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Communication

Genetic Diversity and Phylogeography of Plasmodium vivax Transmission-Blocking Vaccine Candidate Genes pvs47 and pvs48/45 in Honduras

1
Instituto de Investigaciones en Microbiología, Facultad de Ciencias, Universidad Nacional Autónoma de Honduras, Tegucigalpa 11101, Honduras
2
Laboratorio Nacional de Vigilancia de Malaria, Secretaría de Salud de Honduras, Tegucigalpa 11101, Honduras
*
Author to whom correspondence should be addressed.
Parasitologia 2025, 5(3), 36; https://doi.org/10.3390/parasitologia5030036
Submission received: 12 June 2025 / Revised: 14 July 2025 / Accepted: 18 July 2025 / Published: 21 July 2025

Abstract

Plasmodium vivax malaria continues to pose a significant and enduring public health challenge across the Americas. Transmission-blocking vaccines (TBVs), which target gametocyte surface antigens such as Pvs47 and Pvs48/45, are being investigated as promising tools to interrupt transmission and advance toward disease elimination. To investigate the genetic diversity and phylogeographic structure of the pvs47 and pvs48/45 genes in P. vivax, we conducted molecular analyses on samples collected from seven malaria-endemic regions of Honduras using PCR-based sequencing, population genetics, and phylogenetic approaches. This study presents the first complete characterization of the pvs47 gene and expands the available data on pvs48/45 in P. vivax from Honduras. We observed a low level of genetic diversity with no evidence of geographic structuring within the country. At a global scale, Honduran sequences shared variants with other Latin American strains and exhibited region-specific amino acid signatures. These findings suggest that local selective pressures, possibly driven by mosquito vector compatibility, are shaping the evolution of these TBV candidate genes. Our results underscore the importance of regional surveillance to inform the development and deployment of effective transmission-blocking strategies.

1. Introduction

Malaria caused by Plasmodium vivax—the most widespread human malaria species outside Africa—remains a significant public health concern, particularly in some countries of Central and South America [1]. Although less virulent than P. falciparum, P. vivax is associated with substantial morbidity and has proven more difficult to eliminate [2,3] due to its ability to form dormant liver stages (hypnozoites) and its efficient transmission at lower parasitemia levels. In Honduras, ongoing malaria control efforts over the past two decades have led to a decline in reported cases, yet P. vivax continues to circulate, maintaining a persistent risk of transmission and resurgence [1,4].
One promising strategy to interrupt malaria transmission is the development of transmission-blocking vaccines (TBVs), which target sexual-stage antigens to prevent parasite development within the vector [5,6,7,8]. Two of the most studied TBV candidates in this context are Pvs47 and Pvs48/45, members of the conserved 6-cysteine protein family expressed on the surface of gametes [5,8]. Their orthologs in P. falciparum—Pfs47 and Pfs48/45—as well as other TBV candidate antigens, have been extensively characterized, including studies of global sequence diversity and evidence for vector-mediated selection [9,10,11,12,13,14,15]. In general, the sequence diversity in P. falciparum TBV targets is relatively low compared to highly variable asexual-stage antigens, which is encouraging for antibody-based strategies aiming to induce strain-transcending responses [15]. However, non-synonymous mutations do occur and may affect vaccine efficacy; therefore, ensuring heterologous protection remains a key focus of ongoing clinical research [15,16]. In contrast with the growing body of knowledge on TBV antigen diversity in P. falciparum, relatively little is known about the genetic variation in these antigens in P. vivax, especially in Central American populations [17].
Genetic variation in TBV target antigens is a critical consideration in vaccine design, as polymorphisms may influence antigen recognition, transmission-blocking efficacy, and geographic effectiveness. Previous studies have demonstrated that, in P. vivax, pvs47 and pvs48/45 tend to exhibit a low level of nucleotide diversity but a high level of haplotype diversity [17,18,19,20], a pattern often attributed to localized diversification and adaptation to local vector species [9,21]. Also, certain nonsynonymous mutations may serve as markers of adaptation to regional mosquito vectors, highlighting the potential for these genes to reflect both evolutionary pressures and transmission dynamics [22].
Despite their functional relevance, data on pvs47 and pvs48/45 remain limited in Central America. Most global analyses have relied heavily on isolates from Asia [18,19,21] and South America, with few sequences available from Africa or Central American countries such as Honduras [17]. Expanding the geographic representation of these genes in international databases is crucial for enhancing our understanding of regional parasite diversity, tracking evolutionary patterns, and assessing the potential implications for the design and implementation of TBVs.
In this study, we present the first molecular characterization of pvs47 sequences and expand the available data on pvs48/45 from P. vivax isolates in Honduras. Using a targeted amplification and sequencing approach, we analyzed samples collected across seven endemic regions of the country to evaluate genetic diversity, identify putative polymorphic sites, assess patterns of haplotype structure, and investigate phylogeographic relationships with global isolates. This study enhances current knowledge on the genetic variation in TBV candidate antigens in P. vivax.

