 0 0.125 0 0 0 0 0 0 0 0 0 0.00070 0 0 0 0 0 0.00638 0 0 0.02562 0. 0.02403 0.02158 0.00334 0.00630 ± 0.00061 0 0 0 0.00211 ± 0.00135 0.00937 ± 0.00264 0.00531 ± 0.00149 0 0 0.02162 ± 0.00167 0 0.01840 ± 0.00073 0.01669 ± 0.00073 0.00308 ± 0.00164 0.029 ± 0.028 0 0 0 0.095 ± 0.061 0.233 ± 0.063 0.233 ± 0.063 0 0 0.545 ± 0.025 0 0.497 ± 0.02 0.501 ± 0.017 0.083 ± 0.044 T/B T/B B T/B None B B None T MHCIa MHCIb MHCIc MHCIIa MHCIIb 5 16 18 20 28 45 48 54 66 Ia Ib Ic IIa IIb

1 0 0 0 1 3 2 0 0 2 0 1 1 1

1 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 1 3 2 0 0 2 0 1 1 1

1 0 0 0 1 3 2 0 1 2 0 1 1 1

0.029 0 0 0 0.095 0.422 0.239 0 0 0.584 0 0.497 0.501 0.083

2 1 1 1 2 3 3 1 1 3 1 2 2 2

 ± SD H Total no. of mutations Parsimony informative sites Singleton variable sites Segregating sites (S) Epitopea Epitope name

Table 3 Polymorphism observed in the each epitope sequence.

ranged from 0.00214 to 0.00388. A sliding window plot analysis (window length 100 bp, step size 25 bp) using the DnaSP package revealed that  ranged from 0 to 0.0108. The 3 highest peaks of  within the PvDBPII region were identified between nucleotide positions 350 and 575 (Fig. 3A). To examine whether natural selection contributed to generation of this diversity in PvDBPII within the Korean P. vivax population, the rate of non-synonymous (Kn ) to synonymous substitutions (Ks ) was estimated using the Nei and Gojobori’s method (Nei and Gojobori, 1986). The Kn (0.00352) exceeded Ks (0.00071) and the Kn /Ks ratio was 4.958, suggesting that a positive natural selection may be occurring in the PvDBPII of Korean P. vivax isolates (Table 2). We performed Tajima’s D test to more closely explore natural selection in PvDBPII. The Tajima’s D value was estimated to be −0.39146 (P > 0.1), which indicated population size expansion and/or purifying selection. The association between natural selection and host immune pressure was also evaluated by examining the genetic polymorphism in identified or predicted B- and T-cell epitopes and MHC binding regions (Sousa et al., 2010). Seven of 12 tested B- and T-cell epitopes and MHC binding regions were polymorphic, and contained nucleotide diversities (Table 3). Particularly, the high levels of  were predicted in epitopes Ia, Ic, and IIa, which contain 3 polymorphic residues at positions 417, 424, and 503. The Tajima’s D values for the 3 epitopes were all positive, indicating a decrease in population size and/or balancing selection.

K

Fig. 3. Natural selection of PvDBPII. (A) Sliding window plot of nucleotide diversity per site () comparing the level of genetic diversity at PvDBPII. The  values were calculated on DnaSP with a window length of 100 bp and step size of 25 bp. (B) The linkage disequilibrium (LD) plot showing non-random association between nucleotide variants in 70 Korean P. vivax isolates at different polymorphic sites. The R2 values are plotted against the nucleotide distances with two-tailed Fisher’s exact test of significance.

5

Hd ± SD

Kn

Ks

Tajima’s D

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3.3. Recombination The minimum number of recombination events between adjacent polymorphic sites (Rm ) was 7, whereas R between adjacent sites (Ra ) and per gene (Rb ) was 0.0314 and 30.2, respectively. The high value of the recombination parameters suggested that meiotic recombination may occur between sites, resulting in genetic diversity in the PvDBPII. The LD index R2 also declined across the analyzed region, suggesting that intragenic recombination may also be a possible factor contributing the increased diversity of PvDBPII (Fig. 3B).

