Molecular and Biochemical Parasitology 73 (1995) 103-110

MOLECULAR

iYi%HEMlcAL PARAsIToLoGy

Sequence heterogeneity of the C-terminal, Cys-rich region of the merozoite surface protein-l ( MSP-1) in field samples

of Plasmodium falciparum

Yang Kang, Carole A. Long * Department of Microbiology and Immunology, Medical College of Pennsylvania and Hahnemann University, Center City Campus,

Mail Stop 410, Broad and Vine Streets, Philadelphia, PA 19102-1192, USA

Received 13 February 1995; accepted 30 May 1995

Abstract

Recent results with primate plasmodia and rodent models of infection have focused attention on the C-terminal region of the merozoite surface protein-l (MSP-1) as one of the leading candidates for vaccination against the erythrocytic stages of malaria. However, sequence heterogeneity of this region may compromise its use as a vaccine candidate. While the C-terminal region of MSP-1 from the two prototypic alleles of P. fulcipurum has been shown to be relatively conserved in laboratory-maintained strains, little data exist on sequence heterogeneity of this region in field isolates from diverse geographic areas. To address this question, DNA encoding the C-terminal, Cys-rich region of P. falciparum MSP-1 from field samples was analyzed by a polymerase chain reaction (PCR)-direct sequencing method. Sequence data were consistent with those obtained from laboratory-maintained strains. In 15 isolates from Africa, Asia and Latin America, only a few

nucleotide changes were found leading to amino-acid alterations at four positions out of 102 residues. All the variations corresponded to the predicted amino-acid sequence of the other prototype, suggesting that these changes were possibly due

to allelic recombinations. The four changes were E + Q at position 1644 and TSR -+ KNG, or KNG + TSR at positions

1691, 1700 and 1701. Thus, only three patterns of the C-terminal, Cys-rich region of MSP-1, E-TSR, Q-KNG and Q-TSR, were detected. All the Cys residues were conserved. These results support the potential utility of the C-terminal region of MSP-1 as a vaccine candidate.

Keywords: Plasmodium fakiparum; Malaria; Merozoite surface protein-l; Sequence heterogeneity; Vaccine

1. Introduction

Abbreviations: EGF, epidermal growth factor; MSP-1, merozoite

surface protein-l; PCR, polymerase chain reaction; rMSP-I,,,,

secreted 19-kDa C-terminal fragment of merozoite surface pro-

tein-l produced in yeast.

* Corresponding author. Tel.: (l-215) 7624706; Fax: (l-215)

762-8075; e-mail: longc@hal.hahnemann.edu

Malaria, a disease caused by parasites of the genus Plasmodium, is still a very serious problem in

many regions of the world. Current efforts to control malaria include vector control, development of new chemotherapeutic agents and formulation of effica- cious vaccines. We are interested in vaccine develop- ment directed toward blood stages of the parasite,

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0166-6851(95)00102-6

104 Y. Kang, CA. Long/Molecular and Biochemical Parasitology 73 (1995) 103-110

since it is the erythrocytic stages of infection that are responsible for morbidity and mortality associated

with human malaria. One of the most promising

vaccine candidates against blood stage parasites is the major merozoite surface protein-l (MSP-1) [l-7].

The MSP-1 of P. falciparum is a 195~kDa protein

expressed on the surface of the merozoite. It under- goes two steps of proteolytic cleavage during the

maturation of the merozoite [4]. It is first cleaved into four major fragments, and the C-terminal 42-kDa fragment is then further cleaved into two fragments, whose apparent molecular masses are 33 kDa and 19 kDa, prior to invasion of merozoites into erythro-

cytes. While the other fragments are shed from the

surface of the merozoite, the 19-kDa fragment is the only fragment to remain on the surface of the mero-

zoite after invasion [8]. The MSP-1 of P. falciparum is encoded by alle-

les of a single gene which can recombine and gener- ate polymorphism [9]. Recombination between two prototypic alleles of P. falciparum MSP-1, repre-

sented by PfMAD20 and PfKl/Wellcome se- quences, can account for some of the variability seen in different laboratory strains. When MSP-1 amino-

acid sequences from different isolates are compared, this protein can be divided into highly conserved,

semi-conserved and variable regions [4,9]. The C- terminal 1PkDa fragment of MSP-1 is a highly

conserved region [9,10], and contains a series of Cys residues which are conserved among different species

of plasmodia infecting humans, primates and rodents [ll]. It has been suggested that these Cys residues are arranged as two putative epidermal growth factor (EGF)-like domains [12], and this Cys-rich region may play an important role in eliciting protective immune responses against malaria infection [13-211. In rodent models of infection, the passive transfer of

a monoclonal antibody, Mab 302, can protect mice completely against lethal challenge with P. yoelii [14]. The epitope recognized by Mab 302 has been mapped to the C-terminal Cys-rich region of the P.

