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The genome of the malarial parasite Plasmodium falciparum is extremely AT rich. This bias toward a low GC content is a characteristic of several, but not all, species within the genus Plasmodium. We compared 4283 orthologous pairs of proteincoding sequences between Plasmodium falciparum and the less AT-biased Plasmodium vivax. Our results indicate that the common ancestor of these two species was also extremely AT rich. This means that, although there was a strong bias toward A+T during the early evolution of the ancestral Plasmodium lineage, there was a subsequent reversal of this trend during the more recent evolution of some species, such as P. vivax. Moreover, we show that not only is the P. vivax genome losing its AT richness, it is actually gaining a very significant degree of GC richness. This example illustrates the potential volatility of nucleotide content during the course of molecular evolution. Such reversible fluxes in nucleotide content within lineages could have important implications for phylogenetic reconstruction based on molecular sequence data. Key words: malaria, nucleotide bias, biased gene conversion, nucleotide content, molecular phylogeny. Le genome du parasite responsable de la transmission du paludisme, Plasmodium falciparum, est extremement riche en AT. Ce biais en direction d'un faible contenu en GC est caracteristique de plusieurs mais pas toutes les especes au sein du genre Plasmodium. Les auteurs ont compare 4283 paires orthologues de sequences codantes entre le Plasmodium falciparum et le Plasmodium vivax, une espece qui presente un biais plus faible. Les resultats indiquent que l'ancetre commun de ces deux especes etait egalement tres riche en AT. Cela suggere que, bien qu'il ait existe un fort biais en faveur des A+T au cours de l'evolution anterieure de l'ancetre des Plasmodium, il se serait produit un renversement subsequent de cette tendance au cours de l'evolution plus recente chez certaines especes comme le P. vivax. De plus, les auteurs montrent que le genome du P. vivax a non seulement perdu sa richesse en AT, mais encore il a acquis une richesse tres significative en GC. Cet exemple illustre la volatilite potentielle de la composition en nucleotides au cours de l'evolution moleculaire. De tels changements reversibles en composition nucleotidique au sein d'un embranchement pourraient avoir des implications importantes pour la reconstruction de phylogenies sur la base des sequences moleculaires. [Traduit par la Redaction] Mots-cles : paludisme, biais nucleotidique, conversion genique biaisee, composition nucleotidique, phylogenie moleculaire., Introduction Genomic nucleotide content (usually measured as GC content) varies widely among prokaryotic genomes (Jukes and Bhushan 1986; Muto and Osawa 1987; Sueoka 1988; Haywood-Farmer and Otto 2003; Xia 2003) [...]

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (Idh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodiurn gaboni than to the P. falciparum--Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodiurn malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum. ape malaria | human malaria | Plasmodium malagasi | Plasmodium phylogeny doi/ 10.1073/pnas.1005435107

Insect hemocytes mediate important cellular immune responses including phagocytosis and encapsulation and also secrete immune factors such as opsonins, melanization factors, and antimicrobial peptides. However, the molecular composition of these important immune cells has not been elucidated in depth, because of their scarcity in the circulating hemolymph, their adhesion to multiple tissues and the lack of primary culture methods to produce sufficient material for a genome-wide analysis. In this study, we report a genome-wide molecular characterization of circulating hemocytes collected from the hemolymph of adult female Anopheles gambiae mosquitoes--the major mosquito vector of human malaria in subSaharan Africa. Their molecular profile identified 1,485 transcripts with enriched expression in these cells, and many of these genes belong to innate immune gene families. This hemocyte-specific transcriptome is compared to those of Drosophila melanogaster and two other mosquitoes, Aedes aegypti and Armigeres subalbatus. We report the identification of two genes as ubiquitous hemocyte markers and several others as hemocyte subpopulation markers. We assess, via an RNAi screen, the roles in development of Plasmodium berghei of 63 genes expressed in hemocytes and provide a molecular comparison of the transcriptome of these cells during malaria infection. innate immunity | malaria | mosquito | cellular immunity doi/10.1073/pnas.0909463106