2. Materials and Methods

2.1. Sample Collection and Ethical Approval

A total of 31 dried blood spot (DBS) samples were analyzed from patients diagnosed with Plasmodium vivax malaria. These samples were collected through passive case detection at public healthcare facilities under the jurisdiction of the Honduran Ministry of Health, following national guidelines and routine disease surveillance protocols. The samples were obtained between 2023 and 2024 across seven malaria-endemic regions of Honduras (Figure 1). All samples were anonymized before analysis. Initial malaria diagnosis was performed either by light microscopy or rapid diagnostic tests (RDTs), with confirmatory testing conducted using molecular methods. The study protocol (PI 12-2024) was reviewed and approved by the Institutional Ethics Committee (CEI-MEIZ) of the National Autonomous University of Honduras (UNAH).

2.2. DNA Extraction and Molecular Detection of Plasmodium vivax

Genomic DNA was extracted from two 2 mm punches of DBS using the Extracta® DNA Prep for PCR kit (QuantaBio, Beverly, MA, USA), following the manufacturer’s instructions. Purified DNA was stored at −20 °C until further analysis. Plasmodium genus screening was conducted using PET-PCR with primers listed in Table 1. Positive samples were subsequently tested with P. vivax-specific primers. PCR assays were performed in 20 μL reaction volumes containing 10 μL GoTaq® Probe qPCR Master Mix (Promega, Madison, WI, USA), 0.5 μL for both of forward and reverse primers (10 μM), 4 μL nuclease-free water, and 5 μL DNA template (~40 ng/μL). Amplification was performed on a Mic qPCR Cycler (Bio Molecular Systems, Australia) under optimized conditions: initial denaturation at 95 °C for 15 min, followed by 45 cycles of 95 °C (20 s), 63 °C (40 s), and 72 °C (30 s). Fluorescence signals were detected using 6-FAM-labeled (Plasmodium genus) and HEX-labeled (P. vivax) primers. A cycle threshold (Ct) ≤ 40 was considered positive. All runs included positive (reference DNA) and negative (no-template) controls. Data analysis was performed using Mic qPCR Cycler Software v2.10.1.3.

2.3. PCR Amplification and Sequencing of pvs47 and pvs48/45 Genes

2.3.1. Primary and Secondary PCR Amplifications

The initial amplification of pvs47 and pvs48/45 genes was performed by primary PCR reactions. Each 50 μL reaction mixture contained 25 μL of 2× Taq Master Mix (Promega, USA), 2 μL of each primer (10 μM) (Table 1), 2 μL of 10 mg/mL bovine serum albumin (BSA), and 4 μL of genomic DNA. The thermal cycling protocol consisted of an initial denaturation at 95 °C for 5 min; 25 cycles of 95 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min; and a final extension at 72 °C for 10 min.
For secondary amplifications, 1 μL of primary PCR product was reamplified in a 50 μL reaction volume using nested or semi-nested primers (Table 1; Figure 2). The cycling conditions were carried out as follows: initial denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 1 min, annealing at 58 °C (pvs47) or 53 °C (pvs48/45) for 1 min, and extension at 72 °C for 2 min; followed by a final extension at 72 °C for 10 min. Amplicons were visualized on 1% ethidium bromide-stained agarose gels, purified, and sequenced by Psomagen Inc. (Rockville, MD, USA).

