Role of Plasmodium vivax Duffy-binding protein 1 in invasion of Duffy-null Africans

Significance

Duffy-null Africans were thought to be resistant to Plasmodium vivax infection. Recently, P. vivax infection was observed in Duffy-null Africans. This parasite adaptation is potentially a serious public health problem as the majority of African populations are Duffy-null. This article is aimed at understanding whether mutations or DNA expansion in Duffy-binding protein (DBP) contributes to P. vivax Duffy-null infection. Importantly, P. vivax infection in Squirrel monkey has an ability to use an invasion pathway that is independent of the DBPs. Thus, P. vivax may use a different ligand–receptor pair for its infection in Duffy-null Africans, or some Duffy-negative Africans are not null but express a low level of Duffy blood group antigen.

Abstract

The ability of the malaria parasite Plasmodium vivax to invade erythrocytes is dependent on the expression of the Duffy blood group antigen on erythrocytes. Consequently, Africans who are null for the Duffy antigen are not susceptible to P. vivax infections. Recently, P. vivax infections in Duffy-null Africans have been documented, raising the possibility that P. vivax, a virulent pathogen in other parts of the world, may expand malarial disease in Africa. P. vivax binds the Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). To determine if mutations in DBP1 resulted in the ability of P. vivax to bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained from two Duffy-null individuals living in Ethiopia where Duffy-null and -positive Africans live side-by-side. We determined that, although the DBP1s from these parasites contained unique sequences, they failed to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did not account for the ability of P. vivax to infect Duffy-null Africans. However, an unusual DNA expansion of DBP1 (three and eight copies) in the two Duffy-null P. vivax infections suggests that an expansion of DBP1 may have been selected to allow low-affinity binding to another receptor on Duffy-null erythrocytes. Indeed, we show that Salvador (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and perhaps P. vivax in Duffy-null patients may have adapted to use new ligand–receptor pairs for invasion.

In 1975 we identified the failure of Plasmodium knowlesi to invade Duffy-null erythrocytes (1). We assumed that the Duffy blood group null phenotype, a common African phenotype, conferred resistance in Africans to P. vivax, a Plasmodium closely related to P. knowlesi. Subsequent studies demonstrated that African American volunteers who were Duffy-null were resistant to mosquito-transmitted P. vivax (2). In addition, African American soldiers in Vietnam who were infected with P. vivax were all Duffy-positive (3). Furthermore, African Americans in a village in Honduras who were infected with P. vivax were all Duffy-positive whereas those infected with P. falciparum were both Duffy-null and -positive (4). We concluded that Duffy null was the basis of resistance to P. vivax by Africans.

The molecular basis of Duffy null was a single point mutation in the GATA1-binding sequence in the promotor region 5′ to the Duffy blood group ORF (Fig. 1B) that led to Duffy-blood-group–null erythrocytes (5). In vitro studies with P. knowlesi demonstrated that the invasive merozoites were able to bind and reorient apically with Duffy-null erythrocytes but could not form a junction as occurred in Duffy-positive erythrocytes, indicating that the Duffy blood group was required for P. vivax invasion (6). Later, in P. knowlesi parasite culture supernatants, the parasite ligand binding to Duffy blood group antigen was identified as the Duffy-binding protein 1 (DBP1) (7). Subsequently, P. vivax DBP1 was identified by its ability to bind to Duffy-positive but not to Duffy-null erythrocytes (8). Furthermore the domain within DBP1 that conferred Duffy binding was determined to be the cysteine-rich region 2 of DBP1 (9, 10). Later, Duffy blood group antigen was shown to bind DBP1 through a sulfated tyrosine in its first extracellular domain (Fig. S1) (11).

Fig. 1.

