RESUMO
Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.
Assuntos
Carcinogênese/genética , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/genética , Amplificação de Genes , Oncogenes , Aneuploidia , Animais , Carcinogênese/metabolismo , Instabilidade Cromossômica , Aberrações Cromossômicas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cariotipagem Espectral/métodos , Transcrição Gênica , Transcriptoma , Bexiga Urinária/citologiaRESUMO
Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.
Assuntos
Antígenos CD/biossíntese , Neoplasias Colorretais/metabolismo , Glicoproteínas/biossíntese , Antígeno AC133 , Animais , Biópsia/métodos , Células CACO-2 , Linhagem Celular Tumoral , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Citometria de Fluxo/métodos , Genoma , Genômica/métodos , Humanos , Lasers , Camundongos , Camundongos Nus , Microdissecção , Transplante de Neoplasias , Peptídeos , Transcrição GênicaRESUMO
In July 2017, a massive bloom of the potentially toxic cyanobacterial species Planktothrix sp. was observed in the Béni-Haroun Reservoir (Algeria), which was followed by a massive fish death. Many questions were raised in association with the role of cyanotoxins and the fish massive mortality. The objective of this paper is twofold: (1) to investigate the variability of physicochemical and cyanobacterial parameters (chlorophyll-a, phycocyanin, allophycocyanin, and microcystins) throughout the period of July 2017 to June 2018; and (2) to determine the free and total MC levels in viscera and muscle tissues of the common carp (Cyprinus carpio), which are found dead in the considered reservoir in October 2017. Our results showed microcystin (MC) concentrations in water samples (by the protein phosphatase PP2A assay) had reached 651.2 ng MC-LR equiv./L. Total MC levels (free + bound) in the viscera and muscle tissues of sampled dead fish were at 960.24 and 438.54 µg MC-LR equiv./kg dw, respectively. It is assumed that high concentrations of MC observed in the tissues of common carp induced a strong degradation of the visceral contents resulting in the complete lysis of the hepatopancreas, and presumably the massive fish death.
Assuntos
Carpas , Cianobactérias , Proliferação Nociva de Algas , Animais , Argélia , Clorofila , Cianobactérias/patogenicidade , Microcistinas/toxicidade , Fosfoproteínas Fosfatases , Ficocianina , PlanktothrixRESUMO
To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.
Assuntos
Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 8 , Neoplasias Colorretais/metabolismo , Hibridização Genômica Comparativa/métodos , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Cariotipagem Espectral/métodos , Transcrição GênicaRESUMO
The nonrandom positioning of chromosome territories in eukaryotic cells is largely correlated with gene density and is conserved throughout evolution. Gene-rich chromosomes are predominantly central, while gene-poor chromosomes are peripherally localized in interphase nuclei. We previously demonstrated that artificially introduced human chromosomes assume a position equivalent to their endogenous homologues in the diploid colon cancer cell line DLD-1. These chromosomal aneuploidies result in a significant increase in transcript levels, suggesting a relationship between genomic copy number, gene expression, and chromosome position. We previously proposed that each chromosome is marked by a "zip code" that determines its nonrandom position in the nucleus. In this paper, we investigated (1) whether mouse nuclei recognize such determinants of nuclear position on human chromosomes to facilitate their distinct partitioning and (2) if chromosome positioning and transcriptional activity remain coupled under these trans-species conditions. Using three-dimensional fluorescence in situ hybridization, confocal microscopy, and gene expression profiling, we show (1) that gene-poor and gene-rich human chromosomes maintain their divergent but conserved positions in mouse-human hybrid nuclei and (2) that a foreign human chromosome is actively transcribed in mouse nuclei. Our results suggest a species-independent conserved mechanism for the nonrandom positioning of chromosomes in the three-dimensional interphase nucleus.
