Article

Nature 455, 757-763 (9 October 2008) | doi:10.1038/nature07327; Received 18 January 2008; Accepted 8 August 2008

Comparative genomics of the neglected human malaria parasite Plasmodium vivax

Jane M. Carlton1,2, John H. Adams3, Joana C. Silva4,5, Shelby L. Bidwell1, Hernan Lorenzi1, Elisabet Caler1, Jonathan Crabtree1,5, Samuel V. Angiuoli5,8, Emilio F. Merino2, Paolo Amedeo1, Qin Cheng9, Richard M. R. Coulson10, Brendan S. Crabb11,12, Hernando A. del Portillo13,14, Kobby Essien15,16, Tamara V. Feldblyum5, Carmen Fernandez-Becerra13, Paul R. Gilson11, Amy H. Gueye17, Xiang Guo1, Simon Kang'a2, Taco W. A. Kooij18, Michael Korsinczky9,19, Esmeralda V.-S. Meyer20, Vish Nene4,5, Ian Paulsen1,21, Owen White5,6, Stuart A. Ralph22, Qinghu Ren1, Tobias J. Sargeant11,23, Steven L. Salzberg8, Christian J. Stoeckert15, Steven A. Sullivan2, Marcio M. Yamamoto24, Stephen L. Hoffman25, Jennifer R. Wortman5,7, Malcolm J. Gardner1,27, Mary R. Galinski19, John W. Barnwell26 & Claire M. Fraser-Liggett5,7

  1. The Institute for Genomic Research/J. Craig Venter Institute, 9704 Medical Research Drive, Rockville, Maryland 20850, USA
  2. Department of Medical Parasitology, New York University Langone Medical Center, 341 East 25th Street, New York, New York 10010, USA
  3. Department of Global Health, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, Florida 33612, USA
  4. Department of Microbiology and Immunology,
  5. Institute for Genome Sciences,
  6. Department of Epidemiology and Preventive Medicine,
  7. Department of Medicine, University of Maryland School of Medicine, 20 Penn Street, Baltimore, Maryland 21201, USA
  8. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland 20742, USA
  9. Drug Resistance and Diagnostics, Australian Army Malaria Institute, Weary Dunlop Drive, Gallipoli Barracks, Enoggera, Queensland 4051, Australia
  10. Microarray Group, European Bioinfomatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
  11. The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia
  12. Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004, Australia
  13. Barcelona Centre for International Health Research, Hospital Clinic/IDIBAPS, Universitat de Barcelona Roselló 132, 4a planta, 08036 Barcelona, Spain
  14. Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys, 23 08010 Barcelona, Spain
  15. Center for Bioinformatics and Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
  16. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
  17. Hood College, Frederick, Maryland 21701, USA
  18. Department of Parasitology, Heidelberg University School of Medicine, Im Neuenheimer Feld 324, Heidelberg 69120, Germany
  19. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia
  20. Emory Vaccine Center, Yerkes National Primate Research Center and Department of Medicine, Division of Infectious Diseases, Emory University, Atlanta, Georgia 30329, USA
  21. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
  22. Department of Biochemistry & Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia
  23. Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, Australia
  24. Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Lineu Prestes 1374, São Paulo, São Paulo 05508-900, Brazil
  25. Sanaria Inc., 9800 Medical Center Drive, Rockville, Maryland 20850, USA
  26. Malaria Branch, Division of Parasitic Diseases, National Center for Zoonotic, Vector-borne and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA
  27. Present address: Seattle Biomedical Research Center, 307 Westlake Avenue N., Suite 500, Seattle, Washington 98109-5219, USA.

Correspondence to: Jane M. Carlton1,2 Correspondence and requests for materials should be addressed to J.M.C. (Email: jane.carlton@nyumc.org).

This article is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence (http://creativecommons.org/licenses/by-nc-sa/3.0/), which permits distribution, and reproduction in any medium, provided the original author and source are credited. This licence does not permit commercial exploitation, and derivative works must be licensed under the same or similar licence.