P. vivax infection in Duffy-null Ethiopians. (A) The blood film contains P. vivax ring-stage parasites in a Duffy-null Ethiopian. (Right) The third panel shows the irregular amoeboid shape of P. vivax in the erythrocyte. (B) GATA1 transcription factor binds to a specific sequence (CTTATCTT) in the upstream promoter region of the Duffy blood group antigen. Binding of GATA1 transcription factor is required for the expression of Duffy antigen Fya or Fyb on human erythrocytes. A similar GATA1-binding site is observed in both Squirrel and Aotus monkeys’ Duffy blood group antigen. The point mutation at the GATA1-binding site from T to C at position −33 leads to loss of GATA1 binding and results in a homozygous Duffy-null blood type. The heterozygous Duffy blood type individual will have this point mutation (T-33C) in one of the alleles whereas the other allele is wild type (T −33) and will have less Duffy blood group expression on the erythrocyte surface. (C) Nested PCR amplification using P. ovale-specific 18S rRNA gene primers. The gel image is representative of two independent experiments. The sample with P. ovale (control MR4-180) shows a band size of ∼700 bp. In the two Duffy-null samples as well as the remaining control samples, no band was observed for P. ovale. (D) P. vivax- and P. falciparum-specific primers were used in combination in PCR. P. vivax and P. falciparum infection corresponds to a band size of 100 and 200 bp, respectively. Both the Duffy-null Ethiopian patients were specifically infected by P. vivax and not by P. falciparum.

Fig. S1.

Alignment of Duffy blood group antigen. Multiple sequence alignment of Duffy blood group antigen of Squirrel (NCBI: XM_003937900.2), Aotus (NCBI: XM_012449733.1) monkeys, and humans (NCBI: NP_002027.2) shows that they are the same length with minor differences in sequence. The red block shows the epitope region detected by anti-Fy6. The first tyrosine (Y at the 30th position) is important for chemokine binding, and the second tyrosine at the 41st position is sulfated for DBP1 binding. The amino acid at position 42 determines Fya or Fyb blood group, G at the 42nd position determines human Fya blood group antigen, and D at the 42nd position determines Fyb. Duffy null in Africa has a D in position 42 (Fyb), but is not expressed because of a mutation in the GATA1 transcription factor-binding site (Fig. 1B). Squirrel and Aotus monkeys are the Fyb type. Four unique mutations (UM) are observed in the N terminus of the Squirrel monkey sequence. The blood group has seven transmembrane (TM) domains (underlined).

Recently, Duffy-null individuals were found to be infected with P. vivax, both throughout Africa (Kenya, Madagascar, Mauritania, Cameroon, Angola, Equatorial Guinea, Ethiopia, and Sudan) and in South America (12–21), raising the question: How can P. vivax invade in the absence of Duffy blood group expression? Is it possible that mutations in the cysteine-rich region 2 of the P. vivax DBP1 allows binding to another protein on the surface of Duffy-null erythrocytes, unrelated to the Duffy? Alternatively, the widespread duplication of the gene encoding DBP1 observed in Madagascar (14, 22) and the three and eight copies of DBP1 in the two Duffy-null P. vivax-infected patients in Ethiopia (present paper) may allow for low-affinity binding of DBP1 to a new receptor on Duffy-null erythrocytes.

We tested the binding of P. vivax DBP1 in two Duffy-null individuals living in Ethiopia to Duffy-positive and -null erythrocytes and provide evidence that the former possibility was not the case; these DBP1s did not bind Duffy-null erythrocytes. However, concerning the latter possibility, we also provide evidence for DBP1 and DBP2/Erythrocyte-Binding Protein (EBP) (23) independent infection of Squirrel monkeys by P. vivax Salvador (Sal) I.

Results

P. vivax DBP1 Polymorphisms in Binding to Duffy-Null Erythrocytes.

Our study areas in Ethiopia comprise 65% (129 of 200) Duffy-positive and 35% (71 of 200) Duffy-null individuals. The P. vivax infections in Duffy-null Ethiopians is less severe than in Duffy-positive Ethiopians (20) as was observed in Madagascar (14). From the Ethiopian sample collection, we identified two Duffy-null individuals infected with P. vivax. The blood film from one of the Duffy-null individuals with P. vivax shows ring-stage parasites confirming the blood-stage infection in this patient (Fig. 1A). These two Duffy-null individuals had homozygous mutation (C/C) for Duffy-null in the GATA1-binding region upstream of the ORF (Fig. 1B) and had the ribosomal sequence for P. vivax and not for P. falciparum or P. ovale (Fig. 1 C and D).

