- Open Access
Polymorphic patterns of the merozoite surface protein-3β in Korean isolates of Plasmodium vivax
© Kang et al.; licensee BioMed Central Ltd. 2014
- Received: 2 January 2014
- Accepted: 7 March 2014
- Published: 17 March 2014
The merozoite surface protein-3β of Plasmodium vivax (PvMSP-3β) is one of the candidate antigens for blood stage malaria vaccine development. The polymorphisms in PvMSP-3β have been reported in certain P. vivax isolates. However, the diversity of PvMSP-3β throughout its global distribution has not been well understood. In this study, the genetic diversity and the effects of natural selection in PvMSP-3β among P. vivax Korean isolates were analysed.
Blood samples were collected from 95 patients with vivax malaria in Korea. The region flanking full-length PvMSP-3β was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvMSP-3β sequence of each isolate was determined and the polymorphic characteristics and effects of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs.
Five different subtypes of PvMSP-3β were identified based on single nucleotide polymorphisms (SNPs), insertions, and deletions. Although a high level of sequence diversity was observed in the PvMSP-3β gene, the coiled-coil tertiary structure of the PvMSP-3β protein was well conserved in all of the sequences. The PvMSP-3β of Korean isolates is under natural selection. DNA polymerase slippage and intragenic recombination likely contributed to PvMSP-3β diversity in Korean P. vivax isolates.
The PvMSP-3β of Korean P. vivax isolates displayed polymorphisms, with SNPs, insertions and deletions scattered throughout of the gene. These results of parasite heterogeneity are relevant to the development of a PvMSP-3β based vaccine against P. vivax and the implementation of malaria control programmes in Korea.
- Plasmodium vivax
- Merozoite surface protein-3β
- Genetic polymorphism
- Natural selection
Plasmodium vivax is the most widely distributed malaria parasite in worldwide, causing 70–80 million cases annually, and is a major public health problem contributing to the global burden of malaria morbidity in regions outside of Africa . Plasmodium vivax is more difficult to control and eliminate than Plasmodium falciparum mainly because of its tendency to relapse. Moreover, vivax malaria has re-emerged in many temperate regions in recent years, including South Korea, where it had been largely eradicated during the global malaria control campaigns.
Understanding the population structures of the malaria parasites is an important factor in drug-resistance surveillance and estimating the performance of vaccines under development in a particular parasite population . Although the population structures of P. falciparum isolates have been examined on a global scale, those of P. vivax remain largely less understood. With a global distribution that ranges from tropical to temperate regions, P. vivax isolates have exhibited variation in biological characteristics, such as relapse patterns and transmissibility, which can be used to distinguish geographical isolates and even subspecies . Moreover, extensive genetic diversity has also been identified within a local P. vivax population. Therefore, understanding the nature of genetic polymorphisms of P. vivax populations in endemic areas might provide information regarding parasite heterogeneity that is relevant to malaria control efforts.
The P. vivax merozoite surface protein-3 (PvMSP-3) family consists of several related proteins, PvMSP-3α, PvMSP-3β and PvMSP-3γ. Although the level of shared amino acid sequence identity among the proteins is limited, all of them contain a central alanine-rich domain that forms a coiled-coil tertiary structure [4–6]. Limited sequence polymorphism has been observed in the MSP-3 of P. falciparum (PfMSP-3) [7, 8], but the PvMSP-3α is likely to be highly polymorphic and is reported to be a reliable genetic marker for population analysis [9–13]. PvMSP-3β also showed polymorphic patterns among several geographically different P. vivax isolates [14, 15], but its polymorphic pattern in worldwide isolates is poorly understood compared to other vaccine candidate antigens. As PvMSP-3β has been regarded as a potential vaccine target , a more thorough understanding of genetic diversity of the gene is required since genetic polymorphism in the candidate antigen can hamper the efficacy of a vaccine.
Following its re-emergence in Korea in 1993, vivax malaria has persisted, with varying numbers of indigenous cases annually in the country. Several recent studies have suggested that rapid genetic variation has occurred in the Korean P. vivax population in recent years [17–21], which supports settlement of local transmission of vivax malaria in Korea. In this study, the polymorphic nature of PvMSP-3β in Korean P. vivax isolates was analysed. The PvMSP-3β gene is radically polymorphic in Korean P. vivax population, with multiple gene sizes and single nucleotide polymorphisms (SNPs) which are scattered throughout the gene.
