Genotyping of Human parvovirus B19 among Brazilian patients with hemoglobinopathies

Human parvovirus B19 (B19V) infection can be a life-threatening condition among patients with hereditary (chronic) hemolytic anemias. Our objective was to characterize the infection molecularly among patients with sickle cell disease and thalassemia. Forty-seven patients (37 with sickle cell disease, and 10 with beta-thalassemia major) as well as 47 healthy blood donors were examined for B19V infection by anti-B19V IgG enzyme immunoassay, quantitative PCR, which detects all B19V genotypes, and DNA sequencing. B19V viremia was documented in nine patients (19.1%) as two displayed acute infection and the rest had a low titre viremia (mean 3.4 x 10(4) copies/mL). All donors were negative for B19V DNA. Anti-B19V IgG was detected in 55.3% of the patients and 57.4% among the donors. Based on partial NS1 fragments, all patient isolates were classified as genotype 1 and subgenotype 1A. The evolutionary events of the examined partial NS1 gene sequence were associated with a lack of positive selection. The quantification of all B19V genotypes by a single hydrolytic probe is a technically useful method, but it is difficult to establish relationships between B19V sequence characteristics and infection outcome.

Diversity and Adaptation of Human Respiratory Syncytial Virus Genotypes Circulating in Two Distinct Communities: Public Hospital and Day Care Center

HRSV is one of the most important pathogens causing acute respiratory tract diseases as bronchiolitis and pneumonia among infants. HRSV was isolated from two distinct communities, a public day care center and a public hospital in Sao Jose do Rio Preto - SP, Brazil. We obtained partial sequences from G gene that were used on phylogenetic and selection pressure analysis. HRSV accounted for 29% of respiratory infections in hospitalized children and 7.7% in day care center children. On phylogenetic analysis of 60 HRSV strains, 48 (80%) clustered within or adjacent to the GA1 genotype; GA5, NA1, NA2, BA-IV and SAB1 were also observed. SJRP GA1 strains presented variations among deduced amino acids composition and lost the potential O-glycosilation site at amino acid position 295, nevertheless this resulted in an insertion of two potential O-glycosilation sites at positions 296 and 297. Furthermore, a potential O-glycosilation site insertion, at position 293, was only observed for hospital strains. Using SLAC and MEME methods, only amino acid 274 was identified to be under positive selection. This is the first report on HRSV circulation and genotypes classification derived from a day care center community in Brazil.

Transcription profiling of Prss16 (Tssp) can be used to find additional peptidase genes that are candidates for self-peptide generation in the thymus

Positive selection (PS) in the thymus involves the presentation of self-peptides that are bound to MHC class II on the surface of cortical thymus epithelial cells (cTECs). Prss16 gene corresponds to one important element regulating the PS of CD4(+) T lymphocytes, which encodes Thymus-specific serine protease (Tssp), a cTEC serine-type peptidase involved in the proteolytic generation of self-peptides. Nevertheless, additional peptidase genes participating in the generation of self-peptides need to be found. Because of its role in the mechanism of PS and its expression in cTECs, the Prss16 gene might be used as a transcriptional marker to identify new genes that share the same expression profile and that encode peptidases in the thymus. To test this hypothesis, we compared the differential thymic expression of 4,500 mRNAs of wild-type (WT) C57BL/6 mice with their respective Prss16-knockout (KO) mutants by using microarrays. From these, 223 genes were differentially expressed, of which 115 had known molecular/biological functions. Four endopeptidase genes (Casp1, Casp2, Psmb3 and Tpp2) share the same expression profile as the Prss16 gene; i.e., induced in WT and repressed in KO while one endopeptidase gene, Capns1, features opposite expression profile. The Tpp2 gene is highlighted because it encodes a serine-type endopeptidase functionally similar to the Tssp enzyme. Profiling of the KO mice featured down-regulation of Prss16, as expected, along with the genes mentioned above. Considering that the Prss16-KO mice featured impaired PS, the shared regulation of the four endopeptidase genes suggested their participation in the mechanism of self-peptide generation and PS.

BF Integrase Genes of HIV-1 Circulating in Sao Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naive patients from the Sao Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naive and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

Molecular evolution of a malaria resistance gene (DARC) in primates

Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a 3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS = 0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (FST) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants. (C) 2011 Elsevier Ltd. All rights reserved.

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and beta-thalassemia major and healthy blood donors

Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and beta-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with beta-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the Sao Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection. J. Med. Virol. 84:16521665, 2012. (c) 2012 Wiley Periodicals, Inc.

