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Polymorphism in merozoite surface protein-1 gene in north & northwest Indian field isolates of Plasmodium vivax.

Malaria is an important tropical disease with an estimated global burden of 300 to 660 million cases every year, of which around 90 per cent occur in Sub-Saharan Africa where mortality due to malaria is reported to be higher than elsewhere (1,2). Recently Plasmodium vivax has resurged in several countries including Peru and China (3,4).

Though P. falciparum has shown an increased incidence during the last few decades in India, P. vivax continues to be a predominant species contributing around 60 per cent of total cases of malaria in India (5). Despite its public health importance, the molecular analysis of P vivax has been neglected, mainly because the parasite material is limited to small quantities obtained from either infected patients or primate experimental models and unlike P. falciparum, it usually dose not cause severe disease and only rarely causes complicated malaria.

The surface of invasive merozoites has been one of the major targets for research to develop an effective malaria vaccine. Merozoite surface protein -1 (msp-1) present on the surface is an important candidate for vaccine development against asexual blood stages (6). There is evidence to suggest that MSP-1 can elicit protective immune response against asexual blood stage challenge in animal models (6,7). P. vivax msp-1 (Pvmsp-1) is a 200 KDa glycoprotein (8) comprising either variable, semi variable or conserved blocks (9). The conserved blocks of Pv msp-1 gene show homology with the msp-1 of other Plasmodium species hence these conserved blocks have been designated as interspecies conserved blocks (ICB). The most variable segment of this gene is the one, flanked by interspecies conserved blocks 5 (ICB-5) and 6 and is named as polymorphic block-5 (10). The polymorphic block-5 of Pvmsp-1 gene has been reported to be dimorphic in nature (6,7). Two different alleles of this gene namely Belem (type -1) and Salvador-1 (type-2) were identified initially (6,7). Later, a third type was identified in Sri Lanka (11).

The complete gene sequences of the Brazilian Belem (Bel) and Salvador (Sal-1) strains are known. The analysis of specific gene segments derived from parasite isolates from Sri Lanka (11), Colombia (12), Thailand (13) and India (5,14) has indicated inter-allelic recombination between the sequences typified as Bel and Sal-1, thus supporting the notion that msp-1 polymorphism in P vivax is dimorphic in nature, as is in P falciparum (15).

Information on polymorphism of msp-1 gene in P. vivax isolates from India is limited. In the present study the extent of polymorphism in msp-1 gene among north and northwest Indian isolates of P. vivax was investigated.

Material & Methods

Parasite isolates: All patients with P vivax infection included. Blood samples were collected from symptomatic P. vivax malaria patients (Giemsa stained peripheral blood smear examination) who were attending Nehru Hospital, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, and other different clinics and hospitals in northern India. A total of 25 blood samples were collected during January 1999 to December 2001 from different States of northern India (6 from Delhi, 5 from Uttar Pradesh, 4 each from Chandigarh and Haryana, 2 each from Punjab, Rajasthan and Madhya Pradesh). Three patients showing mixed infection (with P. falciparum and P. vivax) were excluded. From each patient, 3-5 ml of blood was obtained by venepuncture in citrate anticoagulant (consisting of 38 mM citric acid, 74 mM sodium citrate and 88 mM sodium chloride) vials (16). The study protocol was approved by the Institute Ethics Committee of PGIMER, Chandigarh.

Isolation of parasite DNA: Parasite DNA was isolated according to the method described by Foley et al (17) with slight modification. Briefly, 50 [micro]l of parasitized blood was washed thrice with 1 ml of ice-cold 5 mM sodium phosphate (pH 8.0) by mixing well by vortexing and centrifuging at 10000 x g for 10 min. The cell pellet was finally resuspended in 50 [micro]l of sterile distilled water and heated in a boiling water bath for 10 min. It was cooled slowly to room temperature, centrifuged at 10000 x g and 40 [micro]l of supernatant was transferred to a new tube for DNA preparation. For PCR, 10 [micro]l of this was used as DNA template in 50 [micro]l PCR mixture.

Polymerase chain reaction (PCR): All PCR reactions were carried out in 50 [micro]reaction volume to amplify block-5 of Pvmsp-1 gene using specific primers (12) in a thermal cycler (Master cycler gradient, Eppendorf, Germany). Amplification was performed in 50 mM KCl, 10 mM Tris-Cl (pH 9.0), 1.5 mM Mg[Cl.sub.2], 0.01 per cent gelatin, 0.2 mM each of dNTPs, (GIBCO-BRL India Pvt. Ltd, Mumbai, India), 1.25 units of AmpliTaq DNA polymerase (GIBCO-BRL India Pvt. Ltd, Mumbai, India), 250 nM each of sense and antisense primers and 10 [micro]l parasite DNA template. The primers (12) used were, 5'ACT ACT TGA TGG TCC TC-3' and 5' TTG TGA CAT GCG TAA GCG-3' (Bangalore Genei Pvt. Ltd, India). The PCR condition were set for initial denaturation at 95[degrees]C for 5 min followed by 35 cycles of denaturation at 95[degrees]C for 1 min, annealing at 55[degrees]C for 2 min and amplification at 72[degrees]C for 2 min. The final extension was done at 72[degrees]C for 10 min. The DNA extracted from blood samples of P. falciparum infected cases and healthy individuals were used as controls for PCR amplification to check the specificity of the primers.