A total of 95 blood samples used in this study were collected from Korean patients infected with P. vivax in Korea between 2007 and 2012 (2007–2010, n = 15 for each year; 2011, n = 18; 2012, n = 17). 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. The P. vivax infections were identified by microscopic examination of thin and thick blood smears, and confirmed by polymerase chain reaction (PCR) [10, 20]. About 5 ml of blood was collected from each individual. The blood was separated into packed cells and plasma and then stored at −80°C until use. Informed consent was obtained from all of the patients before blood collection. The study protocol was approved by the Ethics Committee of the Inha University School of Medicine.
Genomic DNA extraction and amplification of PvMSP-3β
Genomic DNA was extracted from 200 μl of whole blood sample using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). The full-length region encoding PvMSP-3β was amplified using two rounds of PCR. The primers were designed based on PvMSP-3β sequences of Sal I (XM_001613146) and Belem (AF099662) strains deposited in GenBank. The primers used for the first round of PCR were 5′-TTCGCAACACTCGCCTTATTTCGCTCAACG-3′ and 5′-CCCCCAATTCGTCACCAATTTGTTTAGCAT-3′. The primers used for the nested PCR were 5′-TTTCGCTCAACGCGCGCATCTAAAATG-3′ and 5′-TTAGCATATTTTCTTCCGCCTCCTTTA-3′. The following thermal cycling conditions were used for both amplifications: 94°C for 5 min; 30 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 3 min, and a final extension at 72°C for 10 min. The PCR product was analysed on a 1.2% agarose gel, purified from the gel, and ligated into the T&A cloning vector (Real Biotech Corporation, Banqiao City, Taiwan). Each ligation mixture was transformed into Escherichia coli DH5α competent cells and positive clones with the appropriate insert were selected by colony PCR. The nucleotide sequences of the cloned insert were analysed by automatic DNA sequencing using the vector primers, M13 forward and M13 reverse primers. Sequencing analyses with two additional specific internal primers (5′-AGCAAAAACAGAAGCAGAAACAGCACA-3′ and 5′-GGAAATTTTCAGCTTCCGTTTTTGCTT-3′) were also performed to obtain the sequence of central region of the PvMSP-3β. At least two clones from each isolate were sequenced to ensure accuracy, and some isolates underwent three-fold sequence coverage to confirm the existence of rare polymorphisms. The nucleotide sequences reported in this study have been deposited in the GenBank database under the accession numbers JX667768-JX667772.
Sequence and phylogenetic analysis of PvMSP-3β
The nucleotide and deduced amino acid sequences of PvMSP-3β were analysed using EditSeq and SeqMan in the DNASTAR package (DNASTAR, Madison, WI, USA). The phylogenetic tree was constructed using the neighbour-joining method in MEGA4 computational program . Bootstrap proportions were used to assess the robustness of the tree with 1,000 bootstrap replicates. The coiled-coil motifs in each of the sequences were predicted using the Paircoil2 structural analysis program .
Sequence polymorphism analysis
DNA sequence polymorphism analysis was performed on the 95 PvMSP-3β sequences. The number of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and the average number of pair-wise nucleotide differences within the population (K) were estimated using the DnaSP ver. 5.10.00 . The π was calculated to estimate the step-wise diversity throughout the entire PvMSP-3β based on a sliding window of 100 bases with a step size of 25 bp. The rates of synonymous (dS) and non-synonymous (dN) substitutions were estimated and were compared using the Z-test (P <0.05) in MEGA4 program  using Nei and Gojobori’s method  with the Jukes and Cantor correction. To evaluate the neutral theory of evolution, the Tajima’s D test  was performed with the DnaSP ver. 5.10.00 . Fu and Li’s D and F statistics  were also analysed using the DnaSP ver. 5.10.00 .
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 determined using the DnaSP ver. 5.10.00 . The linkage disequilibrium (LD) between the different polymorphic sites was computed based on the R2 index.