yoelii MSP-1 [l&16]. This epitope depends on the correct configuration of disulfide bonds and has been localized to the first EGF-like domain [16,17]. More- over, when expressed as a fusion protein with glu- tathione S-transferase (GST) from Schistosoma japonicum, the two EGF-like domains appear to retain their native structure and can immunize mice

against an otherwise lethal challenge infection [18]. This finding was confirmed by Ling et al. [19]. In the

case of P. falciparum MSP-1, rabbit antisera against a recombinant 42-kDa protein from the C terminus

of MSP-1, including both a variable region (block

16) and the highly conserved Cys-rich region (block 171, can extensively cross-react with heterologous alleles of MSP-1 and is equally effective in inhibit-

ing in vitro growth of parasites of both prototypic alleles [20,21]. Moreover, monoclonal antibodies

which can inhibit P. falciparum invasion in vitro can also recognize the first EGF-like domain of this region [13]. All these data suggest that the conserved

C-terminal region may be a strong subunit vaccine

candidate. Most of the sequence data of the C-terminal

region of P. falciparum MSP-1 have been obtained from laboratory-maintained strains, and little data

exist on sequence heterogeneity of this region in field samples from diverse geographic areas other than analysis of sequence data on samples obtained from Thailand [22]. It is, therefore, possible that data obtained from cultured parasites may not reflect the

heterogeneity prevalent in field samples. To address this question, DNA encoding the C-terminal, Cys-rich

region of MSP-1 of P. falciparum was amplified using PCR and the products were sequenced directly

from 15 field samples from Africa, Asia and Latin America.

2. Materials and methods

2.1. Origin of samples and preparation of parasite DNA

To assess the sequence heterogeneity of the C- terminal region of P. falciparum MSP-1 in field

isolates, DNA encoding this region was analyzed in

blood samples from different geographic areas by direct sequencing of PCR products. There are several advantages to this strategy: (i) Finger prick samples are easily obtained and transported. No special equipment is needed in the field and these samples can be kept at room temperature for a long period of time. (ii) This strategy does not require culturing of parasites, so that both culture-adapted and non- adapted isolates can be sequenced. (iii) The PCR-di-

Y. Kang, CA. Long/Molecular and Biochemical Parasitology 73 (1995) 103-110 105

rect sequencing method does not rely on cloning the

PCR products into vectors and transforming bacteria so that possible errors introduced in propagation of plasmodial DNA in E. coli are minimized. Some

difficulties have been reported in maintaining ge- nomic clones of parasite DNA in bacterial hosts [23]. This problem of clone instability is thought to be a result of the parasite’s unusually A + T-rich DNA

[241. 15 finger-prick samples of P. falciparum-infected

blood were collected from different countries in Africa, Asia and Latin America (Table 2). They were

dried on glass-fiber filter paper and later subjected to

Chelex 100 extraction 125,261. Briefly, 200 ~1 of 5% (w/v) Chelex (Bio-Rad, Richmond, CA, USA) solu-

tion in sterile water at pH 10.5, adjusted by the addition of 1 M NaOH, was added to a 1.5-ml microcentrifuge tube and placed in a heat block at

100°C for 10 min. Each filter paper with a single blood dot was excised with clean, sterile scissors and

soaked in 0.5% saponin/PBS (0.14 M NaCl/O.Ol M Na,HPO,, pH 7.2) for 20 min on ice and then washed with 1 ml PBS. The samples were then gently vortexed for 30 s, returned to the heat block for 10 min and then microcentrifuged at 12500 t-pm

for 1.5 min. 100 ~1 of supematant fluid was trans-

ferred to a new microcentrifuge tube and spun again. The parasite DNA in the supemate was used as

template for PCR reactions, and amplification was performed as soon as possible after Chelex extrac-

tion. To minimize contamination, each sample was kept in a separate plastic bag, and forceps and scis- sors used in the process were sterilized after prepara-

tion of each sample.

