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IN KOREAN ISOLATES OF PLASMODIUM VIVAX
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ABSTRACT |
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(PvMSP-3) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3 locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the Republic of Korea, P. vivax malaria was endemic until the 1970s; this indigenous malaria had not been reported since 1984.3,4 However, indigenous malaria re-appeared in a soldier in 1993,3 and has subsequently become a serious public health threat in the Republic of Korea.4,5 The number of reported cases has increased from 107 in 1995 to 3,930 in 1998, and then slightly decreased to 1,140 in 2003, totaling 19,164 cases by the end of 2003.4,6
Among the proteins of the erythrocytic stages in the life cycle of Plasmodium, the merozoite surface proteins (MSP), including MSP-1, MSP-2, and MSP-3, have been shown to be involved in protective mechanisms of the host7–10 and studied as potential targets for vaccine development.7,8,10 The MSP-3 of P. vivax (PvMSP-3) is a protein with a molecular weight ranging from 148 to 150 kD, an alanine-rich central domain, and a series of heptad repeats that were predicted to form a coiled-coil tertiary peptide structure.11 The PvMSP-3 gene family consists of three intra-specific genes, PvMSP-3, PvMSP-3ß, and PvMSP-3
; these exhibited diversity when P. vivax isolates from diverse geographic localities and origins were compared.11–13 The deduced peptide sequences and structures, corresponding to the PvMSP-3, PvMSP-3ß and PvMSP-3, have similarities with the MSP-3 of P. falciparum (PfMSP-3).9,12–14 PfMSP-3 is a target antigen in the mediation of antibody-dependent cellular immunity,9 which partially protects monkeys against a lethal P. falciparum infection, via immunization.10 Moreover, antibodies to PfMSP-3 were found to decrease parasitemia in immunocompromised mice infected with P. falciparum.15 Therefore, PfMSP-3 is considered as a P. falciparum vaccine candidate.9,14–15 Similarly, PvMSP-3, particularly PvMSP-3, is regarded as a candidate for inclusion in a P. vivax vaccine.11,13
PvMSP-3 was also used as a polymorphic marker in epidemiologic studies of worldwide geographic strains and isolates using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis.16–18 The length and sequence polymorphisms of PvMSP-3 were analyzed, and the basis for its use in the RFLP protocol has been reported.13,18 However, in the case of the re-emerging Korean P. vivax, no studies have been performed on the genetic diversity of PvMSP-3. Therefore, the present study was performed to provide information on the polymorphism of the PvMSP-3 locus among the re-emerging Korean isolates.
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MATERIALS AND METHODS |
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Extraction of the genomic DNA. The whole blood of 28 patients was either directly frozen at –80°C or separated into packed cells and plasma, and then frozen at –80°C. The parasite DNA was extracted using the proteinase K digestion technique, followed by extraction with phenol-chloroform, or using the QIAamp DNA Mini Kit® (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. After precipitation with ethanol, the DNA was re-dissolved in TE buffer (10 mM EDTA, 10 mM Tris-HCl, pH 8.0) and stored at –20°C until used.
Sequencing of the PvMSP-3 gene.
The primer sequences were derived from the MSP-3 gene of the Belem strain (Brazil) of P. vivax (GenBank accession number: AF093584).11 The DNA from each of the 28 patients was amplified by a PCR using oligonucleotide primers that allow amplification of the complete coding region:13 L1 (5'-CTATTCGCACCGA-ACAGTCA-3') and L2 (5'-CATCACCCCAATTTGT-CGTA-3'). These primers bind at positions –72/+86 (L1/L2) of the Belem MSP-3 coding sequence. The primers were used at a final concentration of 0.1 µM in 100 µL of the reaction mixture (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP) containing 10 µL of DNA and 2.5 units of the Taq polymerase (Roche, Mannheim, Germany). The following thermal cycling conditions were used: L1/L2: 95°C for five minutes, then 32 cycles at 94°C for 30 seconds, 59°C for 40 seconds, and 72°C for 60 seconds, followed by a final five-minute extension step at 72°C in a DNA thermal cycler (GeneAmp® PCR System 9600; PE Applied Biosystems, Foster City, CA). After gel purification of the amplified fragment using the QIAEX II gel extraction kit® (Qiagen), part of the gene was re-amplified by repeating the primary PCR.