The next set of data was the sequence of the P. vivax DBP1 in Duffy-blood-group–null Ethiopians. DBP1s in the two Duffy-null patients were different (Fig. 2A). The two sequences in pRE4 expressed on the COS-7 cell surface did not bind Duffy-null erythrocytes but did bind to Duffy-positive erythrocytes (Fig. 2 B and C). Despite a unique sequence for the DBP1 from the Duffy-positive Ethiopian sample, the region expressed in pRE4 bound strongly to Duffy-positive erythrocytes, but did not bind to Duffy-null erythrocytes (Fig. 2 B and C).

Fig. 2.

Mutations observed in region II of DBP1 from different isolates do not bind Duffy-null erythrocytes. (A) The observed mutations in DBP1 region II in Madagascar (22) and Ethiopia field isolates and in India VII (PVIIG_04680.1) and Brazil I (PVBG_05060.1) strains that were grown in Squirrel monkeys are compared with Sal I (PVX110810). Importantly, Ethiopia Duffy (+) is the P. vivax Duffy-positive sample, and Ethiopia Duffy (−) 1 and 2 are the P. vivax samples from two Duffy-null/negative (−) individuals. The amino acid number for each mutation site is given based on the Salvador I sequence from PlasmoDB. Between position 429 and 430 is an Indel for leucine (L) in the Indian VII and Brazil I sequence. (B) Mutated DBP1 sequences from these isolates were cloned and expressed in COS-7 cells using the pRE4 vector. The binding assay was performed using both Duffy-positive and Duffy-null erythrocytes. The binding assays show that, regardless of mutations in DBP1, the mutants still bind only Duffy-positive and not Duffy-null erythrocytes. EBP/DBP2 (GenBank: KC987954) binds both Duffy-positive and -null erythrocytes at low frequency. The PfRH5 (PlasmoDB ID: PF3D7_0424100) full-length extracellular domain (64–1578 bp), which is cloned in pRE4 vector, does not bind to either Duffy-positive or -null erythrocytes. In addition, no plasmid-binding control is also plotted. The data are plotted as a log scale on the y axis. At least three independent experiments with replicates were performed. The error bars indicate the SD. (C) Microscopy images show the erythrocyte rosette formation in COS-7 expressing DBP1 from Salvador 1, Ethiopia, Madagascar, India VII, Brazil I, and EBP/DBP2 from Cambodia. Note that the rosettes are smaller in EBP/DBP2. Blue (Hoechst) stain the COS-7 cell nuclei. (Scale bar, 10 µM.)

We tested the P. vivax sequence from Madagascar that was found in Duffy-positive and heterozygote Duffy positive/null individuals (Fig. 2A) (22). The DBP1 from Madagascar was duplicated, and both sequences were identical in the DBP1 domain except for a single amino acid difference in the signal sequence. The Madagascar DBP1 region 2 inserted into pRE4 and expressed on COS-7 cells bound Duffy-positive erythrocytes but failed to bind Duffy-null erythrocytes (Fig. 2 B and C).

The DBP1 domains were highly mutated in India VII and Brazil I compared with the original P. vivax Sal I sequence. In addition to the mutations, the codon for leucine was inserted in these isolates (Fig. 2A), which was unique in the DBP1 sequences. Despite the variety of sequences, all bound Duffy-positive erythrocytes; none bound Duffy-null erythrocytes (Fig. 2 B and C).

Copy Number Expansion of DBP1 in P. vivax-Infected Duffy-Null Patients.

Recently, the whole-genome sequencing of P. vivax from a Duffy-positive patient in Madagascar has revealed copy number expansion of Duffy-binding protein 1, and the existence of these two DBP1 copies next to each other was confirmed by PCR (22). A recent publication describes the gene copy number variation of DBP1 in P. vivax from Western Thailand (2 and 3 copies), Western Cambodia (2 copies), Papua Indonesia (2 and 3 copies) (24). In our study, to minimize potential issues with primer binding because of different boundaries and heterogeneity of DBP1 duplication lengths, we performed quantitative real-time PCR targeted to DBP1 and found three and eight copies in the two Duffy-null samples from Ethiopia (Fig. 3) compared with the controls: one copy of PvDBP1 in Sal I (25); one and two copies of PvDBP1 in Cambodia by whole-genome sequencing (24); and one copy of aldolase from Sal I. The sample was negative in the no-DNA control. In addition, the sequences from each DBP1 from the two Duffy-null patients were identical in all of the expanded copies (sequenced in 32 and 36 DBP1 clones from the two Duffy-null patients).