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The human malaria parasite Plasmodium vivax is responsible for 25–40% of the approx515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Plasmodium vivax is the major cause of malaria outside Africa, mainly afflicting Asia and the Americas1. Principally a disease affecting poor people living on the margins of developing economies, vivax malaria traps many societies in a relentless cycle of poverty. Intermittent transmission makes protective immunity rare, and the disease strikes all ages. Repeated acute febrile episodes of debilitating intensity can occur for months. In children this can lead to life-long learning impairment, whereas incapacitation of adults has tremendous direct economic consequences through lost productivity and depletion of meagre financial reserves. Drug resistance in P. vivax is spreading, hindering management of clinical cases, and reports of severe pathology, including respiratory distress and coma, are challenging the description of P. vivax malaria as 'benign' (ref. 1).

Several biological characteristics underlie the distinct pathogenic and epidemiological nature of vivax malaria. In contrast to P. falciparum, P. vivax is only capable of infecting reticulocytes, causing severe anaemia by dyserythropoiesis and destruction of infected and uninfected erythrocytes despite much lower parasitaemias. P. vivax cannot infect Duffy-blood-group-negative reticulocytes (a trait shared with the closely related monkey malaria parasite P. knowlesi), and is thus absent from West Africa where Duffy negativity predominates2. Differences in Anopheles mosquito dynamics allow P. vivax transmission in temperate climates not tolerated by P. falciparum. In such regions P. vivax infects hepatocytes but may persist as dormant hypnozoites for months or years before initiating blood-stage infections (relapses) during another transmission season.

Because P. vivax kills infrequently and is not amenable to continuous in vitro culture, it has been relatively little studied in comparison to P. falciparum. The P. vivax genome sequence we report here, and comparative analyses with sequenced malaria parasites P. falciparum3, the rodent parasite P. yoelii yoelii4, 5, and the primate parasite P. knowlesi6 (an excellent model for in vivo studies of human malaria), provide important insights into the biology of this neglected parasite.

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Genome sequencing and characteristics

The approx26.8-megabase (Mb) nuclear genome sequence of P. vivax (Salvador I) was sequenced by whole-genome shotgun methods to tenfold coverage, with targeted gap closure and finishing, and manual curation of automated annotation. Details of these and other methods are given in Supplementary Information. Large contigs totalling approx22.6 Mb were assigned to the 14 P. vivax chromosomes; approx4.3 Mb of small subtelomeric contigs remain unassigned due to their repetitive nature (Supplementary Table 1). P. vivax chromosomes are unique among human Plasmodium species in exhibiting a form of isochore structure7, with subtelomeric regions of low G+C content and chromosome internal regions of significantly higher G+C content. We finished the subtelomeric ends of several P. vivax chromosomes, allowing us to define their isochore boundaries (Fig. 1).

Figure 1: Synteny maps showing the comparative organization of Plasmodium chromosomes.
Figure 1 : Synteny maps showing the comparative organization of Plasmodium chromosomes. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

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Putative orthologues were computed between P. falciparum (Pf), P. vivax (Pv), P. knowlesi (Pk) and P. y. yoelii (Py) proteomes and used to define blocks of synteny (shaded regions) between Py–Pk, Pv–Pk and Pv–Pf chromosomes. Genes on contigs that could not be assigned to chromosomes are not shown (see Supplementary Information). The composite rodent malaria parasite (cRMP) chromosomes generated in ref 18 are shown. Plots below the Pv chromosomes display the following: MS, the position of polymorphic microsatellites; (G+C)-skew, the base composition ((G-C)/(G+C)) within each strand in a 5-kb window; G+C, the percentage of bases in a 2-kb window that are G and C (%). Tracks within the Pv–Pk bracket show two evolutionary parameters: dS (number of synonymous substitutions per synonymous site; top) and omega (ratio of dN (non-synonymous substitutions per non-synonymous site) to dS; bottom). Horizontal red lines on the G+C (%), dS and omega tracks indicate the average value for that chromosome. ad, Distribution of selective constraints (omega) for the gene ontology classifications biological process (a), molecular function (b) and cellular component (c). Selective constraint is also shown for several motifs (d): proteins containing predicted transmembrane domains (TM) and/or signal peptides (SP); GPI-anchored proteins; and proteins predicted to be exported (exportome). Each grey box represents the interquartile range, which contains the sample's 25% to 75% range (quartiles Q1 to Q3, respectively), and the median is indicated (black horizontal bar within the interquartile range). Horizontal tick marks outside of the interquartile range show the range of all elements within Q1 - 1.5 times interquartile range and Q3 + 1.5 times interquartile range (approx99.3% interval of a normal distribution).