Assuntos
Núcleo Celular/fisiologia , Posicionamento Cromossômico , Cromossomos Humanos/fisiologia , Expressão Gênica/fisiologia , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Cromossomos Humanos/genética , Cromossomos Humanos/ultraestrutura , Expressão Gênica/genética , Genoma/genética , Genoma/fisiologia , Humanos , CamundongosRESUMO
Gravitactic bioconvective patterns created by Tetrahymena pyriformis in a Hele-Shaw apparatus were realized and compared with theoretical results. There were found to be two thresholds for bio-convection development: the first indicates the transition from the diffusion to the steady convection state; the second corresponds to the transition from the steady to the unsteady convection state. The results showed that the Hele-Shaw apparatus may be used as a physical analogy of porous media to study 2D bioconvection, with possible extensions to larger scale biological systems where population growth and distribution are driven by similar bio-physical interactions.
Assuntos
Gravitropismo , Movimento , Tetrahymena pyriformis/fisiologia , Animais , Modelos BiológicosRESUMO
Hepatotoxic microcystins (MCs) are the most widespread class of cyanotoxins and the one that has most often been implicated in cyanobacterial toxicosis. One of the main challenges in studying and monitoring MCs is the great structural diversity within the class. The full chemical structure of the first MC was elucidated in the early 1980s and since then, the number of reported structural analogues has grown steadily and continues to do so, thanks largely to advances in analytical methodology. The structures of some of these analogues have been definitively elucidated after chemical isolation using a combination of techniques including nuclear magnetic resonance, amino acid analysis, and tandem mass spectrometry (MS/MS). Others have only been tentatively identified using liquid chromatography-MS/MS without chemical isolation. An understanding of the structural diversity of MCs, the genetic and environmental controls for this diversity and the impact of structure on toxicity are all essential to the ongoing study of MCs across several scientific disciplines. However, because of the diversity of MCs and the range of approaches that have been taken for characterizing them, comprehensive information on the state of knowledge in each of these areas can be challenging to gather. We have conducted an in-depth review of the literature surrounding the identification and toxicity of known MCs and present here a concise review of these topics. At present, at least 279 MCs have been reported and are tabulated here. Among these, about 20% (55 of 279) appear to be the result of chemical or biochemical transformations of MCs that can occur in the environment or during sample handling and extraction of cyanobacteria, including oxidation products, methyl esters, or post-biosynthetic metabolites. The toxicity of many MCs has also been studied using a range of different approaches and a great deal of variability can be observed between reported toxicities, even for the same congener. This review will help clarify the current state of knowledge on the structural diversity of MCs as a class and the impacts of structure on toxicity, as well as to identify gaps in knowledge that should be addressed in future research.
Assuntos
Microcistinas , Animais , Humanos , Microcistinas/biossíntese , Microcistinas/química , Microcistinas/toxicidadeRESUMO
Toxic cyanobacteria (CB) blooms are being reported in an increasing number of water bodies worldwide. As drinking water (DW) treatment can be disrupted by CB, in addition to long term management plans, short term operational decision-making tools are needed that enable an understanding of the temporal variability of CB movement in relation to drinking water intakes. In this paper, we propose a novel conservative model based on a Eulerian framework and compare results with data from CB blooms in Missisquoi Bay (Québec, Canada). The hydrodynamic model considered the effects of wind and light intensity, demonstrated that current understanding of cell buoyancy in relation to light intensity in full-scale systems is incomplete and some factors are yet to be fully characterized. Factors affecting CB buoyancy play a major role in the formation of a thin surface layer that could be of ecological importance with regards to cell concentrations and toxin production. Depending on velocities, wind contributes either to the accumulation or to the dispersion of CB. Lake recirculation effects have a tendency to create zones of low CB concentrations in a water body. Monitoring efforts and future research should focus on short-term variations of CB throughout the water column and the characterization of factors other than light intensity that affect cell buoyancy. These factors are critical for understanding the risk of breakthrough into treatment plants as well as the formation of surface scums and subsequent toxin production.