Fig. 3.

Copy number expansion in Duffy nulls from Ethiopia. Real-time quantitative PCR plot of relative fluorescent units against the number of threshold cycles (Ct) of P. vivax DBP1 (red) in relation to the single-copy P. vivax aldolase (blue). The green line represents the cutoff where the optimal Ct value of each gene for each sample is determined. The difference in the optimal Ct values (∆Ct; *) between the targeted and reference genes reflects variation in gene copy number among the samples. The tabular material shows the qPCR data for DBP1 gene copy number for all of the isolates from two independent runs (four replicates each). The double asterisk (**) indicates the DBP1 copy number from the whole-genome sequence (WGS) of the two Cambodian samples (24) and the Sal I parasites (25).

DBP2 of P. vivax Structurally Related to DBP1 Binds Duffy-Null Erythrocytes.

Recently, a new ligand, called EBP/DBP2, was identified in a Cambodian isolate and in other African isolates (23). This protein shares a domain structure similar to DBP1. We expressed the protein in COS-7 cells and found that region II of DBP2 binds both Duffy-positive and Duffy-null erythrocytes at low frequency (Fig. 2 B and C). EBP/DBP2 may be a ligand for invasion of Duffy-null erythrocytes.

Invasion of Squirrel Monkey Erythrocytes Is Independent of DBP1 and DBP2.

It was previously shown that P. vivax infects Squirrel monkeys despite the failure of DBP1 to bind Squirrel erythrocytes (8, 26, 27). The Duffy blood group of Squirrel monkeys has the GATA1-binding domain that expresses the Duffy blood group, and previous studies have shown that anti-Fy6, an antibody to a domain upstream of the Duffy-binding region, binds Squirrel monkey erythrocytes (28) (Fig. 1B and Fig. S1). It is surprising to know that, despite the high similarity between the Duffy blood group antigens from Squirrel monkeys, Aotus monkeys, and humans (Fig. S1), Sal I P. vivax DBP1 does not bind Squirrel monkey erythrocytes (Fig. 4 A and B). Furthermore, we tested DBP1 from different P. vivax isolates (India VII and Brazil I) and found that DBP1 from India VII and Brazil I formed rosettes with Squirrel monkey erythrocytes at the same frequency as Aotus erythrocytes (Fig. 4 A and B). No duplication of DBP1 occurred in the P. vivax Sal I (Fig. 3) (25). DBP2, found in the subtelomeric region of chromosome 2, was deleted in P. vivax Sal I.

Fig. 4.

Salvador I P. vivax infection in the Squirrel monkey is independent of DBP1. (A) Salvador I DBP1 expressed in COS-7 cells does not bind Squirrel monkey erythrocytes but binds Aotus monkey erythrocytes. However, DBP1 mutations observed in India VII and Brazil I strains are capable of binding equally to both Squirrel and Aotus monkey erythrocytes. These data indicate that Salvador I infection in the Squirrel monkey is independent of DBP1 and can be a model system to study Duffy-null P. vivax infection that occurs in Africans. The data are plotted as a log scale on the y axis. At least three independent experiments with replicates were performed. The error bars indicate the SD. (B) Fluorescent microscopy images show the erythrocyte rosette formation on COS-7 expressing DBP1 from Salvador I, India VII, and Brazil I using Squirrel and Aotus monkey erythrocytes. Blue (Hoechst) stains the COS-7 cell nuclei. (Scale bar, 10 µM.)