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In many aspects, the genomes of mammalian Plasmodium species (P. falciparum, P. knowlesi, P. vivax, P. y. yoelii) are uniform, ranging from 23 to 27 Mb across 14 chromosomes, and comprising approx5,500 genes, most of which (approx51%) contain at least one intron (Table 1). However, differences in nucleotide bias can be extreme (for example, P. vivax and P. falciparum average a G+C content of approx42.3% and approx19.4%, respectively), and a large gene family found in P. y. yoelii raised its gene count to approx5,880 (ref. 4). A remarkable 77% of genes are orthologous between the four species (Supplementary Fig. 1); almost one-half of these encode conserved hypothetical proteins of unknown function. In P. falciparum, the high incidence of tandem repeats and low complexity regions (LCRs) in proteins, especially antigens, has led researchers to propose that LCRs are involved in immune evasion mechanisms, such as antigen diversification8 and reducing the host's antibody response to critical epitopes by acting as a 'smokescreen'9. We found that LCRs tend to constitute a smaller proportion of P. vivax proteins on average (39%) than P. falciparum proteins (60%; Supplementary Fig. 2), and that LCR expansion partly accounts for the slightly larger size of P. falciparum proteins (Supplementary Table 2), but how this relates to differences in immune evasion mechanisms between P. vivax and P. falciparum is unclear.


Notwithstanding the recent functional characterization of the apicomplexan AP2 family of transcriptional regulators in Plasmodium10, the parasite seems to lack most of the standard eukaryotic transcriptional machinery, such as transcription-associated proteins (TAPs)11, but is rich in regulatory sequences12, fostering the idea that gene expression regulation in Plasmodium is complex and unusual. Our initial studies found no significant differences in the TAP repertoire between P. falciparum, P. vivax and P. knowlesi, indicating that transcriptional mechanisms are similar in all three species (Supplementary Table 3). Genes encoding messenger RNA stability proteins containing a CCCH-zinc finger were abundant in all three species, affirming the importance of post-transcriptional regulation in the control of gene expression across Plasmodium. A genome scan of P. vivax for known core promoter elements such as TATA and CAAT boxes identified some candidates, but many of them lacked positional specificity. Similarly, a search for novel promoter elements in regions upstream of approx1,800 mapped transcription start sites (5' UTRs), and for RNA binding elements in approx1,300 3' UTRs, also failed to produce convincing candidates (data not shown). To determine whether binding sites are conserved between P. falciparum and other primate Plasmodium species, we searched for over-represented nucleotide 'words' in regions upstream of clusters of potentially co-regulated genes conserved in P. vivax, P. falciparum, P. knowlesi and P. y. yoelii (Supplementary Information). Seven putative novel regulatory binding sites conserved across at least two species were identified (Supplementary Table 4). These binding sites were associated with core eukaryotic processes such as dephosphorylation and with parasite-specific functions such as cell invasion. Independent support for two of our predicted sites comes from a recent report of the sporozoite-associated motif 5'-TGCATGCA-3' and the merozoite invasion-related 5'-GTGTGCACAC-3' motif13. In our analysis these two sites, together with the dephosphorylation-associated motif 5'-GCACGCGTGC-3', were conserved across the four Plasmodium species.

Examination of natural parasite population structure is key to understanding transmission dynamics, the spread of drug resistance, and to design and test malaria control efforts. Many population studies have exploited the abundant polymorphic microsatellites in the P. falciparum genome, primarily simple sequence repeats such as [TA] dinucleotide and polyA/polyT14. We screened the P. vivax genome for microsatellites, identifying approx160 that are polymorphic between eight P. vivax laboratory lines (Fig. 1 and Supplementary Table 5). P. vivax microsatellites average 27.5% G+C, with an average repeat unit length of 3.1 nucleotides and an average copy number of 19.1. We found fewer microsatellites in P. vivax than in P. falciparum (as noted previously15), probably due to the more conventional nucleotide composition of the former. Even so, these genome-wide polymorphic markers are already facilitating studies of P. vivax population structure and genetic diversity16, 17.