10 [micro]l of PCR products were run on 1.5 per cent agarose gel with ethidium bromide, visualized under UV transilluminator and photographed.

Restriction fragment length polymorphism (RFLP): The amplified product of msp-1 gene block-5 region was digested with Mva-1 (CCjWGG) endonuclease (Fermantas Life Sciences, USA). The selection of the enzyme was done by analyzing published sequences of Pvmsp-1 on website 'webcutter 2.0' (http://users. unimi.it/~camelot/tools/cut2.html) and also by using "Gene runner" software programme. Digestion was performed in a 20 [micro]l reaction mixture containing 16 [micro]l of PCR products, 1.5 ml sterile double distilled water, 2 [micro]l 10X digestion buffer and 0.5 [micro]l (5 units) of restriction enzyme. The digestion was carried out at 37[degrees]C for overnight. Finally, 10 [micro]l of each digested PCR product was analyzed on a 2 per cent agarose gel, stained with ethidium bromide, visualized under UV transilluminator and photographed.

Results

All the 25 clinical isolates showed amplification of target gene. No PCR product was obtained in control reactions. PCR amplified products of these isolates showed marked variations in molecular weight ranging from 450 to 550 bp (Fig. 1). On the basis of molecular weight of amplicons, the isolates could be categorized in four allelic types which were designated as type-1 to type-4 (Table I) according to the frequency of the alleles, type-1 being the most frequent and types-3 and -4 the least frequent (16% each). Two (8%) samples showed two allelic types each indicating mixed clone infection. One was mixture of allelic type-1 and -2 and other of type-3 and -4.

[FIGURE 1 OMITTED]

Analysis of PCR products by RFLP showed further differences among the allelic types. In each allelic type, the RFLP pattern seen in the majority of isolates was designated as sub allelic type A and those showing other patterns were grouped under sub allelic types B, C or D in order of decreasing frequency. Enzymatic digestion of allelic types-1 and -3 produced two distinct sub types (A and B, Fig. 2) while type -2 allele produced 4 subtypes (A, B, C and D, Fig. 1, Table II) Type-4 allele did not produce any sub type (Fig. 2). Interestingly, PCR products of a few isolates could not be digested with Mva-1 restriction enzyme (Fig. 1; Table II).

[FIGURE 2 OMITTED]

The RFLP analysis of isolates having mixed clones confirmed the isolates to be mixture of two different clones. The restriction digestion of RJ-6 (isolated and collected from Bikaner, Rajasthan, RJ abbreviation used for Rajasthan, Lane 1 and 2 Fig. 2) isolate (mixture of types-1&2) with Mva-1 produced four fragments of 350, 300, 250 and 150 bp showing the digestion of both the alleles (Fig. 2) while that of CH-3 (isolated from Chandigarh, CH, abbreviation for Chandigarh, Lane 3 and 4, Fig. 2) isolate produced 3 fragments of 450, 275 and 250 bp (Fig. 2) indicating the digestion of one allelic type (type-4) only (Table II). Type 3 clone in this mixed clone isolate was different from sub-allelic types 3A and 3B, as this allele was not digested with Mva-1. This sub-allelic type was designated as 3C (Table II). Thus, overall the 25 isolates of P. vivax could be differentiated into 9 genotypes by RFLP analysis.

Discussion

The use of single copy polymorphic genes like P. vivax msp-1 facilitates identification of heterogeneity in blood stage malaria parasites. In the present study, polymorphic region-5 flanked by interspecies blocks 5 and 6 (ICB 5 and ICB 6) of msp-1 gene was amplified by PCR from total parasite genomic DNA obtained from P. vivax isolates. These isolates showed significant size polymorphism in this region of the gene. This is in agreement with the earlier observation of Putaporntip et al (13) in Thai isolates and Maestre et al (5) in Indian and Colombian isolates, who have also observed significant size variation in their isolates. In the present study, on the basis of PCR, 4 different genotypes among 25 isolates were observed which confirmed highly polymorphic nature of the gene. Proto et al (18) have observed two types of alleles in Brazilian isolates while three allelic types were observed in Colombian isolates (12). In the present study the molecular size of the amplicon of target region of msp-1 of a few isolates of P. vivax (type-3, 450 bp) was close to that of Belem type (454 bp) as reported by Proto et al (18) while the size in some isolates (type 4 allele; 525 bp) was close to that of Sal -1 (517 bp) type (12,18). Premawansa et al (11) have reported the presence of third type of allele in Sri Lankan isolates which had, Sal-1 like block immediately followed by variable numbers of glutamine repeats which were followed by a Belem like sequence indicating that this allele originated as a result of intragenic recombination.