Discussion

Despite different mutations in DBP1 in two Duffy-null Africans in Ethiopia and in Duffy-positive P. vivax infection in different parts of the world (Madagascar, India VII, Brazil I, and Sal I), none can bind Duffy-null erythrocytes when the variable sequences are expressed on COS-7 cells, although all bind Duffy-positive cells (Fig. 2). The basis of DBP1 DNA expansion in Ethiopia is unknown. Studies on DNA expansion in Plasmodium spp. identify different mechanisms. The simplest to understand is the type where the duplication allows for mutations in the second copy and as a result develops a different function. The six-cysteine in the P. falciparum family often has duplications, and the duplicates vary in sequence. For example, Pfs 48/45 is involved in bringing male and female gametes together to assist in fertilization, and its duplicate, Pfs 47, permits the ookinetes to pass through the mosquito midgut epithelial cells without being marked for destruction by nitrosylation (29). Another example is the duplication of CLAG3.1 and 3.2 with the worldwide isolates having the identical seven-amino-acid differences between CLAG 3.1 and 3.2 (30). CLAG 3.1/3.2 have a critical function in Plasmodium spp. in that they form a part of a transporter in the erythrocyte membrane (31). Again there is evidence that they may differ in function from the mutational differences between the copies (32).

The second type of DNA expansion that may be relevant to our finding involves selective pressure such that the gene protects the parasite by increasing its expression by DNA expansion. The DNA expansion can involve multiple genes around the selected gene. One example of this is the introduction of an inhibitor for P. falciparum dihydroorotate dehydrogenase (DHODH) that leads to DNA expansion around the gene and always includes a stretch of A’s (nucleotides) on each end of the expansion (33). Similar gene duplication also occurred in the P. falciparum Multi Drug Resistance 1 (MDR1) gene under pressure from mefloquine (34, 35). In another human infecting parasite Plasmodium knowlesi, duplication of DBPα, the Duffy blood group binding protein, occurred when the parasite was adapted to grow in human erythrocytes (36).

The data are limited on the phenotypic significance of increased DBP1 gene copy numbers in Ethiopia. However, we speculate that the increase in copy number may lead to increased messenger RNA and protein levels. The DNA expansion may facilitate binding to an alternative erythrocyte receptor that has a lower binding affinity to DBP1. The possibility also exists that some Duffy-null individuals may express a low level of Duffy blood group antigen on their erythrocytes despite the inability of GATA1 to bind to the Duffy blood group locus. Three studies have failed to find Duffy blood group antigen on the surface of Duffy-null erythrocytes (14, 37, 38), although extremely low copy number may have been missed. The possibility of low expression of Duffy in P. vivax-infected Duffy-null individuals has not yet been excluded.

The normal growth of Sal I P. vivax in Squirrel monkeys despite the absence of binding of Squirrel monkey erythrocytes to DBP1 (Fig. 4) and the deletion of DBP2 raises questions about the receptor used by the Sal I P. vivax on Squirrel monkey erythrocytes. The same receptor may be used for P. vivax invasion of Duffy-null erythrocytes. In addition to EBP/DBP2 that binds Duffy-null erythrocytes, Reticulocyte Homology (RH) genes that are similar in structure to those described in P. falciparum are found in P. vivax (39, 40). P. falciparum RH5 in the RH family was shown to be critical for invasion of erythrocytes (41), and it is possible that one of the P. vivax RH genes may be involved in invasion of Duffy-null erythrocytes. There are many other ligands that may explain the findings, including those yet to be described. P. vivax is not yet adapted to Duffy-null Africans in that it now causes a less severe infection than in Duffy-positive people (14, 20). The concern is that P. vivax may adapt to infection in Duffy-blood-group–null Africans such that it may become a new cause of severe disease (42–44).

Materials and Methods

DBP1 and DBP2 (EBP) Constructs in pRE4 Vector.

For the expression of DBP1 mutants from different P. vivax strains in COS-7 cells (American Type Culture Collection), the erythrocyte-binding domain (region II) (10) of different DBP1 mutants [Sal I: PVX_110810, one Ethiopia Duffy-positive and two Ethiopia Duffy-null patients; India VII: Broad Institute database ID PVIIG_04680.1, Brazil I: Broad Institute database ID PVBG_05060.1 and Madagascar (22)] was synthesized and cloned into the pRE4 vector (kind gift from Gary Cohen and Roselyn Eisenberg, School of Dental Medicine, University of Pennsylvania, Philadelphia) in unique ApaI and PvuII restriction enzyme sites. The EBP/DBP2 gene sequence (511–1,452 bp) was also cloned in the pRE4 vector. The EBP/DBP2 full-length sequence is available in GenBank (KC987954). For negative control, the PfRH5 (PlasmoDB ID: PF3D7_0424100) gene sequence (64–1,578 bp) without the signal sequence was cloned in the pRE4 vector and does not bind human erythrocytes.