2.2. PCR amplifications

Two allele specific 5’ primers, M5 and K5, were derived from sequences in block 16 of P. falciparum MSP-1 gene (Fig. 1) of two strains: PngMAD20 and Wellcome [lo]. M5 and K5 represent the two proto- typic MSP-1 alleles, PfMAD20 and PfKl/Well- come respectively. The 3’ primer, T3, was derived

from DNA encoding the putative hydrophobic an- chor region (Fig. 1) [22]. The primary PCR reactions were performed by using two pairs of primers, M5 and T3, and K5 and T3, independently on the same sample. All reactions were carried out in a total

123 5 67 10 12 14 ,6 17

WI

iiL 4 --_,

Sl 52 + t te KS s3 S4 T3

Fig 1. Schematic representation of the gene encoding the C-termi-

nal region of P. falciparum MSP-1. The conserved, variable and

semivariable regions of the gene encoding PfMSP-1 are repre-

sented by open, filled and hatched boxes, respectively. The C-

terminal conserved region (block 17) and the variable region

(block 16) which are amplified includes nt 4800 to 5350 after Ref.

9. The location of Cys residues are indicated by vertical lines. The

directions of primers used for PCR and sequencing are indicated

by arrows. Primers M5, K5 and T3 were used in primary amplifi-

cations; M5, K5, T3, S4 and Sl were used for reamplifications:

M5, K5, S2, T3 and S3 were used as sequencing primers.

The sequences of primers are as follows: M5, 5’-

GATACGAAAAAAGATATGCGGC-3; K5, 5’-GCTGATT-

TATCAACAGATTATAACC-3’; T3,5’-TTAAGGTAACATATT-

‘PTAACTCCTAC-3’; Sl, 5’-AATTCTGGATGTITCAGACAT-3’;

S2, 5’-CCAAATCCTACTTGTAACG-3’; S3, 5’-TTCGT-

TACAAGTAGGATTTGG-3’; S4, S’-AGAGGAACTG-

CAGAAAATACC-3’.

volume of 50 ~1, including 2.5 ~1 of extracted DNA

supernatant/40 ng of each primer/O.5 units of Taq polymerase (Promega, Madison, WI, USA). The

thermocycler profile was 1 min 45 s at 94°C 1 min at 55°C and 1 min at 72°C for 45 cycles. The anticipated sizes of the PCR products are 508 bp or 485 bp, depending on the MSP-1 allele. Genomic

DNA from the Dd2 strain of P. falciparum passaged in culture was used as a positive control. Sterile distilled water and uninfected blood treated with

Chelex 100 were used as negative controls in all amplifications.

After analysis by agarose gel electrophoresis, 1 ~1 of each primary PCR product was used as tem- plate, and two pairs of nested primers, M5 or K5 and S4, Sl and T3, were used to reamplify DNA encod-

ing the C-terminal, Cys-rich region of MSP-1 on each template (Fig. 1). The secondary PCR reactions were done in 100 ~1 volume with 35 cycles of amplification (45 s at 94°C 45 s at 50°C and 45 s at 72°C).

106 I’. Kang, CA. Long/Molecular and Biochemical Parasitology 73 (1995) 103-110

2.3. Purification of PCR products

The PCR products from three identical reactions with nested primers were pooled together and sub- jected to 8% polyacrylamide gel electrophoresis. Gels were stained with ethidium bromide and the appro-

priate size bands were excised from the gel and transferred into dialysis bags in 1:50 TAE buffer (1 X TAE: 0.04 M Tris . acetate/O.001 M EDTA,

pH 8.0). Electroelution was then performed in the 1:50 TAE buffer with 10 PA current overnight. The

eluted DNA in the dialysis bag was collected and

treated with Wizard PCR Prep (Promega), following the manufacturer’s instructions. The DNA concentra-

tion was measured by spectrophotometry (A,,,).

2.4. DNA sequencing

2-3 pg of purified PCR products were sequenced directly with the modified dideoxy chain-termination technique [27] using Sequenase version 2.0 kit (US

Biochemical, Cleveland, OH, USA). Sequencing primers used are shown in Fig. 1.