2.3.2. Molecular Characterization and Population Genetic Analyses

Given that both genes were over 1300 nucleotides in length, their full sequence could not be obtained using only the primers from the secondary PCR reactions. Therefore, several internal primers were also employed to assemble each complete sequence (Table 1, Figure 2).
Sequence quality control and editing were performed using Geneious Prime software v.2024.0.5. Initial processing involved trimming sequence ends using the automated “Trim ends” function, followed by manual verification of electropherogram quality, in which all positions showed single unambiguous peaks without evidence of mixed signals. Complete gene sequences were assembled by aligning all primer-derived sequences for each isolate.
We retrieved homologous nucleotide sequences from GenBank exhibiting >98% query coverage and >96% nucleotide identity compared to our reference sequences (Supplementary Table S1). Publicly available sequences from GenBank were aligned with the newly obtained sequences from this study. Nucleotide diversity (π) and haplotype diversity (Hd), segregating sites (S), average nucleotide differences (k), number of haplotypes (H), Tajima’s D test [27], and Fu and Li’s D* and F* tests [28] were calculated in DnaSP v6.12.03.
Also, nucleotide sequences underwent subsequent analysis through multiple sequence alignment using the “Geneious Alignment” tool (default parameters) (Geneious Prime software v.2024.0.5.) and in-frame translation to amino acid sequences. The resulting polypeptide alignments enabled identification of polymorphic sites and calculation of identical residues, pairwise identity percentages, and pairwise positivity percentages. To ensure data accessibility, we deposited representative sequences of all identified haplotypes in GenBank.

2.4. Polymorphism Identification, Phylogenetic Tree Construction, and Haplotype Network Analysis

Polymorphic sites and alternative residues were recorded for each position in the polypeptide alignment. Neighbor-joining trees were generated from the aligned datasets using the Jukes Cantor genetic distance model, with nodal support assessed via bootstrap analysis (1000 replicates) [29]. Orthologous polypeptide sequences of P. cynomolgi or P. coatneyi were used as outgroups. Haplotype network reconstruction of amino acid sequences using the minimum spanning network was calculated in PopArt 1.7.

3. Results

Using a partial sequencing and assembly strategy, we obtained 26 complete coding sequences (CDSs) of pvs47 and 23 pvs48/45 CDSs from samples collected in seven endemic departments of Honduras (Figure 1). The CDSs of pvs47 comprised 1299 nucleotides encoding 433 amino acid residues, while pvs48/45 consisted of 1350 nucleotides encoding 450 amino acids. The 26 pvs47 sequences revealed a single SNP at position 66, resulting in two haplotypes (accession numbers PV700528-9 and PV30102). Seventy-three percent of the sequences showed the 66A genotype, while the remaining displayed the 66C genotype. For pvs48/45, we identified three polymorphic sites at positions 631, 750, and 1057, yielding three haplotypes (CCC, AAG, and CAG) (accession numbers PV700525-7). The CAG haplotype predominated (74% of sequences), while the AAG and CCC haplotypes were less frequent (13% and 8.7%, respectively). Two of the samples displaying the AAG haplotype were collected from migrant patients in transit through Honduras en route to North America, one of whom was of Venezuelan origin (Supplementary Table S1). No distinct pattern of haplotype distribution was observed concerning the patients’ department of origin, suggesting a lack of strong geographic structuring at the sub-national level.
Following a search for homologous sequences of both genes in GenBank, we downloaded all sequences meeting the following criteria: a high identity percentage (>96%), query coverage (>98%), and containing at least 98% of the coding sequence (CDS). This resulted in 48 pvs47 sequences originating from 11 countries and four continents (America, Asia, Oceania, and Africa) and 44 pvs48/45 sequences from seven countries (Supplementary Table S1), which were subsequently aligned with our newly obtained sequences.