COS-7 Cells–Erythrocyte-Binding Assay.

The assay was performed in an eight-well chambered cover glass (LAB-TEK, Thermo Fisher Scientific). COS-7 cells were maintained in DMEM supplemented with 10% (vol/vol) FBS and nonessential amino acids at 37 °C with 5% CO₂. The day before transfection, the COS-7 cells (2.5 × 10⁴ cells) were plated on an eight-well chamber. Later the cells were transfected with 200 ng of DBP1-RII from Salvador I, Ethiopia (one Duffy-positive and two Duffy-null samples), Madagascar, India VII, Brazil I, and EBP/DBP2 (Cambodia) using Lipofectamine LTX plus reagent (Invitrogen) according to the manufacturer’s protocol. The negative control used in this study is the PfRH5 gene cloned into the pRE4 vector, which did not bind to either Duffy-positive or -null erythrocytes. The transfected cells were kept at 37 °C for 48 h for the surface expression of DBP1-RII, EBP/DBP2, and RH5 in transfected COS-7 cells. After 48 h, the transfected COS-7 cells were incubated with human Duffy-positive and Duffy-null erythrocytes or Squirrel monkey or Aotus monkey erythrocytes (10% hematocrit) for 2 h at 37 °C. After incubation, the cells were washed three times with incomplete RPMI. In total, 20 or 64 fields were counted for rosette formation from monkey and human rosettes, respectively. The blood used in this study was received from the Interstate Blood Bank, Memphis, TN, and the NIH blood bank. Monkey erythrocytes were obtained in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee (NIH).

An immunofluorescence assay was performed to determine the transfection efficiency using the ID3 antibody that recognized the N terminus of pRE4 and the DL6 antibody that recognized the extracellular C-terminal end (45). The nuclei were stained with Hoechst stain. Immunofluorescence images were taken using 40× objectives from Leica Epi microscope using LASAF software, and the rosettes were counted using 20× objectives from Carl Zeiss Axiovert 40 CFL microscope.

Diagnosis of P. vivax and Duffy Blood Group Antigen Sequencing.

Clinical samples from Jimma, Ethiopia, were collected to determine P. vivax infection and Duffy blood type. Scientific and ethical clearance was obtained from the institutional scientific and ethical review boards of Jimma University, Ethiopia, and the University of California, Irvine. Written informed consent/assent for study participation was obtained from all consenting heads of households, from parents/guardians (for minors under the age of 18), and from adults for each individual who was willing to participate in the study. For each malaria symptomatic or febrile patient, three to four blood spots, equivalent to ∼50 µL each, were blotted on Whatman 3MM filter paper (Sigma). Parasite DNA was extracted from dried blood spots by the Saponin (Fluka)/Chelex (Bio-Rad) method (46), and genomic DNA was eluted in a total volume of 200 µL TE buffer.

Slides were examined under microscopes using a 100× objective. All slides were read in duplicate by two independent microscopists at the time of sample collection. A nested amplification of the 18S rRNA gene of Plasmodium (P. falciparum, P. vivax, P. malariae, and P. ovale) was performed to identify positive infection and parasite species using published protocols (47, 48). Genomic DNA of each sample was amplified in duplicate for verification. In addition, parasite DNA content was estimated using the SYBR Green qPCR detection method using species-specific primers that targeted the 18S rRNA genes (20, 49). All PCR assays included positive controls of both P. falciparum 7G8 (MR4-MRA-926) and HB3 (MR4-MRA-155) isolates as well as P. vivax Pakchong (MR4-MRA-342G) and Nicaragua (MR4-MRA-340G) isolates (MR4, https://www.beiresources.org/About/MR4.aspx, in addition to negative controls, including uninfected samples and water.