3. Results

Parasite DNAs in field samples from different origins were extracted with Chelex 100 and sub- jected to PCR amplification using allele specific 5’ primers, M5 and K5, and the conserved 3’ primer T3. After 45 cycles of amplification, the products

were analyzed by agarose-gel electrophoresis and

PCR results are shown in Table 1. Of 15 samples tested, 8 showed the positive 485-bp bands only

when M5 and T3 were used as primers; 2 demon- strated 508-bp bands only when K5 and T3 were used as primers; and the other 5 samples showed

bands of the appropriate sizes using both pairs of primers (Table 1). Thus, 20 PCR products were obtained using allele-specific primers M5 and K5, and the conserved 3’ primer T3. This result indicated that in the majority of the samples (10/15), only one

of the two prototypic alleles of MSP-1 could be detected; however, other samples (5/15) might con- tain more than one allele of P. falciparum MSP-1.

Each of the PCR products was reamplified with

two pairs of nested primers, M5 or K5 and S4, and Sl and T3 (Fig. 1). The products of reamplification

were then purified by polyacrylamide gel elec- trophoresis and electroelution. The two purified DNA fragments from each sample, MS-or K5-S4 and Sl- T3, were sequenced directly. To minimize possible PCR errors and confirm the variations found, PCR- amplifications on the same Chelex-treated samples

were repeated using allele-specific primers M5 and K5, and the conserved primer T3. The same proce-

dures were followed but the complementary strand

of the purified PCR products was sequenced. Most of the variations obtained in the primary sequencing

were confirmed and a few uncertain nucleotides were clarified. Eventually, 20 double-stranded se- quences were obtained from 15 field samples, and the deduced amino-acid sequences were compared with the corresponding sequences of the two proto- typic alleles, PfMAD20 and PfKl/Wellcome, repre- sented by PngMAD20 and Wellcome, respectively (Fig. 2)[10]. The amino-acid sequence of the 19-kDa

fragment of the PfKl/Wellcome allele is that of the Wellcome isolate. It represents the same allelic form

Table 1

Results of PCR amplification of the C-terminal conserved region of P. falciparum MSP-1 from field samples

Alleles

(Primers)

Samples

01 02 03 04 05 06 07 08 09 10 11 12 13 14 15

FTMAD20 + - + + - + + + + + + + + + +

(M5 and T3)

PfKl/Wellcome - + + + + + - - + - + - - - -

(KS and T3)

The PCR reactions were performed by using the allele-specific primers from block 16 of the MSP-1 gene, M5 and KS, and the conserved

primer from anchor region, T3 independently on the same sample. PCR products were either 485 or 508 bp, depending on the MSP-1 allele.

+ and - indicate whether bands of the appropriate size were observed using the corresponding primers.

Y. Kang, CA. Long/ Molecular and Biochemical Parasitology 73 (1995) 103-110 107

4/U mm0 Typ.(U) __________ ____-9____ _______-__ ________--

pnp-0 1‘19 ExmBQmQcv -T= maLD8uaac xc%LmmQBO IR- xl/r.llool. Typ.(,) ---------- _----_____ __________ __________

X,13 t/11 HaDlO Typ.,l,) __________ __________ __~_______ -110_ ______

PIW-NIDZO I“9 rum- ammecDn DaTcracDBa (IaauI~Bc mw I 10 xl/m.l~oc.u -VI ---------- ___--_____ _+-__-___ _~~_______

I/T a/7

aDto Typu1, --_____--- -----_____ __

png-ma10 1709 TXQDBIPLFD o*rcaIIaurL O~IIIIIamL lR- n/w.lro.m. _,l, ---------- __-_-_____ __

Fig 2. Amino-acid sequence variations in the C-terminal Cys-rich

region of P. falciparum MSP-1 from field samples. The amino-

acid sequences of parental alleles, Png-MAD20 and WELL-

COME, are shown in the middle two lines [lo]. The sequence of

the PngMAD20 is listed completely, a blank space in the WELL-

COME sequence indicates that the residue is identical to that of

the Png-MAD20. The sequences of MAD20 and Kl/Wellcome

type isolates from field samples are shown in the upper and lower

lines, respectively. Amino acids that are identical to those of the

corresponding alleles are indicated by dashes. The differences are

shown by letters. Only the position of the Png-MAD20 allele is

shown [lo]. The number above or below the variation in the

sequences refers to the frequency of variation. The rate for the

variations at position 1700 and 1701 is the same, thus indicated by

one number.