We calculated molecular diversity indices and neutrality tests, with all values reported in Table 2.
The nucleotide sequences were subsequently translated to amino acid sequences and aligned to identify polymorphisms and determine protein variants or haplotype numbers. Honduran sequences showed a single polymorphism at position 22 (Leu/Phe) in Pvs47 and three polymorphisms in Pvs48/45 at positions 211 (Asn/His), 250 (Asn/Lys) and 353 (Glu/Gln). At a global scale (sequences downloaded from GenBank together with the Honduran sequences obtained in this study), we identified 25 polymorphisms in Pvs47 and 17 in Pvs48/45 (Figure 3). The percentage of identical sites was 94.2% and 96.2%, with pairwise identity values of 99% and 99.3%, respectively. A greater percentage of residue changes occurred in domain 1 of Pvs47; nonetheless, no segments of either gene were entirely free of nonsynonymous substitutions. Among the identified polymorphisms, two amino acid positions—one in each protein—exhibit notable geographic specificity, fixed in Old World isolates while displaying substitutions in strains from the Americas. In Pvs47, position 27 seems to be fixed and is uniformly occupied by glutamic acid (E) in all Old World sequences, whereas most American isolates show a substitution to lysine (K). A small number of Brazilian (n = 3) and Mexican (n = 2) strains retain the ancestral glutamic acid, and two additional Brazilian sequences exhibit an alternative third substitution to arginine (R) (Figure 3). Similarly, in Pvs48/45, residue 250 asparagine (N) is fixed in Old World isolates, but most American sequences display a lysine (K) substitution. A minority of samples, including several from Honduras and seven from Colombia, retain the ancestral asparagine (Figure 3).
Using the same alignments, which used orthologous sequences from other Plasmodium species as outgroups, cladograms were constructed to examine the potential geographic clustering of the strains and to assess the phylogenetic placement of the Honduran sequences relative to those previously reported from other countries. As shown in Figure 4, the Pvs47 cladogram reveals at least five clusters associated with specific continents or geographic regions. Cluster I included sequences reported from Thailand, Indonesia, Korea, and India. Cluster II grouped sequences from Brazil and Mexico. Cluster III comprised sequences from Vanuatu and Korea, while Cluster IV grouped seven of the Honduran sequences with others from Brazil, Mexico, and Colombia. The final cluster included the remaining 19 Honduran sequences along with three sequences from Brazil. In the case of Pvs48/45, the number of clusters was lower. The first two clusters included sequences from Thailand, China, Vanuatu, and Korea. Cluster III grouped two of the Honduran sequences with six sequences from Colombia, while Cluster IV included the remaining 21 Honduran sequences together with three sequences from Colombia.
Additionally, to confirm the evolutionary relationships among the sequences of both proteins, variant networks were constructed based on allelic variants. The Pvs47 network comprised 10 variants, three of which (V2, V5, and V6) included between 16 (V2) and 24 (V5 and V6) sequences. The remaining variants contained between one and three sequences each (Figure 5). Honduran sequences were found within variant V6, along with sequences from Brazil and Mexico, and in variant V2, together with sequences from Brazil, Mexico, and Colombia. Variant V5 included only sequences from Asia, Vanuatu, and Mauritania. In the case of Pvs48/45, the network consisted of six variants. Variant V4 was the most frequent, comprising most Honduran sequences, along with four from Colombia and five from Asia and Africa. Variant V2 included two additional Honduran sequences, together with six from Colombia. In contrast, variant V3 primarily grouped sequences from China and other Asian countries.