The DNA cycle sequencing was performed on 28 PCR-amplified DNA samples using the Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) according to the manufacturer’s instructions. Each reaction contained 4 µL of the Terminator Ready Reaction Mixture, 2 µL of the sequencing primer (1 µM), and 4 µL of the amplicon. The sense-stranded sequencing primers were F1, 5'-GTCAAAGCAGTAGTG GCAAA-3'; F2, 5'-CAACAGG-TAAAAGCAGAAAT-3'; F3, 5'-AAGGCGGAAGTGCT-GAACTC-3'; F4, 5'-CTCATCAAAAGTGGTTAAAA-3'; F5, 5'-ATCGCCGCAGCGAAGAAGGC-3'; and F6, 5'-GACACTTACGATGATATTAA-3'. The anti-sense-stranded primers were R1, 5'-AATGGTAGCTGCCTGGGTTG-3'; R2, 5'-TGCGTCATTTTCCGCTTCTT-3'; R3, 5'-TGCCTTCGC-CACTTCGGCAG-3'; R4, 5'-ATCTCTACGGCTATTTC-CGC-3'; R5, 5'-GGTTAATGGTATCACACTGG-3'; and R6, 5'-TTTTCCTTCTCGGTCACTTG-3'. The samples were run in an automated sequencer (Models 373A and 377; PE Applied Biosystems) and then analyzed using the ABI PRISMTM software (PE Applied Biosystems).
Analysis of DNA and deduced amino acid sequences.
The whole gene sequences of the PvMSP-3 of the re-emerging Korean isolates and the isolates from the imported cases were aligned and compared with the previously published gene sequences from various geographic localities.13 The amino acid and nucleotide sequence alignments were analyzed using Clustal X version 1.81.19 New nucleotide sequence data obtained in this study have been deposited in GenBank with the accession numbers AY266087 (SKOR-67), AY266088 (SKOR-69), AY266089 (MALA-84), AY266090 (PAKI-93), and AY266091 (INDO-94).
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RESULTS |
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showed a large number of amino acid substitutions, insertions, and deletions compared with the well-known Belem strain.13 Sequence variation was pronounced at the coiled-coil heptad repeat region of the central alanine-rich domain (Figure 1
). The extreme N-terminus was relatively well conserved, whereas the central C-terminus displayed some variability. In contrast, the N-terminal half of the alanine-rich domain was diverse, and the C-terminal half of the alanine-rich domain well conserved (Figure 2).
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of the 28 isolates examined (Table 1). The 24 Korean isolates were divided into two types, type I (15 isolates showing the identical sequences) represented by SKOR-67, and type II (9 isolates showing the identical sequences) represented by SKOR-69. The two types showed striking size variations, 2.5 kb and 1.7 kb, respectively, which depended on the deletions in the N-terminus of the central alanine-rich core region (Figure 1
). The deduced amino acid sequences of the allelic type I (e.g., SKOR-67) were generally similar to that of the Belem strain, despite the presence of 39 substitutions and 8 insertions (K108 
E115) (Figure 1). Allelic type II (e.g., SKOR-69) showed 32 substitutions and 257 deletions (G97 A100 and K170 K439, excluding common deletions at amino acid positions 183185 and 244257) when compared with the Belem strain; it was similar to the Chess strain (New Guinea) (Figure 1).
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locus of the four isolates from the imported cases were distinguished from those of the Belem strain and the two Korean types. They could be designated as the allelic types III (MALA-84), IV (INDO-94 and INDI-96 showing the identical sequences), and V (PAKI-93) (Figure 1 and Table 1). In the alanine-rich domain, the MALA-84 showed 49 amino acid substitutions, 153 deletions (L151 A182, E186 Q243, and C258 A320), and 4 insertions (K108 E111). The INDO-94 and INDI-96 showed 35 substitutions and 42 deletions (K263 A269 and K578 L612). The PAKI-93 showed 86 substitutions, 4 deletions (G97 A100), and 14 insertions (A244 K257) in the alanine-rich domain (Figure 1).