Plasmodium falciparum, the causative agent of malignant malaria, is among the most severe human infectious diseases. The closest known relative of P. falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single isolate was previously known. The co-speciation hypothesis suggests that both parasites evolved separately from a common ancestor over the last 5-7 million years, in parallel with the divergence of their hosts, the hominin and chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi, from wild and wild-born captive chimpanzees in Cameroon and Cote d'Ivoire, shows that P, reichenowi is a geographically widespread and genetically diverse chimpanzee parasite. The genetic lineage comprising the totality of global P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. This finding is inconsistent with the co-speciation hypothesis. Phylogenetic analysis indicates that all extant P. falciparum populations originated from P. reichenowi, likely by a single host transfer, which may have occurred as early as 2-3 million years ago, or as recently as 10,000 years ago. The evolutionary history of this relationship may be explained by two critical genetic mutations. First, inactivation of the CMAH gene in the human lineage rendered human ancestors unable to generate the sialic acid Neu5Gc from its precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More recently, mutations in the dominant invasion receptor EBA 175 in the P. falciparum lineage provided the parasite with preference for the overabundant Neu5Ac precursor, accounting for its extreme human pathogenicity. chimpanzees | human evolution | Plasmodium falciparum | Plasmodium reichenowi | zoonosis

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host--pathogen interactions and malaria virulence. dense granule | invasion | malaria | microneme | transfection

Variation in the multiplication rate of blood stage malaria parasites is often positively correlated with the severity of the disease they cause. The rodent malaria parasite Plasmodium yoelii yoelii has strains with marked differences in multiplication rate and pathogenicity in the blood. We have used genetic analysis by linkage group selection (LGS) to identify genes that determine differences in multiplication rate. Genetic crosses were generated between genetically unrelated, fast- (17XYM) and slowly multiplying (33XC) clones of P. y. yoelii. The uncloned progenies of these crosses were placed under multiplication rate selection in blood infections in mice. The selected progenies were screened for reduction in intensity of quantitative genetic markers of the slowly multiplying parent. A small number of strongly selected markers formed a linkage group on P. y. yoelii chromosome 13. Of these, that most strongly selected marked the gene encoding the P. yoelii erythrocyte binding ligand (pyebl), which has been independently identified by Otsuki and colleagues [Otsuki H, et al. (2009) Proc Natl Acad Sci USA 106-10.1073/pnas.0811313106] as a major determinant of virulence in these parasites. In an analysis of a previous genetic cross in P. y. yoelii, pyebl alleles of fast- and slowly multiplying parents segregated with the fast and slow multiplication rate phenotype in the cloned recombinant progeny, implying the involvement of the pyebl locus in determining the multiplication rate. Our genome-wide LGS analysis also indicated effects of at least 1 other locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii. erythrocyte binding ligand protein | rodent malaria | linkage group selection | virulence

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.

Regulated [K.sup.+] transport across the plasma membrane is of vital importance for the survival of most cells. Two [K.sup.+] channels have been identified in the Plasmodium falciparum genome; however, their functional significance during parasite life cycle in the vertebrate host and during transmission through the mosquito vector remains unknown. We hypothesize that these two [K.sup.+] channels mediate the transport of [K.sup.+] in the parasites, and thus are important for parasite survival. To test this hypothesis, we identified the orthologue of one of the P. falciparum [K.sup.+] channels, PfKch1, in the rodent malaria parasite P. berghei (PbKch1) and examined the biological role by performing a targeted disruption of the gene encoding PbKch1. The deduced amino acid sequence of the six transmembrane domains of PfKch1 and PbKch1 share 82% identity, and in particular the pore regions are completely identical. The PbKch1-null parasites were viable despite a marked reduction in the uptake of the [K.sup.+] congener [sup.86][Rb.sup.+], and mice infected with PbKch1-null parasites survived slightly longer than mice infected with WT parasites. However, the most striking feature of the phenotype was the virtually complete inhibition of the development of PbKch1-null parasites in Anopheles stephensi mosquitoes. In conclusion, these studies demonstrate that PbKch1 contributes to the transport of [K.sup.+] in P. berghei parasites and supports the growth of the parasites, in particular the development of oocysts in the mosquito midgut. [K.sup.+] channels therefore may constitute a potential antimalarial drug target. malaria | pathogenesis | mosquito I drug target

Complete DNA sequences of the small subunit ribosomal RNA (SSUrRNA) gene and partial sequences of three other loci were obtained from three variant-type and three classic-type Plasmodium ovale isolates from [...]

