RESUMO
Our understanding of the phylogenetic and structural characteristics of the Merkel Cell Polyomavirus (MCPyV) is increasing but still scarce, especially in samples originating from South America. In order to investigate the properties of MCPyV circulating in the continent in more detail, MCPyV Viral Protein 1 (VP1) sequences from five basal cell carcinoma (BCC) and four saliva samples from Brazilian individuals were evaluated from the phylogenetic and structural standpoint, along with all complete MCPyV VP1 sequences available at Genbank database so far. The VP1 phylogenetic analysis confirmed the previously reported pattern of geographic distribution of MCPyV genotypes and the complexity of the South-American clade. The nine Brazilian samples were equally distributed in the South-American (3 saliva samples); North American/European (2 BCC and 1 saliva sample); and in the African clades (3 BCC). The classification of mutations according to the functional regions of VP1 protein revealed a differentiated pattern for South-American sequences, with higher number of mutations on the neutralizing epitope loops and lower on the region of C-terminus, responsible for capsid formation, when compared to other continents. In conclusion, the phylogenetic analysis showed that the distribution of Brazilian VP1 sequences agrees with the ethnic composition of the country, indicating that VP1 can be successfully used for MCPyV phylogenetic studies. Finally, the structural analysis suggests that some mutations could have impact on the protein folding, membrane binding or antibody escape, and therefore they should be further studied.
Assuntos
Proteínas do Capsídeo/genética , Variação Genética , Poliomavírus das Células de Merkel/classificação , Poliomavírus das Células de Merkel/isolamento & purificação , Filogenia , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Brasil , Carcinoma Basocelular/virologia , Epitopos de Linfócito B/genética , Poliomavírus das Células de Merkel/genética , Mutação de Sentido IncorretoRESUMO
BACKGROUND: In Brazil, two species of Plasmodium have been described infecting non-human primates, Plasmodium brasilianum and Plasmodium simium. These species are morphologically, genetically and immunologically indistinguishable from the human Plasmodium malariae and Plasmodium vivax parasites, respectively. Plasmodium simium has been observed naturally infecting monkeys of the genera Alouatta and Brachyteles in a restricted area of the Atlantic Forest in the south and southeast regions of Brazil. However, its reported geographical distribution and the diversity of its vertebrate hosts may be underestimated, since available data were largely based on analyses by microscopic examination of peripheral blood, a method with limited sensitivity, considering the potential sub-patent feature of these infections. The present study describes, for the first time, the natural infection of P. simium in capuchin monkeys from the Brazilian Atlantic Forest. METHODS: Blood samples from 30 non-human primates belonging to nine species kept in the Primate Centre of Rio de Janeiro were collected. Fragments of spleen and liver from one dead monkey found in the neighborhoods of the Primate Centre were also analysed. Molecular diagnosis was performed by nested PCR (18SSU rRNA) and the amplified fragment was sequenced. RESULTS: Thirty per cent of the captive animals were infected with P. simium and/or P. brasilianum. The dead monkey tested positive for DNA of P. simium. For the first time, Cebinae primates (two specimens of genus Cebus and two of genus Sapajos) were found naturally infected by P. simium. The infection was confirmed by sequencing a small fragment of 18SSU rRNA. CONCLUSION: The results highlight the possibility of infection by P. simium in other species of non-human primates whose impact could be significant for the malaria epidemiology among non-human primates and, if it becomes clear that this P. simium is able to infect monkeys and, eventually, man, also for the maintenance of transmission of human malaria in the context of a zoonosis in areas under influence of the Atlantic Forest.
Assuntos
Cebinae , Malária/veterinária , Doenças dos Macacos/epidemiologia , Plasmodium/fisiologia , Animais , Animais de Laboratório , Brasil/epidemiologia , Feminino , Malária/epidemiologia , Malária/parasitologia , Masculino , Doenças dos Macacos/parasitologia , Reação em Cadeia da Polimerase/veterinária , PrevalênciaRESUMO
Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.