For all P. vivax-positive samples, an ∼500-bp fragment of the human Duffy blood group antigen gene that encompasses the −33rd nucleotide position located in the GATA1 transcription factor-binding site of the gene promoter was amplified and sequenced following published protocols (5, 50). The point mutation T-33C leads to failure of Duffy antigen expression on the surface of erythrocytes, and individuals with homozygous (C/C) are Duffy-null (5).

PCR and Cloning of DBP1 to Identify the Similarities/Differences Between Expanded DBP1 Copies.

Primers of DBP1 region 2 (forward: 5′-GATATTGATCATAAGAAAACGATCTCTAGT-3′; reverse: 5′-TGTCACAACTTCCTGAGTATTTTTTTTAGCCTC-3′) were designed based on the DBP1 region 2 (PVX_110810) reference sequence of P. vivax Sal I. Amplification was conducted in a 20-µL reaction mixture containing 2 µL of genomic DNA, 10 µL of 2×DreamTaqTM Green PCR Master Mix (Fermentas), and 0.5 µM primer. Amplification reactions were performed in a Bio-Rad MyCycler thermal cycler, with an initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 90 s with a final 5-min extension at 72 °C. PCR products were cloned using pDrive vector (Qiagen). For the two Duffy-negative individuals, 32 and 36 clones were sequenced to determine the sequence for the DNA expansion.

Plasmids were sequenced in both forward and reverse directions using the M13 primers (forward: 5′-GTAAAACGACGGCCAGT-3′; reverse: 5′-AACAGCTATGACCATG-3′) on ABI 3100 (Applied Biosystems) automated DNA sequencer with BigDye dye terminator cycle sequencing kits. Sequences were aligned with ClustalX (51) and manually edited in BIOEDIT (52).

Real-Time Quantitative PCR Detection of PvDBP1 Gene Copy Number.

We examined PvDBP1 DNA expansion in the Ethiopian Duffy-null samples using primers (forward: 5′-AGGTGGCTTTTGAGAATGAA-3′; reverse: 5′-GAATCTCCTGGAACCTTCTC-3′) were designed between region II to III of PvDBP1 (PVX_110810) and used along with a reference gene, P. vivax aldolase, which is known to be a single-copy gene (forward: 5′-GACAGTGCCACCATCCTTACC-3′ and reverse: 5′-CCTTCTCAACATTCTCCTTCTTTCC-3′) (53) from P. vivax Sal I to estimate gene copy number by the SYBR Green qPCR detection method. In addition, two samples from Cambodia, one with a single DBP1 copy and another sample with two copies determined by whole-genome sequencing (24), were used as positive control. Water was used as the no-DNA control.

Amplification was conducted in a 20-µL reaction mixture containing 2 µL of genomic DNA, 10 µL 2×SYBR Green qPCR Master Mix (Thermo Scientific), and 0.5 µM primer. Reaction was performed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with an initial denaturation at 95 °C for 3 min, followed by 45 cycles at 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min with a final 95 °C for 10 s. This was then followed by a melting curve step of temperature ranging from 65 °C to 95 °C with a 0.5 °C increment to determine the melting temperature of each amplified product. Each assay included an internal reference gene, the P. vivax aldolase, which is known to be a single copy gene in P. vivax, as well as negative controls (uninfected samples and water). The PvDBP1 copy number (n) in each sample was quantified based on the threshold cycle (Ct) using the follow equation: n = 2^∆∆Ct±SD, where ∆∆Ct = (Ct_pvaldo − Ct_pvdbp1) − (Ct_pvaldocal − Ct_pvdbp1cal). Ct_pvaldo and Ct_pvdbp1 are threshold cycle values for the aldolase gene and the dbp1 gene, respectively, whereas Ct_cal is an average difference between Ct_aldo and Ct_pvdbp1 obtained for the positive control Salvador I that contains a single copy of PvDBP1 and aldolase gene fragments. SD is SD calculated as follows: SD = √(S²_pvdbp1 + S²_pvaldo + S²_cal), where S_pvdbp1 and S_pvaldo are the SDs from the average Ct calculated for three replicates in the pvdbp1 and pvaldo amplifications, and S_cal is an average SD of the ∆Ct values for the calibrator.