Relaxation of Adaptive Evolution during the HIV-1 Infection Owing to Reduction of CD4+T Cell Counts

Background: The first stages of HIV-1 infection are essential to establish the diversity of virus population within host. It has been suggested that adaptation to host cells and antibody evasion are the leading forces driving HIV evolution at the initial stages of AIDS infection. In order to gain more insights on adaptive HIV-1 evolution, the genetic diversity was evaluated during the infection time in individuals contaminated by the same viral source in an epidemic cluster. Multiple sequences of V3 loop region of the HIV-1 were serially sampled from four individuals: comprising a single blood donor, two blood recipients, and another sexually infected by one of the blood recipients. The diversity of the viral population within each host was analyzed independently in distinct time points during HIV-1 infection. Results: Phylogenetic analysis identified multiple HIV-1 variants transmitted through blood transfusion but the establishing of new infections was initiated by a limited number of viruses. Positive selection (d(N)/d(S)>1) was detected in the viruses within each host in all time points. In the intra-host viruses of the blood donor and of one blood recipient, X4 variants appeared respectively in 1993 and 1989. In both patients X4 variants never reached high frequencies during infection time. The recipient, who X4 variants appeared, developed AIDS but kept narrow and constant immune response against HIV-1 during the infection time. Conclusion: Slowing rates of adaptive evolution and increasing diversity in HIV-1 are consequences of the CD4+ T cells depletion. The dynamic of R5 to X4 shift is not associated with the initial amplitude of humoral immune response or intensity of positive selection.

Dengue virus type 3 adaptive changes during epidemics in São Jose de Rio Preto, Brazil, 2006–2007

Global dengue virus spread in tropical and sub-tropical regions has become a major international public health concern. It is evident that DENV genetic diversity plays a significant role in the immunopathology of the disease and that the identification of polymorphisms associated with adaptive responses is important for vaccine development. The investigation of naturally occurring genomic variants may play an important role in the comprehension of different adaptive strategies used by these mutants to evade the human immune system. In order to elucidate this role we sequenced the complete polyprotein-coding region of thirty-three DENV-3 isolates to characterize variants circulating under high endemicity in the city of São José de Rio Preto, Brazil, during the onset of the 2006-07 epidemic. By inferring the evolutionary history on a local-scale and estimating rates of synonymous (dS) and nonsynonimous (dN) substitutions, we have documented at least two different introductions of DENV-3 into the city and detected 10 polymorphic codon sites under significant positive selection (dN/dS > 1) and 8 under significant purifying selection (dN/dS < 1). We found several polymorphic amino acid coding sites in the envelope (15), NS1 (17), NS2A (11), and NS5 (24) genes, which suggests that these genes may be experiencing relatively recent adaptive changes. Furthermore, some polymorphisms correlated with changes in the immunogenicity of several epitopes. Our study highlights the existence of significant and informative DENV variability at the spatio-temporal scale of an urban outbreak.

Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia)

Araucaria angustifolia, a unique species of this genus that occurs naturally in Brazil, has a high socio-economic and environmental value and is critically endangered of extinction, since it has been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi. Ninety-seven strains were isolated and subjected to chemical tests. All isolates presented at least one positive feature, characterizing them as potential PGPR. Eighteen isolates produced indole-3-acetic acid (IAA), 27 were able to solubilize inorganic phosphate, 21 isolates were presumable diazotrophs, with pellicle formation in nitrogen-free culture medium, 83 were phosphatases producers, 37 were positive for siderophores and 45 endospore-forming isolates were antagonistic to Fusarium oxysporum, a pathogen of conifers. We also observed the presence of bacterial strains with multiple beneficial mechanisms of action. Analyzing the fatty acid methyl ester (FAME) and partial sequencing of the 16S rRNA gene of these isolates, it was possible to characterize the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the species Ewingella americana as a PGPR. (C) 2011 Elsevier GmbH. All rights reserved.

Adjuvant chemoradiotherapy after d2-lymphadenectomy for gastric cancer: the role of n-ratio in patient selection. results of a single cancer center

Background: Adjuvant chemoradiotherapy is part of a multimodality treatment approach in order to improve survival outcomes after surgery for gastric cancer. The aims of this study are to describe the results of gastrectomy and adjuvant chemoradiotherapy in patients treated in a single institution, and to identify prognostic factors that could determine which individuals would benefit from this treatment. Methods: This retrospective study included patients with pathologically confirmed gastric adenocarcinoma who underwent surgical treatment with curative intent in a single cancer center in Brazil, between 1998 and 2008. Among 327 patients treated in this period, 142 were selected. Exclusion criteria were distant metastatic disease (M1), T1N0 tumors, different multimodality treatments and tumors of the gastric stump. Another 10 individuals were lost to follow-up and there were 3 postoperative deaths. The role of several clinical and pathological variables as prognostic factors was determined. Results: D2-lymphadenectomy was performed in 90.8% of the patients, who had 5-year overall and disease-free survival of 58.9% and 55.7%. The interaction of N-category and N-ratio, extended resection and perineural invasion were independent prognostic factors for overall and disease-free survival. Adjuvant chemoradiotherapy was not associated with a significant improvement in survival. Patients with node-positive disease had improved survival with adjuvant chemoradiotherapy, especially when we grouped patients with N1 and N2 tumors and a higher N-ratio. These individuals had worse disease-free (30.3% vs. 48.9%) and overall survival (30.9% vs. 71.4%). Conclusion: N-category and N-ratio interaction, perineural invasion and extended resections were prognostic factors for survival in gastric cancer patients treated with D2-lymphadenectomy, but adjuvant chemoradiotherapy was not. There may be some benefit with this treatment in patients with node-positive disease and higher N-ratio.

Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.