Abstract:parasite causes the largest number of malaria infection in some malarious areas of the world including Iran. Considering transfer and genetic dynamics of the parasite population in a specific area can help us to predict the spread of the infection either emergence of new cases or drug resistance in the context of elimination program in the malarious areas. Study on the genetic diversity of common alleles in a given geographical area, for vaccine and immune level studies can be important. The purpose of this st… Show more
“…Residue position 410 was multivariant—although its K410N form was found to be exclusive to Iran (figure 15 a , b ). A leucine insertion was also observed between V429 and P430 in subdomain 3, away from Site1 and Site2—as in India and Brazil [41,73,76,77]. Figure 15( a ) Graph showing Pv DBL SAAP profile within Iran.…”
Plasmodium vivax
(
Pv
) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in
Pv
DBL globally, with an emphasis on India as it remains a major contributor to the global
Pv
malaria burden. Based on 1358
Pv
DBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of
Pv
DBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the
Pv
DBL subdomain 2 (
α
1–
α
6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of
Pv
DBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in
Pv
DBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.
“…Residue position 410 was multivariant—although its K410N form was found to be exclusive to Iran (figure 15 a , b ). A leucine insertion was also observed between V429 and P430 in subdomain 3, away from Site1 and Site2—as in India and Brazil [41,73,76,77]. Figure 15( a ) Graph showing Pv DBL SAAP profile within Iran.…”
Plasmodium vivax
(
Pv
) malaria continues to be geographically widespread with approximately 15 million worldwide cases annually. Along with other proteins, Duffy-binding proteins (DBPs) are used by plasmodium for RBC invasion and the parasite-encoded receptor binding regions lie in their Duffy-binding-like (DBL) domains—thus making it a prime vaccine candidate. This study explores the sequence diversity in
Pv
DBL globally, with an emphasis on India as it remains a major contributor to the global
Pv
malaria burden. Based on 1358
Pv
DBL protein sequences available in NCBI, we identified 140 polymorphic sites within 315 residues of
Pv
DBL. Alarmingly, country-wise mapping of SAAPs from field isolates revealed varied and distinct polymorphic profiles for different nations. We report here 31 polymorphic residue positions in the global SAAP profile, most of which map to the
Pv
DBL subdomain 2 (
α
1–
α
6). A distinct clustering of SAAPs distal to the DARC-binding sites is indicative of immune evasive strategies by the parasite. Analyses of
Pv
DBL-neutralizing antibody complexes revealed that between 24% and 54% of interface residues are polymorphic. This work provides a framework to recce and expand the polymorphic space coverage in
Pv
DBLs as this has direct implications for vaccine development studies. It also emphasizes the significance of surveying global SAAP distributions before or alongside the identification of vaccine candidates.
“…Recent developments in high-throughput sequencing technology has enabled Insertion-deletions (INDEL)-based gene polymorphisms to construct high-resolution genetic maps and evaluate parasite population structures (33). The approach has been used for investigating polymorphisms in genes encoding gametocyte antigen, duffy binding protein and merozoite surface proteins (msp) (34)(35)(36).…”
Background
Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for P. malariae , an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics.
Methods
Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N=37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, genetic richness index, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity ( H d ) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed.
Results
Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), genetic richness index (0.050-0.250) and absence of linkage disequilibrium ( I A S = 0.03, p-value > 0.05 ) (N=37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.092, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10 -4 and was further reduced to 1.66 × 10 -4 when pmmsp1 polymorphisms were included.
Conclusions
Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.
“…Recent developments in high-throughput sequencing technology has enabled Insertion-deletions (INDEL)based gene polymorphisms to construct high-resolution genetic maps and evaluate parasite population structures [33]. The approach has been used for investigating polymorphisms in genes encoding gametocyte antigen, duffy binding protein and merozoite surface proteins (msp) [34][35][36]. Previous studies in P. vivax and P. falciparum have utilized gene polymorphisms in msp and circumsporozoite protein to study the parasite diversity [37][38][39].…”
Background: Molecular genotyping in Plasmodium serves many aims including providing tools for studying parasite population genetics and distinguishing recrudescence from reinfection. Microsatellite typing, insertion-deletion (INDEL) and single nucleotide polymorphisms is used for genotyping, but only limited information is available for Plasmodium malariae, an important human malaria species. This study aimed to provide a set of genetic markers to facilitate the study of P. malariae population genetics. Methods: Markers for microsatellite genotyping and pmmsp1 gene polymorphisms were developed and validated in symptomatic P. malariae field isolates from Myanmar (N = 37). Fragment analysis was used to determine allele sizes at each locus to calculate multiplicity of infections (MOI), linkage disequilibrium, heterozygosity and construct dendrograms. Nucleotide diversity (π), number of haplotypes, and genetic diversity (H d) were assessed and a phylogenetic tree was constructed. Genome-wide microsatellite maps with annotated regions of newly identified markers were constructed. Results: Six microsatellite markers were developed and tested in 37 P. malariae isolates which showed sufficient heterozygosity (0.530-0.922), and absence of linkage disequilibrium (I A S =0.03, p value > 0.05) (N = 37). In addition, a tandem repeat (VNTR)-based pmmsp1 INDEL polymorphisms marker was developed and assessed in 27 P. malariae isolates showing a nucleotide diversity of 0.0976, haplotype gene diversity of 0.698 and identified 14 unique variants. The size of VNTR consensus repeat unit adopted as allele was 27 base pairs. The markers Pm12_426 and pmmsp1 showed greatest diversity with heterozygosity scores of 0.920 and 0.835, respectively. Using six microsatellites markers, the likelihood that any two parasite strains would have the same microsatellite genotypes was 8.46 × 10 −4 and was further reduced to 1.66 × 10 −4 when pmmsp1 polymorphisms were included. Conclusions: Six novel microsatellites genotyping markers and a set of pmmsp1 VNTR-based INDEL polymorphisms markers for P. malariae were developed and validated. Each marker could be independently or in combination employed to access genotyping of the parasite. The newly developed markers may serve as a useful tool for investigating parasite diversity, population genetics, molecular epidemiology and for distinguishing recrudescence from reinfection in drug efficacy studies.
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