Anti-malaria vaccines and drugs could be greatly improved if we knew which phases of Plasmodium falciparum development in red blood cells are major inducers and which are major targets of natural immune responses. This information should focus attention on relevant immunogens and prove useful in developing immune-based therapies. Here we explore the hypothesis that innate immune responses mediate synchronization between the replication cycles of parasites in different red blood cells which is reflected in periodic fevers. Based on a recently developed, rather general mathematical model, we find that periodicity is highly sensitive to the position of both the inducing phase interval and the target phase interval in the parasite replication cycle. In addition, the degree of variability in the length of the replication cycle also strongly affects periodicity. To produce synchronization, the inducing and the target phase intervals must be developmentally distant from each other. We developed a computer program which prompts for information based on measurements of the numbers of erythrocytes in two replication cycle intervals chosen by the researcher, tests our model, and predicts the two phase intervals most critical to the synchronizing immune response. The program can be obtained from the authors.

Members of a multigene family in the rodent malaria parasite Plasmodium yoelii yoelii code for 235-kilodalton proteins (Py235) that are located in the merozoite apical complex, are implicated in virulence, and may determine red blood cell specificity. We show that distinct subsets of py235 genes are expressed in sporozoites and hepatic and erythrocytic stages. Antibodies to Py235 inhibited sporozoite invasion of hepatocytes. The switch in expression profile occurred immediately after transition from one stage to another. The results suggest that this differential expression is driven by strong biological requirements and provide evidence that hepatic and erythrocytic merozoites differ., Invasive stages (ookinete, sporozoite, and merozoite) of the malaria parasite penetrate specific host cell types at different stages of the life cycle. The 235-kD rhoptry proteins (Py235) of the rodent [...]

Proteins sequestered within organelles of the apical complex of malaria merozoites are involved in erythrocyte invasion, but few of these proteins and their interaction with the host erythrocyte have been characterized. In this report we describe MAEBL, a family of erythrocyte binding proteins identified in the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei. MAEBL has a chimeric character, uniting domains from two distinct apical organelle protein families within one protein. MAEBL has a molecular structure homologous to the Duffy binding-like family of erythrocyte binding proteins located in the micronemes of merozoites. However, the amino cysteine-rich domain of MAEBL has no similarity to the consensus Duffy binding-like amino cysteine-rich ligand domain, but instead is similar to the 44-kDa ectodomain fragment of the apical membrane antigen 1 (AMA-1) rhoptry protein family. MAEBL has a tandem duplication of this AMA-1-like domain, and both of these cysteine-rich domains bound erythrocytes when expressed in vitro. Differential transcription and splicing of the maebl locus occurred in the YM clone of P. yoelii yoelii. The apical distribution of MAEBL suggested localization within the rhoptry organelles of the apical complex. We propose that MAEBL is a member of a highly conserved family of erythrocyte binding proteins of Plasmodium involved in host cell invasion.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, [F.sub.2] progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.

Evolutionary trees are often estimated from DNA or RNA sequence data. How much confidence should we have in the estimated trees? In 1985, Felsenstein [Felsenstein, J. (1985) Evolution 39, 783-791] suggested the use of the bootstrap to answer this question. Felsenstein's method, which in concept is a straightforward application of the bootstrap, is widely used, but has been criticized as biased in the genetics literature. This paper concerns the use of the bootstrap in the tree problem. We show that Felsenstein's method is not biased, but that it can be corrected to better agree with standard ideas of confidence levels and hypothesis testing. These corrections can be made by using the more elaborate bootstrap method presented here, at the expense of considerably more computation.

A study of the evolutionary relationships of different species of Plasmodium by analyzing the small subunit rRNA gene sequences of 11 Plasmodium species reveals that the most potent human parasite, Plasmodium falciparum, is related to the chimpanzee parasite P. reichenowi. The times of divergence between human and chimpanzee lineages and between their respective parasites are similar. Two other human parasites P. vivax and P. malariae are slightly related to each other and to the falciparum-reichenowi clade, indicating the phylogenetic independence of the parasitic relationship of the Plasmodium species with their hosts.

A study of the small subunit rRNA sequences of Plasmodium cynomolgi, P. falciparum and P. cynomolgi confirms that sexually expressed type B and asexually expressed type A genes interact to a degree that eliminates stage-specific sequence signature they might have procured in the last shared ancestor. Independent evolution is absent for the two gene types. A single copy contains the type B gene.

A genetic characterization was carried out on several parasitic protozoa representing species of Trypanozoma, Leishmania and Plasmodium using the random amplified polymorphic DNA (RAPD) technique. The resulting relationships acquired by the RAPD method correlated well with previous results using multilocus enzyme electrophoresis. These results demonstrate the applicability of the RAPD technique in establishing genetic and evolutionary relationships among these organisms.