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Chromosome synteny and genome evolution

Previous studies have indicated significant conservation of gene synteny between Plasmodium parasites4 in direct proportion to their genetic distance. We generated a synteny map of P. vivax, P. knowlesi, P. falciparum, and the rodent malaria parasites P. y. yoelii, P. berghei and P. chabaudi (considered as a single lineage18 owing to their virtually complete synteny; Fig. 1). The P. vivax and P. knowlesi chromosomes are highly syntenic except for microsyntenic breaks at species-specific genes (in particular the P. knowlesi kir and SICAvar genes; see ref. 6); a previous study identified such breaks as foci for the evolution of host–parasite interaction genes18. The karyotypes of P. vivax and P. knowlesi correspond to the most parsimonious reconstruction of the ancestral form of the six species; the karyotypes of P. falciparum and the rodent malaria parasites can be reconstructed from this form through nine and six chromosomal rearrangements, respectively (Supplementary Fig. 3). No 'hotspots' of synteny breakage were identified, indicating that intersyntenic breakpoints were not 'reused' during the divergence of the species, and no obvious motifs except for (A+T)-rich regions and LCRs were identified in regions of the P. vivax genome predicted to have recombined to give single P. falciparum chromosomes. Of the 3,336 orthologues between all six species, 3,305 (99%) were found to be positionally conserved (Supplementary Table 6).

We used 3,322 high-quality P. vivax/P. knowlesi orthologues to obtain maximum likelihood estimates of the rate of substitution at synonymous (dS) and non-synonymous (dN) sites, as well as omega (dN/dS; Supplementary Table 7 and Fig. 1). P. vivax chromosomes differ significantly in their average values for both dS and dN, but the two variables are strongly correlated within and between chromosomes (Supplementary Fig. 4). The chromosomes also differ significantly in average %GC4 (the G+C content in third codon positions of fourfold degenerate amino acids). This variable is positively correlated with average dS and inversely correlated with chromosome length, such that synonymous sites in genes on the smallest chromosomes (approx1 Mb) evolve approx1.5 times faster than genes on the two largest (approx3 Mb) chromosomes (Supplementary Fig. 5). These observations strongly indicate the existence of heterogeneous mutation rates across the genome. It is unclear if this is due to cytosine-to-thymine deamination, which is more probable in (G+C)-rich regions, as it is not known whether DNA methylation occurs in P. vivax. The degree of selective constraint (omega) also varies across classes of genes. Genes encoding glycosylphosphatidylinositol (GPI)-anchored proteins, cell adhesion proteins, exportome proteins (proteins predicted to be exported) and proteins with transmembrane or signal peptide motifs, all of which are at least partly extracellular, were found to evolve significantly faster than genes involved in, for example, carbohydrate metabolism, enzyme regulation and cell structure (Fig. 1a–d and Supplementary Table 8). The host immune system, by targeting extracellular peptides, seems to have strongly influenced evolutionary rate variation between gene classes in Plasmodium.

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A highly conserved Plasmodium metabolome

We found that key metabolic pathways, housekeeping functions and the repertoire of predicted membrane transporters are highly conserved between the P. vivax and P. falciparum3 proteomes (Supplementary Table 9), suggesting that the two species have much the same metabolic potential. Conservation of metabolic processes also extends to the apicoplast, an apicomplexan plastid secondarily acquired from an ancient cyanobacterium. The apicoplast has lost photosynthetic function, but is essential to the parasite's metabolism, hosting nuclear-encoded proteins that are targeted to the apicoplast lumen by a conserved bipartite amino-terminal presequence. The complete genome sequence of P. vivax offers an opportunity to update and improve the apicoplast proteome that was predicted in silico3. Apicoplast-targeted proteins conserved in P. vivax participate in major metabolic processes previously recognized in P. falciparum19, such as complete type II fatty acid synthesis, isopentenyl diphosphate and iron sulphur cluster assembly pathways, and a fragmented haem synthesis pathway distributed between the apicoplast and mitochondria. Conservation of these pathways in P. vivax is important because synthetic pathways for type II fatty acid synthesis and isopentenyl diphosphates are targets for antimalarial chemotherapeutics20. The revised Plasmodium apicoplast proteome (Supplementary Table 10) also clarifies the localization of two important processes. We show thiamine pyrophosphate biosynthesis, previously thought to take place in the apicoplast19, to be cytosolic. Conversely, we confirm a glyoxalase pathway in the apicoplast, with glyoxalase I and glyoxalase II enzymes being targeted there21; both enzymes are potential drug targets. Thus, comparison of overall apicoplast metabolic capabilities shows very few differences between P. vivax and P. falciparum.