Assuntos
Cianobactérias/fisiologia , Água Potável/microbiologia , Eutrofização , Hidrodinâmica , Lagos/microbiologia , Luz , Modelos Teóricos , Quebeque , Análise Espaço-Temporal , Purificação da Água , VentoRESUMO
BACKGROUND: Identification of single nucleotide polymorphisms (SNPs) associated with gene expression levels, known as expression quantitative trait loci (eQTLs), may improve understanding of the functional role of phenotype-associated SNPs in genome-wide association studies (GWAS). The small sample sizes of some previous eQTL studies have limited their statistical power. We conducted an eQTL investigation of microarray-based gene and exon expression levels in whole blood in a cohort of 5257 individuals, exceeding the single cohort size of previous studies by more than a factor of 2. RESULTS: We detected over 19,000 independent lead cis-eQTLs and over 6000 independent lead trans-eQTLs, targeting over 10,000 gene targets (eGenes), with a false discovery rate (FDR) < 5%. Of previously published significant GWAS SNPs, 48% are identified to be significant eQTLs in our study. Some trans-eQTLs point toward novel mechanistic explanations for the association of the SNP with the GWAS-related phenotype. We also identify 59 distinct blocks or clusters of trans-eQTLs, each targeting the expression of sets of six to 229 distinct trans-eGenes. Ten of these sets of target genes are significantly enriched for microRNA targets (FDR < 5%). Many of these clusters are associated in GWAS with multiple phenotypes. CONCLUSIONS: These findings provide insights into the molecular regulatory patterns involved in human physiology and pathophysiology. We illustrate the value of our eQTL database in the context of a recent GWAS meta-analysis of coronary artery disease and provide a list of targeted eGenes for 21 of 58 GWAS loci.
Assuntos
Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genômica , Locos de Características Quantitativas , Adulto , Idoso , Alelos , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , NavegadorRESUMO
The emerging technology of microarray analysis allows the establishment of molecular portraits of prostate cancer and the discovery of novel genes involved in the carcinogenesis process. Many novel genes have already been identified using this technique, and functional analyses of these genes are currently being tested. The combination of microarray analysis with other recently developed high-throughput techniques, such as proteomics, tissue arrays, and gene promoter-methylation, especially using tissue microdissection methods, will provide us with more comprehensive insights into how prostate cancer develops and responds to gene-targeted therapies. Animal models of prostate cancer are being characterized by high throughput techniques to better define the similarities and differences between those models and the human disease, and to determine whether particular models may be useful for specific targeted therapies in pre-clinical studies. Although profiling of mRNA expression provides important information of gene expression, the development of proteomic technologies will allow for an even more precise global insight into cellular signaling and structural alterations during prostate carcinogenesis. Not only will the "omic" revolution change basic science, but it will lead to a new era of molecular medicine.
Assuntos
Biomarcadores Tumorais/metabolismo , Perfilação da Expressão Gênica , Genômica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Animais , Biomarcadores Tumorais/genética , Humanos , Masculino , Neoplasias da Próstata/metabolismoRESUMO
The sudden appearance of toxic cyanobacteria (CB) blooms is still largely unpredictable in waters worldwide. Many post-hoc explanations for CB bloom occurrence relating to physical and biochemical conditions in lakes have been developed. As potentially toxic CB can accumulate in drinking water treatment plants and disrupt water treatment, there is a need for water treatment operators to determine whether conditions are favourable for the proliferation and accumulation of CB in source waters in order to adjust drinking water treatment accordingly. Thus, a new methodology with locally adaptable variables is proposed in order to have a single index, f(p), related to various environmental factors such as temperature, wind speed and direction. The index is used in conjunction with real time monitoring data to determine the probability of CB occurrence in relation to meteorological factors, and was tested at a drinking water intake in Missisquoi Bay, a shallow transboundary bay in Lake Champlain, Québec, Canada. These environmental factors alone were able to explain a maximum probability of 68% that a CB bloom would occur at the drinking water treatment plant. Nutrient limitation also influences CB blooms and intense blooms only occurred when the dissolved inorganic nitrogen (DIN) to total phosphorus (TP) mass ratio was below 3. Additional monitoring of DIN and TP could be considered for these source waters prone to cyanobacterial blooms to determine periods of favourable growth. Real time monitoring and the use of the index could permit an adequate and timely response to CB blooms in drinking water sources.