Invasion of erythrocytes by malaria parasites involves the recognition of Duffy blood group antigen and sialic acids of erythrocyte glycophorin by different binding proteins expressed by different Plasmodium species. The P. knowlesi gene family coding for the Duffy binding protein was cloned and characterized. Comparisons to the genetic structures of homologous genes in P. falciparum and P. vivax showed that the proteins are members of the same gene family. Sequence conservation in these genes may indicate that these regions code for the receptor binding function.

To the Editor: We report a case of Plasmodium malariae-related multiple organ dysfunction syndrome (MODS) in a healthy immunocompetent patient. Despite extensive investigation, P. malariae was the only pathogen identified. [...]

The rodent malaria model Plasmodium chabaudi was used to test the hypothesis that the extent of competitive release (total fitness gain for resistant parasites) could be restricted further by reducing drug concentrations in short duration regimens while still alleviating the clinical symptoms of disease. The higher dose treatment was shown to result in stronger and more prolonged positive selection for resistance compared to the low-dose treatment and hence milder drug dosages might achieve better resistance management.

The structures of N-terminally truncated (residues Met30-Arg396) dihydroorotate dehydrogenase (DHODH) in complex with two inhibitors as well as the first structure of the enzyme to be characterized without any bound inhibitor are described. The studies have provided new insights into the dynamic features of the DHODH reaction and have provided new approaches to the design of inhibitors against DHODH.

The properties of Hs-18Pf-SAHH, human enzyme with plasmodial helix 18 and Tc-18Hs-SAHH with trypanosomal enzyme with human helix 18 are studied. The stability in helix 18 in SAHHs, estimated from the helix propensities is observed to be greater in case of Hs-SAHH than Tc-SAHH and Pf-SAHH.

Report is presented on discovery of a mechanism that accounts for a conserved protein required for gamete fusion using gene discovery methods. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene.

This review discusses the unusual features of Plasmodium genome as exhibited by the highly A+T rich rodent malarial parasites. The genome is characterized by displaying large amount of low complexity inserts in their protein globular domains, differences in its transcription machinery, its DNA repair system with vulnerability for mutations, and 'animal-like' adhesion molecules on the cell surface.

Most studies of gene expression in Plasmodium have been concerned with asexual and/or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) [is less than or equal to] [10.sup.-4]), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.

Malaria has been invoked perhaps more than any other infectious disease as a force in the selection of human genetic polymorphisms. Evidence for genome shaping interactions can be found in the geographic and ethnic distributions of the hemoglobins, blood group antigens, thalassemias, host cell membrane molecules, HLA classes and cytokines. Human genetic variations and immuno-protective defenses correspondingly influence the structure and polymorphisms of Plasmodium parasite populations, notably in genes that affect the success and virulence of infection. Coevolutionary adaptations likewise occur with the mosquitoes responsible for the transmission of malaria parasites in human populations. Four Plasmodium species are responsible for the usual human malarias. The species responsible for the most acute and deadly form of malaria, P. falciparum, is related to a avian parasite and may be a recent introduction into humans. The three other parasite species have emerged from longstanding evolutionary relationships with primates, which may explain their tendency to produce less severe forms of the disease. In Africa, where the selective force from P. falciparum is strongest, the severity of malaria and its manifestations vary in prevalence among human populations. Understanding the malaria-protective factors and susceptibility determinants in these populations should provide insights into pathogenesis and perhaps provide a foundation for advances against the disease.

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.

Investigation of a genetically attenuated malaria parasite--which infects but does not kill its host--provides insight into how to develop a malaria vaccine (pages 954-958). Malaria kills more than 1 million [...]

Plasmodium, the mosquito-borne parasites responsible for malaria, have developed a range of molecular strategies to avoid a host blockade against invasion. New research in this area has identified an unusual and very interesting method of clonal phenotypic variation used by the Plasmodium species P. yoelii to avoid blockade by its host, a small African rodent. This molecular variation takes place during the development of the invasive merozoite parasite form. Researchers analyzed p235 transcripts in individual parasites, and now believe that a single p235 gene is transcribed in each nucleus that is replicated during schizogony.

Two fundamental assumptions underlying the evolutionary trade-off model are tested using the rodent malaria Plasmodium chabaudi. These assumptions, that higher replication rates lead to higher levels of virulence and that higher replication rates lead to higher transmission, are shown to be justified in malaria parasites. This was demonstrated by measuring virulence and transmission in eight parasite clones isolated from the wild, thereby creating a system that allows across-clone genetic relationships between replication rate, virulence and transmission to be examined.