P. vivax can form hypnozoites, a latent hepatic stage responsible for patent parasitaemia relapses months or even years after an initial mosquito-induced infection22. Hypnozoites survive most drugs that kill blood-stage parasites; complete elimination of P. vivax infections (radical cure) requires primaquine, the only licensed drug that can kill hypnozoite stages. However, resistance to the drug is spreading23, and its use is contra-indicated in pregnant women or patients with glucose-6-phosphate dehydrogenase deficiency, which is common in malaria-endemic regions. After an initial examination of P. vivax-specific proteins failed to identify leads (Supplementary Table 11), we hypothesized that the genetic switch for hypnozoite formation may involve P. vivax homologues of dormancy genes. Analysis of the predicted P. vivax proteome revealed some candidates (Supplementary Table 12). However such an association remains speculative, and investigation of hypnozoite formation and activation will require continued development of in vitro systems for culturing P. vivax liver stages24.

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Gene families shape Plasmodium biology

Plasmodium lineages display differential gene family expansion that has shaped the specific biology of each species. Phenotypes illustrating this include parasite invasion of red blood cells and antigenic variation. Invasion of erythrocytes by extracellular Plasmodium merozoites, crucial to the development of malaria in an infected individual, depends on specific interactions between merozoite ligands and erythrocyte surface receptors (Fig. 2). Plasmodium species-specific mechanisms act mostly during the preliminary phases of invasion (for example, merozoite attachment and orientation). In P. vivax, but not P. falciparum, invasion is restricted to Duffy-positive reticulocytes2. P. vivax Duffy-binding protein (DBP25) and reticulocyte-binding proteins (RBPs26) are the archetypes of two distinct Plasmodium families of cell-binding proteins involved in erythrocyte selection (referred to as the Duffy-binding-like (DBL) and reticulocyte-binding-like (RBL) families, respectively). Homologues of rbp1 and rbp2, two genes originally identified in P. vivax, include the P. falciparum rh/nbp genes (reviewed in ref. 27) and the Py235 family in P. y. yoelii (reviewed in ref. 28). Unexpectedly, we identified additional rbp genes in the P. vivax genome (Supplementary Table 13), including multiple rbp2 genes, which could provide P. vivax with a diversity of invasion mechanisms comparable to that of P. falciparum. This finding dispels a view that P. vivax has a relatively uncomplicated erythrocyte invasion mechanism. Instead, P. vivax probably has alternative invasion pathways, as differential expression of rbp homologues in P. falciparum29 and P. y. yoelii30 is closely linked to switching of invasion pathways (Fig. 2). All rbp2 loci occur in the subtelomeric regions of P. vivax chromosomes: non-syntenic, dynamic regions of the genome in which species-specific genes are generated (Supplementary Fig. 6).

Figure 2: Predicted erythrocyte invasion pathways and dominant ligands of Plasmodium species.
Figure 2 : Predicted erythrocyte invasion pathways and dominant ligands of Plasmodium species. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

RBL and DBL invasion families predicted from several Plasmodium proteomes are shown above a Plasmodium merozoite colliding and re-orientating on the red blood cell surface. Species-specific RBL families interact with an array of species-specific DBL proteins that use both alternative (crossed arrows) and fixed (straight arrows) pathways with known or predicted receptors on the surface of erythrocytes. Blocking these receptor–ligand interactions offers a potential mechanism to prevent clinical malaria. DARC, Duffy antigen receptor for chemokine-dependent (+) and -independent (-) pathways; GPA/B/C, P. falciparum glycophorin A/B/C receptors; RH SA+/-, rhesus sialic-acid-dependent (+) and -independent (-) pathways; 'X', 'Y', predicted receptors. The asterisk indicates that the presence of this pathway is controversial.