4. Discussion Tertian malaria caused by P. vivax infection is one of the most important health-threatening infectious diseases in Korea. Since its re-emergence in 1993 (Chai et al., 1994), the outbreak has persisted until now, with increasing or decreasing annual numbers of indigenous cases, and a total case number of up to 30,600. During the early period of re-emergence, most malaria cases were reported among military personnel, and the geographic distribution was highly restricted to the Demilitarized Zone (DMZ) and areas adjacent to the DMZ where no civilians reside (Park et al., 2003). However, despite the significant decrease of malaria cases among military personnel, that is mainly due to aggressive chemoprophylaxis to control malaria, the numbers of malaria cases have been increasing among the civilian population and the geographic distribution also expanding into adjacent cities and counties (Park et al., 2009), which indicates settlement of local transmission. In this study, we analyzed genetic polymorphism and natural selection of PvDBPII among 70 Korean P. vivax isolates collected during 2005–2010 to understand the nature and genetic polymorphism of P. vivax Korean isolates. Sequence variation in PvDBPII among P. vivax isolates that were collected during the early period of re-emergence (1996–1998) in Korea has been previously analyzed (Kho et al., 2001; Suh et al., 2001); however, our results are significantly different from those of the 2 previous studies. Sequence analysis revealed that a total of 17 SNP were identified at 17 positions in PvDBPII of the 70 Korean isolates and the sequences were classified into 13 different haplotypes; 11 of them were novel and had not been previously reported. Among the 17 SNP, 14 were non-synonymous mutations that resulted in amino acid changes and the other 3 were synonymous mutations. Seven of the 14 non-synonymous changes were previously reported, whereas the other 7 changes (V299I, K320I, F381L, N396S, W420R, V474A, and N493S) were unique to Korean isolates, which had not been previously identified. The 7 most commonly identified substitutions among P. vivax populations, K371E, D384G, R390H, N417K, L424I, W437R, and I503K, were also found both in Korean isolates analyzed in this study and in 2 previous studies. L424I was identified in all the Korean isolates, but the frequencies of other substitutions were different between this study and the 2 previous studies. Two substitutions, E385K and K386N, which were identified with low frequencies in previous studies, were not found in our study. Moreover, the substitutions, R287I, Q289H, L290F, K293S, E294D, L295H, D303Y, I322V, E328K, L335S, R348T, T362A, M364L, G368R, K371N, E374Q, I380V, K386Q, Q389H, R391H, R391P, Q393P, W394L, W395L, Y445S, Q454P, S504R, Y520N, E526K, E538G, and N560K, which were previously reported in Korean isolates (Suh et al., 2001), were not identified in the isolates analyzed in this study. Interestingly, F306L, which has only been reported in Asian isolates, including isolates from Thailand (Gosi et al., 2008), Iran (Babaeekho et al., 2009), Sri Lanka (Premaratne et al., 2011), and Myanmar (Ju et al., 2012), was not identified in any of the Korean isolates. Although polymorphic