In contrast to findings of Maestre et al (5) showing absence of Belem type of sequence amongst 9 Indian isolates, in the present study we found both Belem as well as Sal-1 type of alleles amongst 25 isolates. However, we found larger numbers of other allelic types (types 1 & 2), probably due to frequent intragenic recombination in P. vivax. Maestre et al (5) also reported the event of intragenic recombination (44%) among Indian and Colombian Isolates. These findings indicate that recombinations are main source of genetic diversity in pvmsp-1 as suggested by other studies (11-13,29). In another study, Putaporntip et al (13) observed large size variation in 15 isolates within the same region of the gene and classified these into four types on the basis of sequence analysis. Our study also showed the presence of 4 allelic types on the basis of size of PCR product of same polymorphic region.

RFLP analysis of PCR amplified products of this polymorphic region revealed more differences among the isolates. With endonuclease Mva-1, 2-4 sub allelic types in three allelic type were obtained. However, PCR amplified product of three isolates belonging to different allele types could not be digested by repeated attempts, suggesting the loss of restriction site for Mva-1 enzyme. Similarly, three alleles of pvmsp-1 gene have been reported from China using PCR-RFLP (20). The variation in restriction site for endonuclease within the sequence of different alleles could be generated by intragenic recombination and/or additions or deletions of nucleotides in sequences (13). It is believed that the intragenic recombination frequently occur in P. vivax field isolates (5,21,22). A number of recombinant alleles have been identified among the Indian and Colombian (5), Korean (23) and Brazilian (19) isolates. Further, Kolakovich et al (10) identified that the hybrid sequence of allele showed more than one intragenic recombination.

Although several studies have reported higher degree of polymorphism in msp-1 gene in isolates from highly endemic areas but in the present study, isolates originating from areas with low to moderate endemicity also showed marked polymorphism in this gene. Since the exact degree of endemicity of each area from where the isolates were received is not known, the result could not be analyzed according to the degree of endemicity of malaria. Kim et al (14) have reported that the P. vivax parasite population is highly diverse in Kolkata, India, despite the low level of transmission.

Our results also showed that the use of PCR-RFLP technique to genotype P. vivax isolates on the basis of msp-1 gene might be a powerful tool to identify novel genotypes and could be applied in epidemiological studies and genetic surveys where sequencing is not possible or affordable. The limitation of this method is that it cannot detect some sequence changes like single point mutations, substitution and small deletions which can be detected by sequencing.

In conclusion, the study showed that extensive genetic polymorphism exists in msp-1 gene among the north and northwest Indian isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

Acknowledgment

Authors thank Dr C.R. Pillai, Emeritus Scientist, National Institute of Malaria Research, Delhi, for providing isolates of P. vivax and acknowledge the Indian Council of Medical Research (ICMR), New Delhi for financial support.

Received September 19, 2008

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Umar Farooq, N. Malla & M.L. Dubey

Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh, India

Reprint requests: Dr M.L. Dubey, Professor, Department of Parasitology, Postgraduate Institute of Medical Education & Research Chandigarh 160 012, India e-mail: mldubey@gmail.com
Table I. Allelic types of P. vivax based on molecular weight of PCR
products of MSP-1 gene (Polymorphic block-5) (n=25)

Approx. size of Allelic type No. of Allelic
PCR products (bp) P. vivax frequency
 isolates (%)

550 1 8 32
500 2 7 28
450 3 4 16
525 4 4 16
Mixed clone -- 2 8

Table II. Sub-allelic types of Pvmsp-1 gene (Polymorphic block-5)
based on RFLP pattern of PCR products (n=25)

 Allelic type on the Suballelic types on the basis of RFLP
 basis of PCR

Size (bp) Allelic type Approx. size of Sub-allelic
 digested products (bp) types

550 1 300 & 250 1A
 550 (Not digested) 1B
500 2 300 & 200 2A
 250 & 250 2B
 350 & 150 2C
 500 (Not digested) 2D
450 3 300 & 150 3A
 250 & 200 3B
525 4 275 & 250 -
450, 525 3 & 4 450 (Not digested) 3C
(Mixed clone) 275 & 250 4
550, 500 1 & 2 300 & 250 1A
(Mixed clone) 350 & 150 2C

Allelic type Suballelic types on
on the basis the basis of RFLP
of PCR

Size (bp) No. of Frequency of
 isolates allele (%)

550 6/8 75
 2/8 25
500 3/7 42.85
 1/7 14.28
 2/7 28.57
 1/7 14.28
450 3/4 75
 1/4 25
525 4/4 100
450, 525 1 --
(Mixed clone)
550, 500 1 --
(Mixed clone)
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Author:Farooq, Umar; Malla, N.; Dubey, M.L.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Dec 1, 2009
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