Based on the sequence data, diagrammatic representations of the full PvMSP-3 gene have been constructed for the five major allelic types (Figure 2). Allelic type I (2.53 kb) included the Belem strain and 15 Korean isolates including SKOR-67, and allelic type II (1.78 kb) included the Chess strain and 9 Korean isolates including SKOR-69. Allelic type III (2.09 kb) was found in the MALA-84 isolate, allelic type IV (2.42 kb) in the INDO-94 and INDI-96 isolates, and allelic type V (2.56 kb) in the PAKI-93 isolate (Figure 2).
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DISCUSSION |
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Genetic studies of re-emerging P. vivax malaria have been performed on several parasitic proteins, including the circumsporozoite surface protein (CSP),20,21 Duffy-binding protein (DBP),22 apical membrane antigen-1 (AMA-1),23,24 MSP- 125,26, and 18S ribosomal RNA.27 The CSP gene of the Korean isolates showed two haplotypes, i.e., SK-A and SK-B, which were similar to the Chinese isolate CH-5 and the North Korean (NK) strain, respectively; both haplotypes could be classified as the East Asian group.20 The AMA-1 gene of the re-emerging Korean P. vivax could also be classified into two allelic types, similar to the Chinese CH-5 and CH-10 isolates, respectively.23 With regard to the DBP locus, two types were also shown to coexist among the Korean isolates.22 However, these allelic types appeared as different combinations in each isolate, and as a whole, the isolates could not be divided into two or three big genotype groups.
MSP-3 is known to be another useful marker for genetic polymorphism of malaria parasites in endemic areas; thus, studies have been performed on this genetic locus.13,16–18 In addition to its epidemiologic significance, this locus is also known to be a potential candidate for vaccine development; the potential vaccines should concentrate on the C-terminus (the nucleotide sequence positions 1,300–2,058) of the alanine-rich domain and the acidic C-terminal region because this region is highly conserved across a range of geographically distinct P. vivax isolates.13 No information was available on the genetic characteristics of PvMSP-3 of the Korean isolates; thus, studies at this antigenic site were required.
In the present study, PvMSP-3 of the re-emerging Korean isolates was classified into two distinct allelic types, I and II. The gene size of PvMSP-3 of allelic type I was similar to that of the Belem strain, although at positions 97–116, their amino acid sequences were different: AAKPEAALEEQK-KEELQKEL in type I (SKOR-67) and GPNAEPNAEQI in the Belem strain (Figure 1). The gene size of PvMSP-3 of type II was similar to those of the Chess isolate and Br69-1.7 (Brazil), although at positions 558–563 and 605–612, their amino acid sequences were different: MSELEK and TAAN-VVKD in type II (SKOR-69), LSKLEE and KEATAAKL in the Chess isolate, and LSKLEE and TAANVVKD in the Br69-1.7 isolate (Figure 1). The sequence homology between type I and Belem (93.0%), type I and Br69-2.4 (94.0%), type II and Chess (99.2%), and type II and Br69-1.7 (97.8%) were relatively high. However, both the allelic types I (SKOR-67) and II (SKOR-69) appeared to be different from the North Korean strain (the NK strain),13 particularly in the sequence of the coiled-coil heptad repeat region of the alanine-rich domain of the PvMSP-3 (Figure 1). The sequence similarity between the allelic type I and NK was 87.1%, and that between the allelic type II and NK was 90.7%. The allelic types I, II, and III in this study were similar to the allelic types A, C, and B, respectively, reported from western Thailand.18 The allelic types IV and V are reported for the first time in the present study.