residues were widely distributed throughout the PvDBPII sequence, polymorphisms at residues 417, 437, and 503, either in single or in multiple combinations, can affect the efficacy of inhibitory antibodies against erythrocyte binding (VanBuskirk et al., 2004b; Hans et al., 2005; McHenry et al., 2011). As these residues form a critical discontinuous epitope in PvDBP, which might be the main target for inhibitory antibodies, these polymorphisms could be subject to immune pressure responsible for parasite escape from the host immune system. This strong immune pressure forces greater diversity of PvDBP by generating new PvDBP variants that are able to bind erythrocytes, but become resistant to inhibitory antibodies (ColeTobian and King, 2003; VanBuskirk et al., 2004a,b; Hans et al., 2005; Sousa et al., 2010). Although the variants N417K (44.3%), W437R (42.9%), and I503K (42.9%) were identified with lower frequencies when compared to other variations, all 3 variations were commonly found in 29 isolates (41.4%), which suggests a strong association between N417K, W437R, and I503K is found in PvDBPII of Korean P. vivax isolates. Phylogenetic analysis revealed that the 70 Korean PvDBPII sequences are clustered into 3 distinct clades, which differs from a previous study showing that Korean isolates are clustered into 2 groups, SK-1 and SK-2 (Kho et al., 2001). Our result clearly showed that a new group, namely SK-3, is found in Korean isolates. Moreover, SK-3 (39/70, 55.7%) is more prevalent than SK-1 (7/70, 10.0%) and SK-2 (24/70, 34.3%), which also differed from result of the previous study (SK-1, 33.3% and SK-2, 66.6%) (Kho et al., 2001). Considering the high rate of recombination rate of Korean isolates, intragenic recombination may influence in emergence of new haplotypes. Polymorphic analyses based on PvDBPII sequences from P. vivax Korean isolates collected at an early phase of re-emergence suggested that PvDBPII of Korean isolates showed similar or higher level of genetic polymorphism comparable to other malaria endemic countries, including Papua New Guinea and Colombia (Cole-Tobian and King, 2003; Sousa et al., 2011). Although an accurate intensity of malaria transmission in Korea has not been determined, it seems likely that the intensity might be strictly limited, because transmission is restricted to the summer season (May to October) and the transmission ability of mosquito vector seems to be low (Sim et al., 1997; Ree, 2000; Park et al., 2009). Considering these epidemic characteristics of malaria, the genetic change may occur more slowly in Korea compared to other highendemic areas. The Hd and  for all 70 Korean PvDBPII sequences were 0.775 ± 0.040 and 0.00299 ± 0.00018, respectively, and these values are much lower than those of previous studies that predicted using the previously reported sequences (Cole-Tobian and King, 2003; Sousa et al., 2011). The 3 highest peaks of  within PvDBPII in 70 Korean P. vivax isolates were identified between C5 and C7, which was consistent with previous observations (Tsuboi et al., 1994; Xainli et al., 2000; Cole-Tobian and King, 2003). These results imply certainty that PvDBPII in Korean isolates shows a polymorphic nature, but the diversity is much lower than previously reported. The ratio of Kn and Ks is a useful indicator for the action of natural selection in most coding gene sequences. Previous studies on PvDBPII diversity indicate that the high Kn relative to Ks reflects positive selection pressure (Ampudia et al., 1996; Xainli et al., 2000; Cole-Tobian and King, 2003; Martinez et al., 2004; Premaratne et al., 2011). The positive value of Kn /Ks (4.958) for 70 PvDBPII sequences suggest that the region in Korean P. vivax isolates is under positive natural selection. The direction of Tajima’s D is potentially informative concerning the evolutionary and demographic forces that a population has experienced. The negative value of Tajima’s D (−0.39146, P > 0.10) for PvDBPII of Korean isolates implies an excess of rare polymorphisms in the population, which indicates population size expansion and/or purifying selection (Tajima, 1989). Polymorphism in B- and T-cell epitopes and MHC binding regions of

Please cite this article in press as: Ju, H.-L., et al., Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates. Acta Trop. (2012), http://dx.doi.org/10.1016/j.actatropica.2012.09.016