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The final phase of invasion, merozoite entry into an intraerythrocytic vacuole, uses an intracytoplasmic molecular motor (components of which are highly conserved between Plasmodium species) coupled to simultaneous shedding of crucial merozoite surface proteins (MSPs). There are at least ten distinct MSPs (Supplementary Table 14), and P. vivax genome analysis reveals two particularly interesting MSP families, MSP3 and MSP7. Eleven members of the msp3 gene family occur in tandem on a approx60-kb region of P. vivax chromosome 10 (Supplementary Fig. 7), and show weak similarity to four msp3 gene family members on P. falciparum chromosome 10 and to two P. knowlesi msp3 genes located on different chromosomes. Thus, there has been a significant expansion of the msp3 gene family in P. vivax, perhaps as a means to enhance immune evasion, as P. falciparum and P. vivax msp3 gene family members have been shown to be antigenic and to partially immunize non-human primates against blood-stage parasites31. In P. falciparum, MSP6 (a member of the MSP3 family that lacks heptad repeats) non-covalently binds with MSP1, but there is no counterpart to MSP6 in P. vivax. MSP7, another P. falciparum antigen that binds to MSP1 on the surface of merozoites, has also been expanded in P. vivax, with eleven copies on chromosome 12, compared to six and three members in P. falciparum and P. y. yoelii, respectively; it is not known if any P. vivax MSP7 proteins bind to MSP1.

The surface coats of merozoites and extracellular forms of Plasmodium parasites are composed largely of GPI-anchored proteins, many of which are important targets of protective immune responses and thus constitute promising vaccine candidates. When we predicted the GPI-anchored proteome of P. vivax and compared it to validated P. falciparum GPI-anchored proteins32, 29 of the 30 GPI-anchored proteins identified in P. falciparum had counterparts in P. vivax (Supplementary Fig. 8), an extraordinary level of conservation. MSP2 (the second most abundant merozoite surface protein in P. falciparum) is absent in the P. vivax genome, and P. vivax contains one additional GPI-anchored protein that seems to be a member of the 'six cysteine' apicomplexan-specific gene family33. Both the P. vivax and P. knowlesi genomes encode an apparently paralogous gene next to msp1, which is the largest and most abundant protein on the P. falciparum merozoite surface. P. vivax MAP1 is not closely related to MSP1 (11% identity, 22% similarity), although their sizes, a predicted GPI-attachment site, and structural features such as a carboxy-terminal double EGF module are similar.

A second notable parasite phenotype is antigenic variation: the ability to vary surface proteins during the course of an infection to evade the host's immune response. In P. falciparum, antigenic variation is mediated by species-specific gene families such as var, members of which are expressed clonally and regulated epigenetically34. In P. vivax, the largest multigene family vir, part of the pir (Plasmodium interspersed repeats) superfamily found in several Plasmodium species5, has been implicated in antigenic variation; 35 gene copies were previously identified35. We identified 346 vir genes in the P. vivax genome located within (A+T)-rich subtelomeric regions of chromosomes (Fig. 1). Structurally, vir genes vary greatly, ranging from 156 to 2,316 bp in length and containing 1–5 exons. VIR proteins were previously classified into six subfamilies (A–F) on the basis of sequence similarity35, and representatives of these subfamilies were identified in patient isolates36. Clustering the VIRs in the Salvador I genome yielded six new subfamilies (G–L) and we confirmed gene expression for several of these in natural infections (Supplementary Table 15). Motif analysis of the total VIR repertoire (Fig. 3) showed that approximately one-half (171) contain a transmembrane domain, and one-half (160) contain a motif similar to the PEXEL/VSP sequence linked to export of parasite proteins37, 38. Introns from 25 vir genes contain a conserved motif proximal to the donor splice site, suggesting possible functionality of the sequence in the control of vir gene expression, as has been shown for P. falciparum var introns39. Motif-shuffling among the sequences is apparent, particularly among large VIR proteins that have undergone an expansion of some motifs at the amino terminus. Similarly to P. falciparum var genes, in situ hybridization analysis has shown that P. vivax chromosome ends localize to the nuclear periphery40, where ectopic recombination favours the generation of variants and gene expansion. Although the repeat structure of P. vivax subtelomeric regions is not as extensive as that seen in P. falciparum6, P. vivax probably uses chromosomal exchange as a mechanism for generating antigenic diversity. VIR proteins represent an extremely diverse family, members of which currently appear more divergent than members of other partially characterized PIR families such as the P. chabaudi CIR (135 members) and the P. berghei BIR (245 members) families (Supplementary Fig. 9). Shared structural characteristics have been shown between VIR subfamily D proteins and the P. falciparum Pfmc-2tm family located at Maurer's clefts, and VIR subfamily A proteins and the P. falciparum SURFIN family found on the surface of infected erythrocytes41. We speculate that the extreme diversity and sub-structuring of VIR proteins indicate members' different subcellular localizations and functions, including immune evasion.