The PvMSP-3 allelic types of the Korean isolates and the isolates from the imported cases were compared with the allelic types of the PvMSP-1 gene (Han ET and others, unpublished data); close correlations were shown between the two genetic loci (Table 1). All 15 Korean isolates having allelic type I for PvMSP-3 showed allelic type A for PvMSP-1(Table 1). Seven of the nine isolates with allelic type II for PvMSP-3 were matched with allelic type B for PvMSP-1; the remaining two isolates showed different combinations of allelic type II for the PvMSP-3 and allelic type A for PvMSP-1, respectively (Table 1). In the isolates from the imported cases, allelic type III of the PvMSP-3 correlated with allelic type C of PvMSP-1, allelic type IV with allelic types D and E, and allelic type V with allelic type F (Table 1).
Nevertheless, it was difficult to determine the epidemiologic and clinical significance of each allelic type of the PvMSP-3 locus. The types did not clearly correlate with the two different incubation periods of the patients (short and long), which is well known in re-emerging Korean P. vivax malaria.4 Similarly, no direct relationships were shown between the allelic types of the CSP, DBP, AMA-1, and MSP-1 of the Korean isolates and the regularity of the fever intervals, the incidence of anemia or thrombocytopenia, or the frequency of relapses.20–25
Without firm evidence, North Korea was suggested to be the origin of the reemerging P. vivax malaria in the Republic of Korea.3,4 Previous studies on the genetic characteristics of CSP20,21 and AMA-123,24 support this suggestion. In the present study, neither allelic types I and II of the re-merging Korean isolates corresponded with the NK strain in terms of the sequence of the polymorphic region of the PvMSP-3 locus. However, it can be speculated that more than three allelic types coexist in North Korea. Conversely, the di-allelic nature of PvMSP-3 in the Korean isolates may suggest a likely origin of the re-emerging malaria confined to a small number of introductions from a limited geographic area of North Korea.
The polymorphic nature of the PvMSP-3 gene can be used as a marker in the discrimination of multiple infections in epidemiologic studies.16–18 In previous studies, the percentage of multiple infections was 23% in Papua New Guinea16 and 35.6% in Thailand.18 In our study, the PvMSP-3 gene showed the presence of five allelic types among the 24 Korean isolates and the 4 isolates from imported cases. However, by direct sequencing, no cases of mixed infections, with more than two allelic types, were observed. To detect any coexisting minor sequence variants, which may not be detected by direct sequencing, PCR fragments from five isolates each of allelic types I and II and one each of types III, IV, and V were cloned and sequenced, but no isolates showed such minor variants.
To conclusively determine the genetic diversity of re-emerging Korean P. vivax, the nucleotide sequences of the PvMSP-3 full-length gene were studied. The results showed that two allelic types, each having distinct sequences, coexist among the reemerging Korean isolates.
Received March 9, 2004. Accepted for publication July 2, 2004.
Financial support: This study was supported by a grant from the Ministry of Health and Welfare (2003; grant no. 03-PJ1-PG1-CH01-0001) and BK21 Human Life Sciences, Ministry of Education, Republic of Korea.
Authors’ addresses: Eun-Taek Han, Department of Parasitology, Kangwon National University College of Medicine, Chunchon 200-701, Korea, Telephone: 82-33-250-7941, Fax: 82-33-242-7571. Tae-Eui Song, Jae-Hwan Park, Eun-Hee Shin, Sang-Mee Guk, and Jong-Yil Chai, Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and the Institute of Endemic Disease, Seoul National University Medical Research Center, Seoul 110-799, Korea, Telephone: 82-2-740-8342, Fax: 82-2-765-6142. Tae-Yun Kim, Institute of Malariology and Department of Parasitology, Inje University College of Medicine, Pusan 614-735, Korea, Telephone: 82-51-890-6731, Fax: 82-51-890-6792.
Reprint requests: Jong-Yil Chai, Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Disease, Seoul National University Medical Research Center, Seoul 110-799, Korea, E-mail: cjy{at}snu.ac.kr.
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REFERENCES |
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share structural similarities with P. vivax merozoite surface protein-3 and define a new gene family. Mol Biochem Parasitol 115: 41–53.[Web of Science][Medline]
is limited to specific domains. Parasitology 125: 393–405.[Medline]
locus of Plasmodium vivax: global and local diversity. Am J Trop Med Hyg 61: 518–525.[Abstract]
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