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parasite antigens may well enable parasites to escape host immune responses, because a polymorphism in the epitopes can up- or down-regulate T-cell responses to the index peptide or completely arrest an immune response, thus assisting escape of the parasite from the host immune system (Tanabe et al., 2007; Sousa et al., 2010). High scores of Hd and  and a high ratio of Kn /Ks were found in the regions that are identified or predicted B- and T-cell epitopes and MHC binding regions of Korean PvDBPII sequences. Meanwhile, two peptides (28 and 54), which did not contain known epitopes (Cole-Tobian et al., 2003), had lower nucleotide diversity. Overall  values for these epitopes were also greater than that for the entire PvDBPII. In particular, high scores of  were identified in peptides Ia, Ic, and IIa, which are predicted to be exposed to the surface of the PvDBP molecule (Sousa et al., 2010). The putative changes in protein structure may alter antibody binding efficacy of a particular epitope, thereby allowing escape from the host protective immune response (Cole-Tobian et al., 2002; Sousa et al., 2010). Positive Tajima’s D values for these epitopes also suggest that a balancing natural selection preferentially acted on the epitopes in PvDBPII of Korean isolates. These results collectively suggest that strong natural selection, probably by host immune selection pressure, occurs at PvDBPII in the Korean isolates. Many factors may contribute to genetic diversity in malaria populations, including mutations, intragenic recombination, natural selection, gene flow between different regions, and population size. Although it remains controversial, recombination is likely to be an important contributor to diversity in P. vivax and may be a critical source of variation in the PvDBPII (Martinez et al., 2004; Sousa et al., 2010). The existence of recombination events and the decline in the LD with increasing distance between nucleotide sites suggest that meiotic recombination may also contribute to maintain the diversity of PvDBPII among Korean P. vivax isolates, similar to previous reports in isolates from Brazil, Colombia, Myanmar, and Sri Lanka (Sousa et al., 2010; Premaratne et al., 2011; Ju et al., 2012). In conclusion, our results suggest that the PvDBPII of Korean P. vivax isolates shows a polymorphic nature and the population is under natural selection, which is similar to results from 2 previous studies on genetic variation of PvDBPII in Korean isolates (Kho et al., 2001; Suh et al., 2001). However, our results are significantly different from those of the 2 previous studies in that PvDBPII of P. vivax Korean isolates showed a limited genetic polymorphism at restricted positions. Furthermore, the PvDBPII of P. vivax Korean isolates showed a lower genetic diversity than found in previous studies that analyzed genetic diversity of PvDBPII based on previously reported sequences (Cole-Tobian and King, 2003; Sousa et al., 2011). Considering that tertian malaria has been transmitted in highly restricted and narrow areas of Korea in summer season (Park et al., 2003, 2009), it seems not likely that these differences were induced by geographical differences for blood collections and annual changes of mosquito vectors. It may have resulted from artifacts in sequencing techniques (e.g. PCR or sequencing errors) in the 2 previous studies, because the Korean isolates were sequenced directly from PCR products without cloning. Moreover, the numbers of samples analyzed in the previous studies were too small to fully understand the genetic polymorphism of PvDBPII among Korean isolates. Recent studies on several major antigens, including circumsporozoite protein, merozoite surface protein-1 (MSP-1), and MSP-3␣, and microsatellite loci suggest that P. vivax Korean isolates were genetically homologous until 2000, but genetic diversity was rapidly disseminated thereafter (Choi et al., 2010; Honma et al., 2011; Kang et al., 2012; our unpublished data). It is not certain whether these patterns would be found in PvDBPII. However, our results clearly suggest that the polymorphic nature of PvDBPII among recent Korean isolates is differs from that among the isolates in an early phase of re-emergence, which indicates that possible genetic change, could occur in PvDBPII of the Korean P. vivax

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population. Therefore, more careful approaches using larger numbers of samples covering all epidemic years from re-emergence to the current time would be necessary to provide an in-depth understanding of the nature and genetic polymorphism of P. vivax Korean isolates. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (2011-0028135). References Adams, J.H., Sim, B.K., Dolan, S.A., Fang, X., Kaslow, D.C., Miller, L.H., 1992. A family of erythrocyte binding proteins of malaria parasites. Proceedings of the National Academy of Sciences of the United States of America 89, 7085–7089. Ampudia, E., Patarroyo, M., Patarroyo, M., Murillo, L., 1996. Genetic polymorphism of the Duffy receptor binding domain of Plasmodium vivax in Colombian wild isolates. Molecular and Biochemical Parasitology 78, 269–272. Babaeekho, L., Zakeri, S., Djadid, N.D., 2009. 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Please cite this article in press as: Ju, H.-L., et al., Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates. Acta Trop. (2012), http://dx.doi.org/10.1016/j.actatropica.2012.09.016