Figure 3: VIR protein motifs and organization.
Figure 3 : VIR protein motifs and organization. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

The structure of an archetypal vir gene is shown at the top, followed by VIR motifs in each of the subfamilies A–E and G–L (subfamily F is not shown), arranged from the N terminus (left) to the C terminus (right). Consensus motif sequences numbered in decreasing order of statistical significance are shown colour coded below the figure. Motif 2, transmembrane (TM) domain; motif 3, PEXEL/VSP-like motif; all remaining motifs are predicted to be exposed globular domains. The overall organization and order of the motifs is maintained, with the central core motifs 9, 1, 3, 6 and 10 followed by C terminus motifs 7, 2, 4, 8 and 5 embedded in a variant-sized portion of the molecule. Motifs are listed in the Supplementary Information.

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We identified eight novel gene families (Pv-fam-a to Pv-fam-e and Pv-fam-g to Pv-fam-i; Supplementary Table 16) in the P. vivax genome, most of which are located in subtelomeric regions (Fig. 1). Of particular interest are (1) the PvTRAG (Pv-fam-a) gene family (36 genes), one member of which was previously identified (it encodes a protein localized to the caveola–vesicle complex of infected erythrocytes, and has been shown to elicit a humoral immune response during the course of natural infections42); and (2) the Pv-fam-e family (Supplementary Fig. 10), 36 copies of which are found in two loci on either side of the predicted centromere on chromosome 5, with one 10-gene locus present in a 47% G+C region, and a second 26-gene locus present in a 36% G+C region. Whereas P. vivax proteins have a fairly balanced codon composition, using all 61 sense codons almost equally (effective number of codons, Nc = 54.2), their orthologues in P. falciparum are more biased (Nc = 37.5), with G- and C-ending codons nearly absent from fourfold degenerate amino acids (Supplementary Table 17). However, P. vivax gene families, which are predominantly located in (A+T)-rich regions, have a codon composition of Nc = 47. This pattern suggests a strong influence of local mutation pattern on the nucleotide composition of genes and indicates a potential for differential gene expression.

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Plasmodium drug interaction genes

The sexual stages of P. vivax are produced before the onset of clinical symptoms, permitting mosquito transmission early in an infection. Such early parasite transmission may delay development of resistance to many of the antimalarial drugs used to treat vivax malaria, despite the extensive long-term use of these drugs in regions endemic for both P. vivax and P. falciparum43. Nevertheless, P. vivax can develop resistance to most of the current antimalarial drugs. To understand the interactions between antimalarial drugs and the parasite proteins implicated in drug binding and resistance, we examined crystal structures and developed homology models for several P. vivax proteins in the predicted proteome, and compared the predicted binding sites and reported mutations with those of their P. falciparum orthologues (Table 2).


Currently, the most efficacious novel antimalarial drugs are derivatives of artemisinin (qinghaosu) and atovaquone, used predominantly in combination therapies. Arteminsinin derivatives, the most potent drugs recommended for treatment, may target a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA)-type protein, ATPase6 (ref. 44). We constructed homology models of P. vivax and P. falciparum ATPase6 and identified two residues in the putative active sites for artemisinin that differ between the two species (P. vivax A263 and S1008, equivalent to L263 and N1039 in P. falciparum). A change in residue 263 from leucine to alanine results in a threefold increase in susceptibility to artemisinin by Xenopus laevis expressing PfATPase6 (ref. 44), although the IC50 (concentration required to inhibit parasite growth by 50%) for some P. vivax field isolates appears higher than the IC50 for P. falciparum45. However, it should be noted that clinical resistance of any human Plasmodium species to artemisinin derivatives has yet to be documented. Atovaquone, used in combination with the antifolate proguanil, selectively inhibits mitochondrial electron transport at the cytochrome bc(1) complex; mutations in the cytochrome b (cytb) gene can interfere with this inhibition, causing resistance. We constructed a homology model of P. vivax CYTB and compared it to the P. falciparum CYTB homology model46, revealing almost identical structures, including the predicted atovaquone active sites. Although there are no reports of atovaquone treatment failures for P. vivax infections, our studies indicate that should resistance arise, the same sites in P. vivax CYTB may be implicated.