DNA sequence polymorphisms in each domain of PvMSP-3β among Korean isolates
Segregating sites (S)
Singleton variable sites
Parsimony informative sites
Total no. of mutations
Hd ± SD
π ± SD
Fu and Li’s D
Fu and Li’s F
0.773 ± 0.028
0.07271 ± 0.00240
3.15439 (P < 0.01)
2.53522 (P < 0.02)
3.36192 (P < 0.02)
0.733 ± 0.028
0.03040 ± 0.00179
2.27420 (P < 0.05)
2.39279 (P < 0.02)
2.81226 (P < 0.02)
Based on the occurrence of small indels in the sequence and the presence of degenerate repeat on either side of the indels, DNA polymerase slippage has been suggested as a possible mechanism generating diversity of PvMSP-3β . Similar small indels and flanking degenerate repeats were also identified in PvMSP-3β sequences of Korean P. vivax isolates, which suggested slipped-strand mispairing during DNA replication may have contributed to PvMSP-3β diversity. The 11 amino acid-length repeated elements that were identified in the large-scale insertion  were also conserved in the end of the N-terminal domain and the end of insert A of PvMSP-3β of Korean P. vivax isolates. The diversity of Plasmodium antigens, including PvMSP-3α, is also known to be generated by intragenic recombination events, and is maintained by balancing selection [9, 10, 20, 28]. Similarly, it has been postulated that recombination is another major factor generating allelic diversity of PvMSP-3β . To understand the role of the recombination event in PvMSP-3β in Korean P. vivax isolates, the recombination parameters and the linkage disequilibrium in the gene were analysed. For the N-terminal domain of PvMSP-3β, the minimum number of recombination events between adjacent polymorphic sites (Rm) was 13, whereas the R between adjacent sites (Ra) and per gene (Rb) was 0.0036 and 3.6, respectively. In the case of C-terminal domain of PvMSP-3β, the Rm was 4 with a Ra of 0.0029 and a Rb of 3.1. These high recombination parameter values suggested that recombination may have occurred between sites, contributing to genetic diversity in PvMSP-3β gene. The linkage disequilibrium index, R2, was also declined across the analysed region, which further indicates that intragenic recombination might have contributed to the diversity observed in both the N-terminal and C-terminal domains of PvMSP-3β in Korean P. vivax isolates (Figure 5B).
The genetic diversity of malaria parasites is closely associated with the levels of endemicity and intensity of transmission [29, 30]. The P. vivax population in hyperendemic areas, such as Papua New Guinea, is highly diverse and shows multiplicity of infections [31, 32]. However, complex genetic structures have also been identified in P. vivax populations in hypo-endemic areas, such as China, Myanmar and Thailand [10, 33–38]. Several recent studies have suggested that genetic diversity within the P. vivax population in Korea has increased in recent years [17–21]. Considering the low endemicity and transmission intensity in Korea, it remains unclear how genetic diversity of P. vivax in Korea is rapidly disseminated and maintained in recent years. An increased influx of international travellers and foreign workers might have contributed to the increasing allelic variation in the Korean P. vivax population through the introduction of malaria parasites from other endemic areas . It is also possible that the Korean P. vivax population has evolved under evolutionary pressure, most probably related to host immune response. Further studies of genetic diversity in the Korean P. vivax population in Korea are warranted to clarify the patterns of genetic variation and the biological relevance of increasing P. vivax diversity.
Radical genetic polymorphism was identified in the PvMSP-3β gene of Korean P. vivax isolates. The size of the PvMSP-3β varied, as a result of large and small insertions/deletions, and numerous SNPs were observed throughout the gene. Despite the high level of sequence diversity, the coiled-coil tertiary structure of the protein was well conserved in all of the Korean P. vivax isolates, suggesting that the protein is under functional constraints. The genetic diversity of the PvMSP-3β of Korean P. vivax isolates has been influenced by natural selection, particularly in the N-terminal and C-terminal domains. Polymerase slippage during DNA replication and intragenic recombination might have contributed to the genetic diversity of PvMSP-3β in the Korean P. vivax population. These findings of parasite heterogeneity are relevant to the development of a PvMSP-3β-based vaccine against P. vivax and the implementation of malarial control programmes in Korea.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2011–0028135).
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