Assuntos
Antígenos Virais de Tumores/genética , Vírus BK/classificação , Vírus BK/genética , Variação Genética , Mutação de Sentido Incorreto , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Vírus BK/isolamento & purificação , DNA Viral/química , DNA Viral/genética , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.
Assuntos
Cloroquina/farmacologia , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos/genética , Variação Genética , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/genética , Brasil/epidemiologia , Doenças Endêmicas/estatística & dados numéricos , Marcadores Genéticos , Humanos , Malária Vivax/sangue , Malária Vivax/epidemiologia , Testes de Sensibilidade Parasitária , Fenótipo , Reação em Cadeia da Polimerase , Distribuição AleatóriaRESUMO
The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.
Assuntos
Humanos , Cloroquina/farmacologia , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos/genética , Variação Genética , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/genética , Brasil/epidemiologia , Doenças Endêmicas/estatística & dados numéricos , Marcadores Genéticos , Malária Vivax/sangue , Malária Vivax/epidemiologia , Testes de Sensibilidade Parasitária , Fenótipo , Reação em Cadeia da Polimerase , Distribuição AleatóriaRESUMO
Plasmodium falciparum with reduced sensitivity to artemisinin derivatives has been observed in endemic areas, but the molecular mechanisms for this reduced sensitivity remain unclear. We evaluated the association between in vitro susceptibility of P. falciparum isolates obtained from southwest Nigeria and polymorphisms in selected putative transporter genes (PFE0775C, PF13_0271, pfmrp1, pfcrt, and pfmdr1). Modified schizont inhibition assay was used to determine the in vitro parasite susceptibility to artemether (ATH). Polymorphisms in selected genes were detected by polymerase chain reaction followed by direct DNA sequencing. The half-maximal inhibitory concentration (IC(50)) geometric mean (GM) for all P. falciparum isolates was 1.78 nM (range, 0.03-10.43 nM). Polymorphisms at codons 241, 86, and 76 of PFE0775C, pfmdr1, and pfcrt genes, respectively, were associated with reduced susceptibility to ATH. A new S263P single-nucleotide polymorphism on the PFE0775C gene was also detected in 27% of the isolates. Patient isolates harboring V241L or S263P polymorphisms on the PFE0775C gene showed increased IC(50) (GM: 3.08 nM and 1.79 nM, respectively). Plasmodium falciparum isolates harboring mutant Y86 pfmdr1 and P263 PFE0775C alleles showed a 2.5-5.5-fold increase in ATH IC(50.) This study shows that polymorphisms on the PFE0775C and pfmdr1 genes are associated with reduced sensitivity to ATH in fresh isolates of P. falciparum from Nigeria.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Proteínas de Transporte/genética , Resistência a Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético , Artemeter , Proteínas de Transporte/metabolismo , Criança , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: Anti-malarial drug resistance has emerged as one of the biggest challenges confronting the worldwide effort to control malaria. The appearance of chloroquine and multi-drug resistance had devastating effects on therapeutic efficacy of former first-line agents. Artemisinin has proven to be an excellent therapeutic alternative to fill the void in chemotherapeutic options left by resistance mechanisms. At the time of introduction, no resistance to artemisinins had been recorded, and artemisinins demonstrated excellent parasite reduction rates. In an attempt to protect artemisinin efficacy, the World Health Organization (WHO) made artemisinin-based combination therapy (ACT) its official first-line treatment recommendation for uncomplicated Plasmodium falciparum in 2006. In Brazil, artemether/lumefantrine became the Brazilian Malaria Control Programme's official treatment recommendation in 2007. The sarco/endoplasmic reticulum Ca2+ - ATPase ortholog of P. falciparum (pfatp6) has been suggested as one of the targets of artemisinins. Consequently, pfatp6 gene polymorphisms are being investigated as markers of artemisinin resistance elsewhere. The goal of this work was to describe the molecular profile of pfatp6 in P. falciparum isolates from different localities in the Amazonas State. METHODS: DNA polymorphisms of the pfatp6 gene in 80 P. falciparum isolates from 11 municipalities of the Amazonas State (Western Brazilian Amazon), before and after the introduction of ACT in the Brazilian anti-malarial guidelines, were analysed by automatic sequencing. Mutations in the pfatp6 gene were searched using Mutation Surveyor v3.25 software. RESULTS: The P. falciparum pfatp6 gene presented polymorphisms at codons 37, 630 and 898. The R37K mutation was found in 16% of the samples, A630S in 32% and I898I in 52%. No S769N mutation, however, was detected in the analysed samples. CONCLUSION: Despite the small number of samples, data presented here provide baseline information about polymorphisms of pfatp6 gene before and after exposure to ACT in a low transmission area, which will help to infer drug selection pressure in this area in the future.