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Towards a policy shift for P. vivax malaria

Despite the insights into parasite biology provided by the P. vivax genome, many important questions remain that can only be addressed by functional studies. For example, we were unable to find differences in the predicted P. vivax proteome that might explain the rheological behaviour of P. vivax-infected erythrocytes, which remain flexible and can repeatedly pass through the spleen, unlike P. falciparum-infected reticulocyte, the rigidity of which facilitates cytoadherence and avoidance of splenic clearance47. Studies of the hypnozoite transcriptome, although technically challenging, would radically increase our inadequate knowledge of the biology of this dormant form. Studies are currently underway to develop new in vitro culture systems48, which could provide badly needed biological material for such functional studies.

The malaria research and control communities were challenged recently to establish once again the eradication of malaria as a policy goal49. Given the significant contribution of P. vivax to the global malaria situation1, 43, it is imperative that these efforts include elimination of P. vivax as well as P. falciparum. Elimination of P. vivax presents special challenges, in particular the parasite's production of dormant hypnozoites that enables relapses long after the initial parasitaemia has cleared. Indeed, an important aspect of P. vivax eradication will be the development of new drugs to replace primaquine for radical cure. Although the development of new drugs targeting P. vivax liver stages is a formidable task, recent developments offer hope that this goal can be accomplished50.

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Methods Summary

Genome sequencing, assembly, mapping and annotation

Saimiri boliviensis boliviensis monkeys were infected with the Salvador I strain of P. vivax isolated from a patient from El Salvador. Extracted parasite DNA was used to make genomic DNA libraries for shotgun sequencing. Reads were assembled into scaffolds, inter-scaffold gaps closed, and scaffolds assigned to P. vivax chromosomes through hybridization of scaffold-specific probes to pulsed-field gel separated chromosomes. Gene prediction algorithms were used to predict gene models, and each model was manually checked for structural inconsistencies. Gene function was assigned using an automated annotation pipeline with subsequent manual curation.

Genome analysis

Methods for the in silco analysis of the genome sequence are described in the Supplementary Information.

Studies requiring laboratory experimentation

For polymorphic microsatellite identification, primers flanking 333 microsatellites identified from the genome sequence and designed for field studies where access to capillary electrophoresis equipment may not be possible were used to amplify the loci from eight worldwide P. vivax laboratory strains adapted to growth in monkeys (Brazil I, Miami II, Pakchong, Panama I, Nica, Thai II, Vietnam IV and Indonesia XIX). Amplicons were separated by electrophoresis on agarose gels and scored for size differences. For vir gene expression studies, cDNA was generated from total RNA extracted from the Salvador I isolate and from three patient isolates from Brazil. Primers were designed to eight vir gene subfamilies and used to amplify the loci.

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Supplementary Information

Supplementary information accompanies this paper.

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Acknowledgements

We thank the P. vivax research community for their support, and in particular M. Gottlieb and V. McGovern for facilitating financial support. Funding came from the following sources: P. vivax sequencing, assembly and closure, US Department of Defense and National Institute of Allergy and Infectious Diseases; genome mapping, Burroughs Wellcome Fund; and selective constraint analysis, National Institute of General Medical Sciences. We wish to thank TIGR's SeqCore, Closure and IFX core facilities, E. Lee, J. Sundaram, J. Orvis, B. Haas and T. Creasy for engineering support, R. K. Smith Jr for annotation support, E. Lyons and H. Zhang for technical assistance, H. Potts for statistical analysis, T. McCutchan for rDNA sequence annotation, and S. Perkins for the Plasmodium phylogeny.

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Author Information

Sequence and annotation data for the genome are deposited in GenBank under the project accession number AAKM00000000 and are also available at the Plasmodium genome sequence database PlasmoDB (http://plasmodb.org). A minimal tiling path of clones covering each chromosome is available through the malaria repository MR4 (http://www.mr4.org), and a long-oligo array through the Pathogen Functional Genomics Resource Center (http://pfgrc.jcvi.org).

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