Assuntos
Cianobactérias , Água Potável/microbiologia , Microbiologia da Água , Tempo (Meteorologia) , Lagos/microbiologia , Modelos LogísticosRESUMO
In the process of human hematopoiesis, precise regulation of the expression of lineage-specific gene products is critical for multiple cell-fate decisions that govern cell differentiation, proliferation, and self-renewal. Given the important role of microRNAs (miRNAs) in development and differentiation, we examined the global expression of miRNA in CD34(+) cells during lineage specific hematopoiesis and found 49 miRNAs to be differentially expressed, with functional roles in cellular growth and proliferation, and apoptosis. miR-18a was upregulated during erythropoiesis and downregulated during megakaryopoiesis. miR-145 was upregulated during granulopoiesis and down regulated during erythropoiesis. Megakaryopoitic differentiation resulted in significant alteration in the expression of many miRNAs that are believed to play critical roles in the regulation of B and T cell differentiation. Target prediction analyses on three different miRNA databases indicated that TargetScan outperformed microCosm and miRDB in identifying potential miRNA targets associated with hematopoietic differentiation process. An integrated analysis of the observed miRNAs and messenger RNAs (mRNAs) resulted in 87 highly correlated miRNA-mRNA pairs that have major functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/B-catenin and Flt 3 signaling pathways. We believe that this study will enhance our understanding on the regulatory roles of miRNA in hematopoiesis by providing a library of mRNA-miRNA networks.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , MicroRNAs/análise , MicroRNAs/fisiologia , RNA Mensageiro/análise , Diferenciação Celular , Linhagem da Célula , Humanos , Transcriptoma , Tirosina Quinase 3 Semelhante a fms/fisiologiaRESUMO
Analysis of centrosome number and structure has become one means of assessing the potential for aberrant chromosome segregation and aneuploidy in tumor cells. Centrosome amplification directly causes multipolar catastrophic mitoses in mouse embryonic fibroblasts (MEFs) deficient for the tumor suppressor genes Brca1 or Trp53. We observed supernumerary centrosomes in cell lines established from aneuploid, but not from diploid, colorectal carcinomas; however, multipolar mitoses were never observed. This discrepancy prompted us to thoroughly characterize the centrosome abnormalities in these and other cancer cell lines with respect to both structure and function. The most striking result was that supernumerary centrosomes in aneuploid colorectal cancer cell lines were unable to nucleate microtubules, despite the presence of gamma-tubulin, pericentrin, PLK1, and AURKA. Analysis by scanning electron microscopy revealed that these supernumerary structures are devoid of centrioles, a result significantly different from observations in aneuploid pancreatic cancer cell lines and in Trp53 or Brca1 deficient MEFs. Thus, multipolar mitoses are dependent upon the ability of extra gamma-tubulin containing structures to nucleate microtubules, and this correlated with the presence of centrioles. The assessment of centrosome function with respect to chromosome segregation must therefore take into consideration the presence of centrioles and the capacity to nucleate microtubules. The patterns and mechanisms of chromosomal aberrations in hematologic malignancies and solid tumors are fundamentally different. The former is characterized by specific chromosome translocations, whose consequence is the activation of oncogenes. Most carcinomas, however, reveal variations in the nuclear DNA content. The observed genomic imbalances and gross variations in chromosome number can result from unequal chromosome segregation during mitotic cell division. It is therefore fundamental to elucidate mechanisms involved in distribution of the genome to daughter cells. Prior to cell division, the centrosome organizes microtubules and the mitotic spindle. Deciphering the consequences of alterations in centrosome number, structure, and function is an important step towards understanding how a diploid genome is maintained. Although extra centrosomes have now been observed in carcinomas and were correlated with aneuploidy, a careful functional investigation of these structures and their role in generating chromosome imbalances may lead to the identification of distinct mechanistic pathways of genomic instability. Understanding these pathways will also be important in determining whether they are potential molecular targets of therapeutic intervention.
