The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often 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 in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic 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 alternate 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 scientific investigation into this neglected species.

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Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the 'kra' monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated, and it has a close phylogenetic relationship to Plasmodium vivax, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or 'hypnozoite' in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome and other sequenced Plasmodium genomes. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.

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Invasion of erythrocytes by Plasmodium merozoites is an intricate process involving multiple receptor-ligand interactions. The glycophorins and an unknown trypsin sensitive factor are all erythrocyte receptors used during invasion by the major human pathogen Plasmodium falciparum. However, only one erythrocyte receptor, Glycophorin A, has a well-established cognate parasite ligand, the merozoite protein erythrocyte binding antigen-175 (EBA-175). The involvement of several other parasite proteins during invasion have been proposed, but no direct evidence links them with a specific invasion pathway. Here we report the identification and characterization of P. falciparum normocyte binding protein 1 (PfNBP1), an ortholog of Plasmodium vivax reticulocyte binding protein-1. PfNBP1 binds to a sialic acid dependent trypsin-resistant receptor on the erythrocyte surface that appears to be distinct from known invasion receptors. Antibodies against PfNBP1 can inhibit invasion of trypsinized erythrocytes and two P. falciparum strains that express truncated PfNBP1 are unable to invade trypsinized erythrocytes. One of these strain, 7G8, also does not invade Glycophorin B–negative erythrocytes. PfNBP1 therefore defines a novel trypsin-resistant invasion pathway and adds a level of complexity to current models for P. falciparum erythrocyte invasion.

Two related Plasmodium falciparum genes and their encoded proteins have been identified by comparative analyses with Plasmodium vivax reticulocyte binding protein 2 (PvRBP-2). The P. falciparum genes have a structure which suggests that they may be the result of an evolutionary duplication event, as they share more than 8 kb of closely related nucleotide sequence but then have quite divergent unique 3 ends. Between these shared and unique regions is a complex set of repeats, the nature and number of which differs between the two genes, as well as between different P. falciparum strains. Both genes encode large hydrophilic proteins, which are concentrated at the invasive apical end of the merozoite and are predicted to be more than 350 kDa, with an N-terminal signal sequence and a single transmembrane domain near their C termini. Importantly, they also share gene structure and amino acid homology with the Plasmodium yoelii 235-kDa rhoptry protein family, which is also related to PvRBP-2. Together these Plasmodium proteins define an extended family of proteins that appear to function in erythrocyte selection and invasion. As such, they may prove to be essential components of malaria vaccine preparations. P arasites of the genus Plasmodium are estimated to cause between 300 and 500 million cases of malaria, the majority of which are caused by Plasmodium vivax and Plasmodium falciparum (1). Plasmodium parasites have a complex life cycle involving a series of developmental stages in both mosquitoes and mammals, but the clinical manifestations of malaria are all caused by the asexual blood stage. Merozoites, ovoid cells with an apical prominence at one end, invade red blood cells (RBCs), wherein they undergo a growth and multiplication phase (schizogony). The resulting schizont eventually ruptures the RBC, releasing newly formed merozoites for subsequent rounds of invasion.How merozoites identify and invade RBCs has long been a focus of research (2, 3). The merozoite first attaches to a RBC at any point on its surface, and then reorients to bring its apical end into contact with the RBC. The initial attachment stages are reversible, and merozoites can disassociate and attach to a new potential target cell. The subsequent steps are irreversible, and involve the formation of an electron-dense adhesion zone between the apical end of the merozoite and the RBC. This zone then moves around the merozoite toward its posterior end, with a concurrent invagination of the RBC membrane and entry of the merozoite. This cascade of molecular events also involves release of proteins from the rhoptries and micronemes, specialized apical organelles central to the invasion process.The molecular adhesion details behind this tantalizing outline are sketchy. The merozoite surface proteins (MSPs), several of which have been described in a number of species of Plasmodium, together make up a structurally complex coat around the outer membrane of the merozoite and may have a role in the initial reversible adhesive interaction between the merozo...

The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.

Abstract. Allelic diversity at the Plasmodium vivax merozoite surface protein-3␣ (PvMsp-3␣) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3␣ diversity in field samples from children living in a single village in a malariaendemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3␣ alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.Epidemiologic analyses of the population structure of Plasmodium parasites within and between endemic areas is essential for understanding the role of parasite diversity in the transmission of malaria as well as for designing and evaluating malaria vaccines. 1 Several large-scale studies have been conducted for P. falciparum, where the presence and dynamics of either single or multiple polymorphic antigenencoding genes have been investigated. 2-6 Comparable studies using highly polymorphic markers have yet to be reported for P. vivax. Here we present a P. vivax polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol that will facilitate such analyses. Using this protocol, we demonstrate that multiple genotypes of P. vivax are present in an endemic area of Papua New Guinea and provide preliminary evidence for a rapid turnover of P. vivax genotypes within individuals. The analysis is based on the evaluation of the presence and number of P. vivax merozoite surface protein-3␣ (PvMsp-3␣) alleles. 7