The South American Plasmodium falciparum var gene repertoire is limited, highly shared and possibly lacks several antigenic types.

The Plasmodium falciparum var gene family encodes large variant antigens, which are important virulence factors, and also targets of the humoral host response. The frequently observed mild outcomes of falciparum malaria in many places of the Amazon area prompted us to ask whether a globally restricted variant (var) gene repertoire is present in currently circulating and older isolates of this area. By exhaustive analysis of var gene tags from 89 isolates and clones taken during many years from all over the Brazilian Amazon, we estimate that there are probably no more than 350-430 distinct sequence types, less than for any similar sized area studied so far. Detailed analysis of the var tags from genetically distinct clones obtained from single isolates revealed restricted and redundant repertoires suggesting either a low incidence of infective bites or restricted variant gene diversity in inoculated parasites. Additionally, we found a structuring of var gene repertoires observed as a higher pairwise typing sharing in isolates from the same microregion compared to isolates from different regions. Fine analysis of translated var tags revealed that certain Distinct Sequence Identifiers (DSIDs) were differently represented in Brazilian/South American isolates when compared to datasets from other continents. By global alignment of worldwide var DBLalpha sequences and sorting in groups with more than 76% identity, 125 clusters were formed and more than half of all genes were found in nine clusters with 50 or more sequences. While Brazilian/South American sequences were represented only in 64 groups, African sequences were found in the majority of clusters. DSID type 1 related sequences accumulated almost completely in one single cluster, indicating that limited recombination occurs in these specific var gene types. These data demonstrate the so far highest pairwise type sharing values for the var gene family in isolates from all over an entire subcontinent. The apparent lack of specific sequences types suggests that the P. falciparum transmission dynamics in the whole Amazon are probably different from any other endemic region studied and possibly interfere with the parasite's ability to efficiently diversify its variant gene repertoires.

Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates.

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000-2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum.

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P. falciparum populations with low rates of classical meiotic recombination.

Widespread occurrence of antibodies against circumsporozoite protein and against blood forms of Plasmodium vivax, P. falciparum and P. malariae in Brazilian wild monkeys.

BACKGROUND: A survey of malaria antibodies was carried out over 7 years and a total of 777 serum samples from wild monkeys were collected in three distinct ecological areas of Brazil where autochthonous malaria has been reported: the 'Cerrado' (similar to savanna), the Atlantic Forest and the Atlantic Semideciduous Forest. METHODS: We carried out enzyme-linked immunosorbent assay to investigate the presence of IgG antibodies against peptides of the circumsporozoite protein (CSP) repeat region of 'classic'Plasmodium vivax, P. vivax VK247, human P. vivax-like/P. simiovale, P. brasilianum/P. malariae and P. falciparum. We also carried out immunofluorescence assay with asexual forms of P. vivax, P. malariae and P. falciparum. RESULTS: The high prevalence of antibodies against CSP in all areas indicates that the monkeys had intense contact with sporozoites from infected anophelines. The immune response against asexual forms of Plasmodium in the monkeys from the Atlantic Forest indicates the development of the infection. CONCLUSIONS: We discuss the possibility of monkeys being malaria reservoirs in non-endemic areas.

Differential recognition of Plasmodium falciparum merozoite surface protein 2 variants by antibodies from malaria patients in Brazil.

Four variants of merozoite surface protein 2 (MSP-2) of Plasmodium falciparum were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.

Genetic relatedness of Plasmodium falciparum isolates and the origin of allelic diversity at the merozoite surface protein-1 (MSP-1) locus in Brazil and Vietnam.

BACKGROUND: Despite the extensive polymorphism at the merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum, that encodes a major repetitive malaria vaccine candidate antigen, identical and nearly identical alleles frequently occur in sympatric parasites. Here we used microsatellite haplotyping to estimate the genetic distance between isolates carrying identical and nearly identical MSP-1 alleles. METHODS: We analyzed 28 isolates from hypoendemic areas in north-western Brazil, collected between 1985 and 1998, and 23 isolates obtained in mesoendemic southern Vietnam in 1996. MSP-1 alleles were characterized by combining PCR typing with allele-specific primers and partial DNA sequencing. The following single-copy microsatellite markers were typed : Polyalpha, TA42 (only for Brazilian samples), TA81, TA1, TA87, TA109 (only for Brazilian samples), 2490, ARAII, PfG377, PfPK2, and TA60. RESULTS: The low pair-wise average genetic distance between microsatellite haplotypes of isolates sharing identical MSP-1 alleles indicates that epidemic propagation of discrete parasite clones originated most identical MSP-1 alleles in parasite populations from Brazil and Vietnam. At least one epidemic clone propagating in Brazil remained relatively unchanged over more than one decade. Moreover, we found no evidence that rearrangements of MSP-1 repeats, putatively created by mitotic recombination events, generated new alleles within clonal lineages of parasites in either country. CONCLUSION: Identical MSP-1 alleles originated from co-ancestry in both populations, whereas nearly identical MSP-1 alleles have probably appeared independently in unrelated parasite lineages.

Genetic diversity and differentiation in natural Plasmodium falciparum populations inferred by molecular typing of the merozoite surface proteins 1 and 2

Genetic diversity and differentiation, inferred by typing the polymorphic genes coding for the merozoite surface proteins 1 (Msp-1) and 2 (Msp-2), were compared for 345 isolates belonging to seven Plasmodium falciparum populations from three continents. Both loci yielded similar estimates of genetic diversity for each population, but rather different patterns of between-population differentiation, suggesting that natural selection on these loci, rather than the transmission dynamics of P. falciparum, determines the variation in allele frequencies among populations

Genetic diversity and differentiation in natural Plasmodium falciparum populations inferred by molecular typing of the merozoite surface proteins 1 and 2.

Genetic diversity and differentiation, inferred by typing the polymorphic genes coding for the merozoite surface proteins 1 (Msp-1) and 2 (Msp-2), were compared for 345 isolates belonging to seven Plasmodium falciparum populations from three continents. Both loci yielded similar estimates of genetic diversity for each population, but rather different patterns of between-population differentiation, suggesting that natural selection on these loci, rather than the transmission dynamics of P. falciparum, determines the variation in allele frequencies among populations.

Produçäo e caracterizaçäo de alfa-amilase produzida por Rhizopus sp / Production and characterization of alfa-amilase from Rhizopus sp

A strain of Rhizopus sp. screened among more than 800 filamentous fungi showed great ability to produce a thermostable alfa-amylase by solid state fermentation. The best production was obtained with a bran moisture content of 40 per cent when the enzyme activity reached 60 EU/g. of medium. During the purification procedures, a column of DEAE- Sephadex A-50 separeted the enzyme in two fractions and the larger (85 per cent of the total activity) showed optimum pH in a range from 4.0 to 5.6. Optimum temperature was found at 60-65§ C and in this range no loss of activity was observed after 60 min. of treatment in pH 5,0. Its Km and Vm are, respectively, of 5.0 mg/ml of starch and 10,01 uMol of reducing sugar/min./mg. of protein. Its molecular weight was calculated in 64.000 by gel filtration in Sephadex G-200. The dextrinization power of the enzyme was observed preferentialy on substrates compound by chain with higher ramifications, traht is: amylopectin > starch> amylose. Other aspects of the enzyme pattern action are also discussed