Assuntos
Centrossomo , Neoplasias Colorretais/patologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/ultraestrutura , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica/métodosRESUMO
Genomic aberrations on chromosome 8 are common in colon cancer, and are associated with lymph node and distant metastases as well as with disease susceptibility. This prompted us to generate a high-resolution map of genomic imbalances of chromosome 8 in 51 primary colon carcinomas using a custom-designed genomic array consisting of a tiling path of BAC clones. This analysis confirmed the dominant role of this chromosome. Unexpectedly, the position of the breakpoints suggested colocalization with structural variants in the human genome. In order to map these sites with increased resolution and to extend the analysis to the entire genome, we analyzed a subset of these tumors (n = 32) by comparative genomic hybridization on a 185K oligonucleotide array platform. Our comprehensive map of the colon cancer genome confirmed recurrent and specific low-level copy number changes of chromosomes 7, 8, 13, 18, and 20, and unveiled additional, novel sites of genomic imbalances including amplification of a histone gene cluster on chromosome 6p21.1-21.33 and deletions on chromosome 4q34-35. The systematic comparison of segments of copy number change with gene expression profiles showed that genomic imbalances directly affect average expression levels. Strikingly, we observed a significant association of chromosomal breakpoints with structural variants in the human genome: 41% of all copy number changes occurred at sites of such copy number variants (P < 2.2e(-16)). Such an association has not been previously described and reveals a yet underappreciated plasticity of the colon cancer genome; it also points to potential mechanisms for the induction of chromosomal breakage in cancer cells.
Assuntos
Aberrações Cromossômicas , Cromossomos/ultraestrutura , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Família Multigênica , Mapeamento Cromossômico , Feminino , Genoma , Genoma Humano , Histonas/metabolismo , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Polimorfismo GenéticoRESUMO
BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.
Assuntos
Adenocarcinoma/patologia , Aneuploidia , Neoplasias do Colo/patologia , Espaço Intranuclear/ultraestrutura , Adenocarcinoma/genética , Animais , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 7 , Neoplasias do Colo/genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Interfase , Camundongos , Microscopia Confocal , Transcrição Gênica , Trissomia , Células Tumorais Cultivadas/ultraestruturaRESUMO
Parvovirus B19 (B19), currently the only accepted member of the Erythrovirus genus, is the only parvovirus known to be pathogenic in humans. Recently a viral sequence, tentatively termed V9 which showed 11% variability from the published B19 sequences, was described from a patient with aplastic crisis. To search for additional parvovirus variants, we used the new NS1/7.5EC PCR assay whose primers were designed from a conserved region of the B19/V9 sequence and encompasses an MfeI restriction enzyme site that would allow differentiation between B19- and V9-like sequences. Screening of 225 serum and bone marrow samples and 62 plasma pools identified one new atypical parvovirus sequence, A6, from an anemic HIV-positive patient. A6 exhibited 88% similarity to B19 and 92% to V9, compared to >98% correspondence between reported B19 isolates. Based on the genome similarity to B19, an RT-PCR for A6 capsid transcripts was developed and used to test for A6 infectivity of UT7/Epo/S1 cells. Despite high viral titers, A6 viral transcripts were not detected. Thus, although the prevalence of B19 variants probably is low, the true clinical significance remains unknown. Current PCR analyses are unlikely to detect novel variants without the design of specific primers to the A6/V9/B19 common sequences.