4. Discussion

This study provides the first molecular characterization of the pvs47 gene sequences from P. vivax isolates in Honduras and expands the number of available pvs48/45 gene sequences from this region [17]. By employing a targeted amplification and assembly strategy, we successfully obtained complete coding sequences for both genes from seven endemic regions of Honduras. This information provides valuable data to address the limited genomic information available on these TBV candidate antigens in P. vivax, especially relative to their well-studied orthologs in P. falciparum [6,10,11,12,13,14,30,31,32,33]. Given the critical role of these proteins in parasite transmission, understanding their genetic variability is essential for informing vaccine design and regional malaria control strategies.
Our analysis revealed a low level of nucleotide diversity within the Honduran parasite population, evidenced by the detection of only a single SNP in pvs47 and three in pvs48/45, with no apparent geographical clustering at the sub-national level among the isolates. At the protein level, the amino acid sequences encoded by both genes showed a very low level of diversity among Honduran isolates—lower than that reported in Asian populations [18,21,26] and also lower than in strains from Brazil or Colombia. A similar degree of low genetic heterogeneity has been reported in samples collected in Iran, a low-transmission setting characterized by seasonal and unstable malaria [19]. This reduction in genetic diversity in Honduras corresponds with intensified malaria control efforts in the region over the past two decades, which have substantially decreased malaria transmission, from over 35,000 cases in 2000 to fewer than 3,500 cases in 2024 [4,34,35,36].
Despite this limited polymorphism among Honduran populations, the presence of a high level of global haplotype diversity (Hd = 0.918 for pvs47 and 0.900 for pvs48/45) suggests that microevolutionary dynamics or historic demographic events may be shaping local parasite populations. These findings are consistent with previous reports describing a pattern of a low level of nucleotide diversity coupled with a high level of haplotype diversity in P. vivax populations when analyzing candidate genes for TBVs [17]. This suggests that, despite limited sequence variation at the nucleotide level, the accumulation of distinct haplotypes may reflect selective pressures or demographic processes influencing the genetic structure of these loci [14,18,37]. Neutrality tests, although non-significant, yielded negative Tajima’s D and Fu and Li’s statistics, consistent with scenarios of purifying selection or recent population expansion, as also reported in broader global analyses of pvs48/45 and pvs47 sequences [18,21].
Nevertheless, when evaluating global diversity patterns, both pvs47 and pvs48/45 exhibit low levels of polymorphism, particularly in terms of nonsynonymous mutations. This is especially evident when compared to the average number of nonsynonymous SNPs reported in antigens expressed during the asexual blood stage of P. vivax, which are typically more genetically diverse [5]. In pvs47, SNPs predominantly localize to domain 1, a region potentially under selective pressure through host immune responses or vector interaction [12,22,38]. The relatively higher level of diversity observed in domain 1 in our dataset may reflect functional constraints or adaptive pressures linked to its interaction with the mosquito midgut environment, which could drive polymorphism as the parasite adapts to local Anopheles vectors. Additionally, domain 1 may include epitopes exposed to the host immune system, and variation at these sites could contribute to immune evasion by altering antigen recognition. Further studies are needed to clarify the structural and functional role of domain 1 in parasite vector and parasite host interactions.
While TBV candidate antigens generally show a lower level of diversity than blood-stage antigens [5,8,13,14], the observed widespread distribution of polymorphisms, even those at a low frequency, may still contribute to antigenic variation, which is relevant for vaccine efficacy [18].
Phylogenetic analyses revealed clear geographic structuring of Honduran sequences within both the Pvs47 and Pvs48/45 cladograms. Honduran isolates clustered predominantly with strains from Latin American countries such as Brazil, Mexico, and Colombia, indicating regional connectivity and shared ancestry. Similar findings have been reported for pvs48/45 in previous studies analyzing strains from nine countries, including 12 isolates from Honduras [17]. These patterns were corroborated by haplotype network analyses, which demonstrated shared haplotypes between Honduran and other American populations. Notably, predominant haplotypes in these genes were largely restricted to the Americas, while more divergent variants clustered separately in Asia and Oceania. Such structuring mirrors previous global studies [17,21] and highlights the geographic differentiation of these antigens.
The geographic patterns we observed are similar to those previously reported for the 6-cysteine genes pfs47 and pfs48/45 in P. falciparum, which exhibit strong population differentiation likely driven by divergent selection linked to gamete recognition and compatibility [9]. Emerging evidence suggests that Pvs47 and Pvs48/45 in P. vivax may be subject to similar selective pressures, with polymorphisms such as the K27E substitution in Pvs47 acting as markers of adaptation to local mosquito vectors [21,22]. Kuesap et al. recently reported three fixed residues in both Pvs48/45 (N211, N250, and R418) and Pvs47 (L22, L24, and E27) among P. vivax populations isolated in Thailand [18]. These conserved positions may reflect local adaptation or functional constraints critical for the parasite’s survival and transmission in that region. In our study, positions 27 in Pvs47 and 250 in Pvs48/45 stand out as putative geographic signatures differentiating American P. vivax strains from those elsewhere, supporting the hypothesis of vector-driven selection shaping parasite population structure.
Together, these results underscore the utility of pvs47 and pvs48/45 as molecular markers to investigate population structure and evolutionary dynamics in P. vivax. The low level of genetic diversity observed within Honduras likely reflects the impact of declining transmission and possibly recent parasite introductions. The geographic clustering and shared haplotypes with other American populations further suggest that the regional coordination of malaria control and vaccine development could be advantageous across Mesoamerica. Future work should aim to expand sampling across Central and South America, incorporate functional assays to evaluate the antigenic consequences of observed polymorphisms, and assess how these variations influence transmission and vaccine efficacy.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/parasitologia5030036/s1, Table S1: List of Plasmodium vivax samples analyzed in this study, including GenBank accession numbers, country of origin, and corresponding Pvs47 and Pvs48/45 protein variants.