as Thai-Kl, but the DNA sequence is considered

more certain [lo]. The detailed nucleic acid and deduced amino-acid variations of each sample are shown in Table 2. The sequence data demonstrated that the 20 DNA sequences encoding the C-terminal,

Cys-rich region of MSP-1 of P. falciparum from diverse geographic areas were highly conserved with only a few nucleotide changes, leading to amino-acid variations at only four positions 1644, 1691, 1700 and 1701 out of the 102 residues sequenced (using

the numbering system of Ref. 10). All the Cys residues were conserved, and all the nucleotide

changes were non-synonymous, resulting in pre- dicted amino-acid alterations corresponding to the

residues of the other prototypic allele. This suggested

that these variations were possibly caused by allelic recombinations. Insertions or deletions were not de- tected in this region.

These four deduced amino-acid variations oc- curred in two regions. The first alteration (E -+ Q)

was at amino acid 1644, which is between the sec- ond and the third Cys residue in the first putative

Table 2 Nucleotide and putative amino-acid variations of the C-terminal conserved region of P. falciparum MSP-1 from field samples

Sample Geographic Variation

(Allele “) area At 1644 b At 1691 At 1700 At 1701 nt aa nt aa nt aa nt aa

01 (M) West Africa GAA+ CAA E+ Q, _c -

03 CM), 04 (Ml, Indonesia - - _ _

06 (M)

07 (M) Zimbabwe GAA-+CAA E-+Q, - _

08 (MI Guatemala - - _ -

09 (M) Guatemala GAA+CAA E-Q, -

10 (M) Ghana GAA-+CAA E-Q, ACA+AAA T+K AGC+AAC S-tN, AGA-,GGA R+G

11 (M) West Africa - _ _ _

12 (Ml, 13 CM), China _ _ _ _

14 CM), 15 (M)

02 (K), 05 (K> Indonesia GAA+CAA E+Q, - -

03 (K), 04 (K), Indonesia GAA-rCAA E-+Q, ACA+AAA T-K, AGC+AAC S-N, AGA-rGGA R+G

06 (K)

09 (K) Guatemala GAA+CAA E-Q, ACA+AAA T-K, AGC+AAC S-+N, AGA-,GGA R+G

11 (K) WestAfrica GAA+ CAA E+ Q, ACA+AAA T+K, AGC+AAC S-+N, AGA-tGGA R+G

a Refers to the allele-specific oligonucleotide from block 16 used for initial amplification reactions, either M for PfMAD20 allele or K for

PfKl/Wellcome allele.

b The number indicates the amino-acid position at Png-MAD20 [lo]. nt and aa refer to nucleotide and amino-acid, respectively.

’ Dash means that the residue is identical to that of the Png-MAD20 allele [lo].

108 Y. Kang, CA. Long/Molecular and Biochemical Parasitology 73 (1995) 103-110

EGF-like domain. It had the highest frequency of variation among these four changes. The second

group of variations (KNG + TSR, or TSR + KNG) was at positions 1691, 1700 and 1701, which are in the second putative EGF-like domain near the ninth Cys residue. Interestingly, these three amino-acid

variations always correspond to these residues of the alternate prototypic allele (Fig. 2 and Table 2). Over- all, our sequence data revealed three patterns in the

C-terminal conserved region of P. falciparum MSP-1

out of 20 sequences from field samples: E-TSR

(PfMAD20) type (9/20), Q-KNG (PfKl/Well- come) type (6/20) and Q-TSR type (5/20). The E-KNG pattern of this region was not detected in the

samples tested. The PfMAD20 allelic type of MSP-1 in the field

samples tested predominated as opposed to the PfKl/Wellcome allelic type (13:7). While all sam- ples from China showed the PfMAD20 sequence and contained no variations, the sequence patterns in this

region of MSP-1 of samples from other areas seemed

unrelated to geographic origin. Sample 10(M) from Ghana demonstrated allelic variations at all four positions, suggesting that allelic recombination oc-

curred at the whole C-terminal, Cys-rich region. Sample 02(K) and 05(K) demonstrated allelic varia- tions at the three downstream positions 1691, 1700 and 1701, but no variations at 1644 and the upstream

dimorphic region until position 1619 (data not shown); 07 (Ml from Zimbabwe showed the allelic variations at 1644 and the upstream dimorphic re-

gions until position 1620 (data not shown), but no variations at the three downstream positions, indicat-

ing that there could be a possible site of recombina- tion between positions 1644 and 1691 in these three samples. The 01(M) from West Africa and 09(M) from Guatemala demonstrated a single point allelic

variation at position 1644.