Assuntos
ATPases Transportadoras de Cálcio/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Polimorfismo Genético , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Brasil , DNA de Protozoário/química , DNA de Protozoário/genética , Feminino , Humanos , Lactonas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/genética , Mutação , Plasmodium falciparum/classificação , Análise de Sequência de DNA , Adulto JovemRESUMO
The effect of antimalarial drug selection on pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from two distinct geographical locations was determined in 70 and 18 P. falciparum isolates from Nigeria and Brazil, respectively, using nested polymerase chain reaction and direct DNA sequencing approaches. All isolates from Brazil and 72% from Nigeria harbored the mutant SVMNT and CVIET pfcrt haplotype, respectively. The pfcrt CVMNT haplotype was also observed in (7%) of the Nigerian samples. One hundred percent (100%) and 54% of the parasites from Brazil and Nigeria, respectively, harbored wild-type pfmdr1Asn86. We provide first evidence of emergence of the CVMNT haplotype in West Africa. The high prevalence of pfcrt CVIET and SVMNT haplotypes in Nigeria and Brazil, respectively, is indicative of different selective pressure by chloroquine and amodiaquine. Continuous monitoring of pfcrt SVMNT haplotype is required in endemic areas of Africa, where artesunate-amodiaquine combination is used for treatment of acute uncomplicated malaria.
Assuntos
Antimaláricos/uso terapêutico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Alelos , Amodiaquina/uso terapêutico , Artemisininas/uso terapêutico , Brasil/epidemiologia , Variações do Número de Cópias de DNA , Combinação de Medicamentos , Genótipo , Haplótipos , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Família Multigênica , Mutação , Nigéria/epidemiologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNARESUMO
BACKGROUND: Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. METHODS: Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. RESULTS: GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. CONCLUSION: Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.
Assuntos
Predisposição Genética para Doença/genética , Genética Populacional , Glicoforinas/genética , Malária Falciparum/genética , Plasmodium falciparum/fisiologia , Polimorfismo Genético/genética , Alelos , Brasil/epidemiologia , Estudos de Casos e Controles , Doenças Endêmicas , Frequência do Gene , Marcadores Genéticos , Humanos , Malária Falciparum/epidemiologiaRESUMO
We described differential selective pressures along codon sites of the RT and PR genes of HIV-1 from HAART-failing and naïve-treated individuals, through the comparison of the ratio of non-synonymous mutations (d(N)) to synonymous mutations (d(S)) substitution per site. Resistance-associated mutations were found in 1/71 (1.4%) and 109/117 (93.1%) samples from naïve-treated and HAART-failing individuals, respectively, although most of positively selected codons represented polymorphisms in positions 123, 211, 245, 297 in RT and 37, 63 in PR of naïve-treated samples and positions 122, 123, 245, 272, 277, 286, 297 in RT and 10, 15, 20, 35, 37, 62, 63, 64, 71, 72, 77, 93 in PR of HAART-failing samples, except by ARV-resistance codons 74, 184, 215 in RT and 90 in PR exclusively found in HAART-failing group. The number and diversity of sites under selective pressure at populational level also increased in RT but not in PR of treated individuals. Our results demonstrated no evolution of drug-associated codons among untreated individuals, indicating unlikely transmission and adaptation of resistant HIV-1 strains in a free drug environment. Polymorphic sites observed exclusively in HAART-failing group, may contribute to HIV-1 escape and adaptation at individual and populational levels in a drug environment, although those mutually found in HIV-1 despite of previous exposure to ARV treatment, should not be considered accurate resistance markers.