Author Contributions

Conceptualization, G.F.; methodology, K.E., M.E.A., L.C., G.A., G.M. and G.F.; validation, G.M., K.E. and G.F.; formal analysis, G.F.; investigation, K.E., M.E.A., L.C., G.A., G.M. and G.F.; resources, G.M. and G.F.; data curation, K.E. and G.F.; writing—original draft preparation, G.F.; writing—review and editing, K.E., M.E.A., L.C., G.A., G.M. and G.F.; visualization, G.F.; supervision, G.F.; project administration, G.M. and G.F.; funding acquisition, G.F. All authors have read and agreed to the published version of the manuscript.

Funding

Funding for this study was provided by the Genetic Research Center, CIG-UNAH.

Institutional Review Board Statement

The study made secondary use of biological specimens originally collected for malaria diagnosis as per the standard of care in Honduras, to identify parasite species and analyze genes linked to drug resistance, following national regulations for routine malaria surveillance. The ethics committee CEI-MEIZ-UNAH reviewed and approved the study (PI 12-2024).

Informed Consent Statement

Consent to participate was not necessary due to (a) the absence of personal information; (b) the study’s contribution to public health; and (c) the absence of any harm to the participants. The blood samples were anonymized and irrevocably stripped of direct identifiers, so the future re-identification of individuals is not possible.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supplementary File. The newly generated sequences were submitted to the GenBank database under the accession numbers cited in the text.

Acknowledgments

We would like to thank Alejandro Zamora for his support in the use of the network analysis software.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Pvs47 Plasmodium vivax surface protein 47
Pvs48/45Plasmodium vivax surface protein 48/45
TBVsTransmission-blocking vaccines
DBSDried blood spot
RDTsRapid diagnostic tests
PET-PCRPhoto-induced electron transfer polymerase chain reaction