4. Discussion

By aligning published sequences of MSP-1 from a number of laboratory-maintained strains, Miller and colleagues [lo] found that there were amino-acid variations at only four positions (1644, 1691, 1700

and 1701) in the C-terminal, Cys-rich region of P. falciparum MSP-1. These changes resulted in four

possible sequence patterns at these positions: E-KNG, Q-KNG, E-TSR and Q-TSR. They considered the E + Q variation at position 1644 as the most com-

mon variation. Our finding of the three of these patterns in field isolates are therefore consistent with their sequence analysis. In addition to the amino-acid

variations at these four positions, three other amino- acid changes at positions 1669, 1672 and 1716 in

this region were also reported in field samples from Thailand [22]. These investigators used PCR amplifi-

cation followed by subcloning of the PCR products.

These three variations occurred at a relatively low frequencies (l/19, 3/19 and l/19). Of note is that, among the 19 Thailand samples tested, three samples showed the same V + G variation at position 1672. However, none of these alterations were detected in

our samples. The detected variations could be possibly caused

by several different mechanisms. Our data demon- strated that most of the sequences from the field

samples corresponded to one of the two prototypic alleles seen with laboratory isolates (Fig. 2). All

nucleotide variations were non-synonymous and cor- responded to the nucleotides of the other allelic prototype, suggesting that the variations in this re- gion are possibly due to intragenic recombinations

between the two prototypes. Alternatively, point mu- tations or gene conversion might also contribute to the variations [lo]. Our data revealed three patterns

of the 19-kDa fragment of MSP-1 of P. falciparum in the field samples tested. Although the E-KNG pattern was not detected, this pattern has been identi-

fied in the Uganda-PA strain [lo] and wild isolates

from Thailand [22], suggesting that all four patterns of the C-terminal, conserved region of MSP-1 exist

in the field. Recently, these four variations of the C-terminal conserved region of P. falciparum MSP- 1, E-TSR, Q-TSR, E-KNG and Q-KNG, were ex- pressed in yeast (rMSP-1,,,)[28]. Testing the reactiv- ity of a set of monoclonal antibodies to the C-termi- nal conserved region on yeast-derived proteins indi-

cated that both the conserved and variant B-cell epitopes of MSP-1 C terminus were authentically

recreated in rMSP-l,,, [28]. SDS-PAGE analysis of

rMSP-l,,, of the four different products suggested

that the single amino-acid change at 1644 (E + Q) in the first EGF-like domain caused a significant change in mobility of rMSP-l,,,, whereas simultaneous

Y. Kang, CA. Long/Molecular and Biochemical Parasitology 73 (1995) 103-110 109

changes in the other three amino-acid positions (TSR

-+ KNG) in the second EGF-like domain had a relatively small effect on the mobility of this protein [28]. They also showed that protective polyclonal antibodies from monkeys to parasite-produced MSP-1 (E-KNG pattern) recognized all four patterns of

rMSP-1,9, equally well, indicating that the four

amino-acid variations of the C-terminal conserved

region of MSP-1 may not affect the antigenicity of

this protein, Moreover, monoclonal antibody 5B1, which was reported to inhibit invasion in vitro [29],

recognized all four variants, suggesting that at least one potentially protective epitope in this region was

not variant-specific. It remains to be determined how these four variations affect the immunogenicity of the C-terminal, Cys-rich region of P. fulciparum MSP-1. It will also be of interest to determine whether immunization with the C terminus of MSP-1 will promote the selection of additional variants not seen in the field samples tested here.

Acknowledgements

We thank Dr. Kevin C. Kain for generously pro- viding most of the blood samples and for his helpful

suggestions in the method of Chelex extraction and Dr. Zaisong Huang for collecting samples from Hainan Province of China. We also thank Dr. Patrick J. Farley for kindly providing advice on technical and theoretical issues, Ms. Ranjana Srivastava for

assistance with PCR-sequencing and Mr. Paul Calvo, Mr. Tom M. Daly and Mr. Donghui Zhang for

helpful discussions. This work was supported by a grant from NIH (AI-21089) and by the Programme

for Research and Training in Tropical Disease of the World Health Organization.

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