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Figure 1. Map of Honduras showing the number of blood samples analyzed in this study by collection site.
Figure 1. Map of Honduras showing the number of blood samples analyzed in this study by collection site.
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Figure 2. Schematic representation of the genes (a) pvs47 and (b) pvs48/45 showing the primer binding sites used for amplification (red and green) and sequencing (green and blue).
Figure 2. Schematic representation of the genes (a) pvs47 and (b) pvs48/45 showing the primer binding sites used for amplification (red and green) and sequencing (green and blue).
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Figure 3. Schematics showing the alignment of amino acid sequences of the proteins (a) Pvs47 and (b) Pvs48/45. For each alignment, the polymorphic sites and the residue variants observed at each protein position are shown. The polymorphic positions for the sequences obtained in this study (Honduras) are shown in red. Residues highlighted in bold indicate the haplotype found in the isolates from Honduras. Cysteine residues are indicated by red arrows. The two domains (D1 and D2) of each protein are shown at the top of each sequence.
Figure 3. Schematics showing the alignment of amino acid sequences of the proteins (a) Pvs47 and (b) Pvs48/45. For each alignment, the polymorphic sites and the residue variants observed at each protein position are shown. The polymorphic positions for the sequences obtained in this study (Honduras) are shown in red. Residues highlighted in bold indicate the haplotype found in the isolates from Honduras. Cysteine residues are indicated by red arrows. The two domains (D1 and D2) of each protein are shown at the top of each sequence.
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Figure 4. Cladograms of the sequences depicting the phylogenetic relationships of (a) Pvs47 and (b) Pvs48/45 among isolates obtained in this study (Honduras) and sequences reported from other countries. Bootstrap values are shown to the left of each node.
Figure 4. Cladograms of the sequences depicting the phylogenetic relationships of (a) Pvs47 and (b) Pvs48/45 among isolates obtained in this study (Honduras) and sequences reported from other countries. Bootstrap values are shown to the left of each node.
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Figure 5. Haplotype networks of amino acid polymorphisms in (a) Pvs47 and (b) Pvs48/45.
Figure 5. Haplotype networks of amino acid polymorphisms in (a) Pvs47 and (b) Pvs48/45.
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Table 1. List of primers used for the amplification of molecular markers of Plasmodium vivax and P. falciparum.
Table 1. List of primers used for the amplification of molecular markers of Plasmodium vivax and P. falciparum.
Target GeneReactionPrimer NamePrimer Sequence (5′-3′)References
18Sr RNA genePET-PCR for genus PlasmodiumGenus forwardGGC CTA ACA TGG CTA TGA CG[23,24]
Genus reverse6FAM-agg cgc ata gcg cct gg CTG CCT TCC TTA GAT GTG GTA GCT
18Sr RNA genePET-PCR for P. vivaxVivax forwardACT GAC ACT GAT GAT TTA GAA CCC ATT T[25]
Vivax reverseHEX- agg cgc ata gcg cct ggT GGA GAG ATC TTT CCA TCC TAA ACC T
Pvs471st roundCM 000453 FwdCAC ACC ACC GCA AAC AGG[26]
CM 000453 RevGTG CAC ATT CCG CGG TTG
2nd roundnst 1440 FwdGCG GTC CAC CCT AAC TGT AA*
nst 1440 RevTGC TGC AAA CCA CAC ATG T[7]
Sequencing primerspPvs47-FATA TTT CCA ACG AAG CAT TTA TGC
pPvs47-RTTT TCC ATT ATG CTC ACA AAC GC
Pvs47F2GAA GAA AGG GGA GGA CCA AG[26]
Pvs48/451st roundPvs4845FGGA ATA ATT TCG ACC ACT C[17]
887TCA GAA GTA CAA CAG GAG GAG CAC[20]
2nd round866ATG TTG AAG CGC CAG CTC GCC AA
887TCA GAA GTA CAA CAG GAG GAG CAC
Sequencing primerspPvs4845FATG GCC AAA GGA GAG GTC AAG TAC[7]
pPvs48/45-6C-F TCG GCA GAT GCA AGT GAA GGA AGT C
Pvs4845FFTGT AAA ATC TGC GGA CGT GA[26]
Pvs4845RRCGGGTGCTTTAAAAATGGAA
1001FGAATGAGTTGCCCTGGGGAAThis study
1025FTGCCCGAGTGCTTCTTTCAA
249RTCCTGGGATCTTCTTCGGGA
494RAAGGCCACTCTTCCCTTCAC
* Personal communication with Dr. Álvaro Molina-Cruz.
Table 2. Molecular diversity indices and neutrality tests for global pvs47 and pvs48/45 gene sequences.
Table 2. Molecular diversity indices and neutrality tests for global pvs47 and pvs48/45 gene sequences.
Molecular Diversity Indices and Neutrality Testspvs47pvs48/45
Nucleotide diversity (π)0.003740.00258
Segregating sites (S)3321
Average number of nucleotide differences (k)4.875603.49423
Number of haplotypes (h)4532
Haplotype diversity (Hd)0.9180.900
Tajima’s D−1.02707−0.65000
Statistical significanceNot significant,
p > 0.10
Not significant,
p > 0.10
Fu and Li’s D* test statistic−2.08212−2.12009
Statistical significanceNot significant,
0.10 > p > 0.05
Not significant,
p > 0.10
Fu and Li’s F* test statistic−2.01002−1.89449
Statistical significanceNot significant,
0.10 > p > 0.05
Not significant,
p > 0.10
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Euceda, K.; Matamoros, G.; Araujo, M.E.; Chaver, L.; Ardón, G.; Fontecha, G. Genetic Diversity and Phylogeography of Plasmodium vivax Transmission-Blocking Vaccine Candidate Genes pvs47 and pvs48/45 in Honduras. Parasitologia 2025, 5, 36. https://doi.org/10.3390/parasitologia5030036

AMA Style

Euceda K, Matamoros G, Araujo ME, Chaver L, Ardón G, Fontecha G. Genetic Diversity and Phylogeography of Plasmodium vivax Transmission-Blocking Vaccine Candidate Genes pvs47 and pvs48/45 in Honduras. Parasitologia. 2025; 5(3):36. https://doi.org/10.3390/parasitologia5030036

Chicago/Turabian Style

Euceda, Kevin, Gabriela Matamoros, María Esther Araujo, Lesly Chaver, Gloria Ardón, and Gustavo Fontecha. 2025. "Genetic Diversity and Phylogeography of Plasmodium vivax Transmission-Blocking Vaccine Candidate Genes pvs47 and pvs48/45 in Honduras" Parasitologia 5, no. 3: 36. https://doi.org/10.3390/parasitologia5030036

APA Style

Euceda, K., Matamoros, G., Araujo, M. E., Chaver, L., Ardón, G., & Fontecha, G. (2025). Genetic Diversity and Phylogeography of Plasmodium vivax Transmission-Blocking Vaccine Candidate Genes pvs47 and pvs48/45 in Honduras. Parasitologia, 5(3), 36. https://doi.org/10.3390/parasitologia5030036

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